Method for improving breeding efficiency of braconidae splendens

Abstract

The invention provides a method for improving the breeding efficiency of braconus splendens, and belongs to the technical field of insect breeding. According to the method, female bees which are 5-day-old, 6-day-old and 7-day-old and have no egg laying experience after eclosion are used for parasitizing third-year-old Asiatic corn borer larvae. And the parasitism rate, the eclosion offspring number and the female bee number of the braconus splendens are obviously improved. The method is carried out in a small space range, so that the encountering probability of the braconus splendens and the hosts is increased, the time of searching for the hosts by the braconus splendens is shortened, and the parasitic rate of parasitic wasps is increased. The method disclosed by the invention has important significance in efficiently breeding the cotesia macrorrhiza by using the Asiatic corn borer larvae indoors and guiding the field to release the cotesia macrorrhiza.

Description

Technical Field

[0001] The invention relates to the technical field of insect reproduction, and in particular to a method for improving the reproduction efficiency of the belt-necked braconid wasp. Background Art

[0002] The Asian corn borer (Ostrinafurnacalis) is a worldwide pest with a wide distribution range. It is an important pest on corn in my country, seriously affecting the yield and quality of corn. It was included in the list of Class I crop pests and diseases in my country in 2023. At present, the prevention and control methods of the Asian corn borer include chemical control, insect pheromone attraction, and release of trichogrammatids. Chemical control can cause environmental pollution and affect food safety and human health. Insect pheromone attraction mainly kills adult Asian corn borers and has a limited spatial area of ​​effect. Trichogrammatids parasitize the eggs of the Asian corn borer, and are mainly used for prevention and control during the egg stage of the Asian corn borer, and the effect is short-lived. However, there are few reports on biological control methods for the larval stage of the Asian corn borer, when the damage is most serious and lasts the longest.

[0003] Macrocentrus cingulum is the dominant parasitic wasp in the larval stage of the Asian corn borer. The wasp can parasitize 1-4 instar larvae of the Asian corn borer, and the natural parasitism rate in the field can reach 30-40%. The bee has polyembryonic development. The egg laid by the female bee into the host body can develop into dozens to hundreds of embryos after multiple divisions, and finally produce a composite cocoon containing a large number of bee cocoons on the remains of the host. However, when breeding in the laboratory, the number of offspring that can be produced from a single composite cocoon varies greatly, ranging from 20 to 90. Since Macrocentrus cingulum can lay eggs after emergence, the egg-laying period can last for about 7 days, and it can parasitize 1-4 instar larvae of the Asian corn borer, it is particularly important to achieve the optimal indoor breeding system of Macrocentrus cingulum based on the proliferation potential of the parasitic wasp eggs of Macrocentrus cingulum and its preference for the host age.

[0004] Chinese patent CN109874752A discloses a diapause regulation and storage method for the long-bodied belt-shaped braconid. In the embodiment, the diapause induction conditions of the long-bodied belt-shaped braconid and the influence on the offspring emergence rate, etc. are mainly focused on. The efficient reproduction conditions that affect the number of offspring related to the polyembryonic development characteristics of the long-bodied belt-shaped braconid are not involved. Summary of the invention

[0005] The purpose of the present invention is to address the above-mentioned deficiencies in the prior art and provide a method for improving the reproductive efficiency of the long-bodied belt-necked wasp, utilizing the potential of the polyembryonic development of the long-bodied belt-necked wasp in fertility, and breeding as many parasitic wasps as possible with the least number of hosts indoors, thereby providing technical support for the field release technology of the long-bodied belt-necked wasp.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The object of the present invention is to provide a method for improving the reproductive efficiency of the belt-shaped long-bodied braconid, comprising the following steps:

[0008] S1. Select the third-instar larvae of Asian corn borer and place them in a petri dish containing corn leaf fragments as the test host;

[0009] S2, select the fully mated but inexperienced Long-bodied Braconid wasps of the waistline at 5, 6 and 7 days after emergence as parasitic wasps;

[0010] S3, move the pair of long-bodied braconid wasps in step S2, with the ratio of male to female being 1:1, into a container, and feed them with honey water every day;

[0011] S4, moving the Asian corn borer larvae of step S1 into the container of step S3, and after parasitizing for a preset time, taking out the Asian corn borer larvae for single head feeding.

[0012] Furthermore, in step S1, the instars of the Asian corn borer larvae are distinguished by observing the separation of the head shell and the pronotum and the individual size to determine the end of the third instar.

[0013] Furthermore, in step S2, the long-bodied braconid wasps used are individuals of similar size, fully mated and without egg-laying experience.

[0014] Furthermore, in step S3, the size of the container is 9.8 cm in diameter and 9.8 cm in height.

[0015] Furthermore, in step S3, the mass concentration of the honey water is 20% to 25%.

[0016] Furthermore, in step S4, the parasitic time is 4 hours to 4.5 hours.

[0017] Furthermore, in step S4, the parasitized Asian corn borer larvae were raised individually in a plastic tube containing sufficient artificial feed, with a diameter of 2.5 cm and a height of 9.5 cm.

[0018] Furthermore, in step S4, the raising temperature is 26±1°C.

[0019] Furthermore, in step S4, the light cycle of the breeding is 16L:8D.

[0020] Furthermore, in step S4, the relative humidity of the breeding environment is 70±5%.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The present invention uses 5-day to 7-day-old belt-shaped braconids that have no egg-laying experience. At this time, the belt-shaped braconid female bees have a high egg-carrying capacity, and the belt-shaped braconids successfully parasitize more hosts in a short period of time, thereby improving the reproductive efficiency of the belt-shaped braconids;

[0023] (2) The present invention uses larvae of the Asian corn borer at the end of the third instar. Compared with the larvae at the fourth instar, the larvae are smaller and have a shorter initial feeding time, but the number of offspring produced by a single composite cocoon is large, and the proportion of female bees is high. This breaks the traditional perception that larger hosts can produce more offspring;

[0024] (3) The present invention controls the parasitism time of the waistband long-bodied braconid wasp to 4-4.5 hours during the inoculation process. Considering that the time of the Asian corn borer larvae at the end of the third instar is relatively short, it is also necessary to avoid over-parasitism caused by too long a parasitism time;

[0025] (4) The present invention is carried out in a small space, which reduces the time for the waist-belt long-bodied braconid wasp to search for the host, increases the probability of the waist-belt long-bodied braconid wasp meeting the host, and improves the parasitism rate of the waist-belt long-bodied braconid wasp;

[0026] (5) The main way that the long-bodied braconid wasp locates the larvae of the Asian corn borer is through the special smell released by the corn leaves or the excrement of the Asian corn borer after the Asian corn borer feeds on the corn leaves. The long-bodied braconid wasp is attracted by the special smell released by the corn leaves or the excrement of the Asian corn borer. During the parasitism process, the long-bodied braconid wasp is provided with sufficient corn leaf fragments for the larvae of the Asian corn borer, which improves the searching efficiency of the long-bodied braconid wasp.

[0027] (6) The present invention has short duration, simple operation, low cost and is easy to promote. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The parasitism rates of different ages of the long-bodied braconid wasps on the third-instar (3L) larvae of the Asian corn borer are shown in Figure 1. a, b, and c represent significant differences, and n represents the number of repetitions.

[0029] Figure 2 The numbers of offspring and female bees produced by different ages of Longibrachia girdle parasitizing the 3rd instar (3L) larvae of Ostrinia furnacalis. In the figure, a, b, c, and d represent significant differences, and n represents the number of repetitions.

[0030] Figure 3 The parasitism rates of 5-7 days old (5D, 6D, 7D) long-bodied braconid wasps at the end of the 3rd instar (3L), the beginning of the 4th instar (4E), and the end of the 4th instar (4L). In the figure, a and b represent significant differences, and n represents the number of repetitions.

[0031] Figure 4The numbers of offspring and female bees of 5-7 days old (5D, 6D, 7D) parasitized by the 3rd instar (3L), 4th instar (4E) and 4th instar (4L) of the belt-shaped long-bodied braconid wasp are shown in Figure 1. a and b represent significant differences, and n represents the number of repetitions.

[0032] Figure 5 The number of offspring of 5-7-day-old (5D, 6D, 7D) long-bodied braconid parasitized composite cocoons at the end of the 3rd instar (3L), the beginning of the 4th instar (4E) and the end of the 4th instar (4L). In the figure, a, b, c represent significant differences, and n represents the number of repetitions.

[0033] Figure 6 is the parasitism rate under the two breeding conditions;

[0034] Figure 7 are the number of offspring and number of female bees under the two reproductive conditions;

[0035] Figure 8 The number of composite cocoon-eclosion progeny under the two reproductive conditions. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical scheme and advantages of the present invention clearer, the specific embodiments of the present invention are further described in detail below in conjunction with specific examples and drawings. If no specific test method, instrument or condition is specified in the examples, it is carried out according to the technology or condition described in the literature in this field or according to the product instructions. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0037] Example 1

[0038] Effects of parasitism on the third-instar host of different ages of the long-bodied braconid wasp on the parasitism rate and the number of offspring emerging.

[0039] 1. Materials and methods:

[0040] 1.1 Test insects

[0041] The original population of Ostrinia furnacalis was collected from the field, and the laboratory used the new No. 7 artificial diet developed by Zhou Darong et al. in 1970 as the basic formula, which newly added methyl paraben and fumagillin, and reduced the content of agar, sorbic acid and formaldehyde for feeding. The specific preparation method and steps are as follows

[0042] Preparation of artificial feed for Asian corn borer: a. Weigh 32g corn flour, dissolve it in 80ml pure water and soak for half an hour for use; b. Weigh 10g agar powder, 2g sorbic acid, 3g methyl paraben, dissolve in 666ml pure water, mix with the above corn flour solution, and heat to boil. c. Weigh 120g corn flour, 120g soybean powder, 72g yeast powder, 60g white sugar, 4g ascorbic acid and 320ml pure water, stir well. d. Add to the boiled solution, add 1.6ml formaldehyde solution, stir well, wait for the food to cool naturally to 50-60℃, add 0.54g erythromycin and 0.64g fumagillin, stir well, cool, pack into feed boxes, seal well, put in 4℃ refrigerator, and use as needed.

[0043] The belted long-bodied braconid was originally collected from Jiangsu Province. It was reproduced indoors using the fourth-instar larvae of the Asian corn borer as hosts. The parasitized Asian corn borer larvae were placed in an insect box containing artificial feed and continued to be raised. After the cocoons were formed, the cocoons were moved to a culture dish (9 cm in diameter), and after the eclosion, they were moved to a beekeeping cage (30 cm×30 cm×30 cm) for reproduction. They were fed with 20% honey water every day.

[0044] All insects were reared for multiple generations in an insectary at a temperature of 26 ± 1 °C, a relative humidity of 70 ± 5%, and a photoperiod of 16L:8D.

[0045] 1.2 Experimental methods

[0046] (a) A pair of (1♀:1♂) long-bodied braconid wasps with no egg-laying experience at 1 to 7 days after emergence were selected and placed in a plastic box (9.8 cm in diameter and 9.8 cm in height) and fed with 20% honey water.

[0047] (b) Fifteen third-instar larvae of Ostrinia furnacalis were selected and placed in a petri dish (9 cm in diameter) containing corn leaf fragments, and then transferred to a plastic box containing Echinops girdle.

[0048] (c) After 4 hours of parasitism, the larvae of the Asian corn borer were placed individually in a plastic tube (2.4 cm in diameter and 9.5 cm in height) filled with sufficient artificial diet for further feeding. The parasitized larvae of the Asian corn borer were cultured in an incubator at a temperature of 26±1°C, a relative humidity of 70±5%, and a photoperiod of 16L:8D.

[0049] (d) After 10 days of parasitism, check whether the Asian corn borer has successfully parasitized. Wrap the successfully parasitized Asian corn borer larvae with toilet paper to facilitate the cocooning of the larvae of the long-bodied braconid wasp.

[0050] Data collation: record the number of parasites and the number of male and female offspring that have emerged.

[0051] Parasitism rate = (number of parasitized plants / (number of parasitized plants + number of non-parasitized plants)) × 100.

[0052] The number of offspring refers to the total number of offspring produced by a single L. girdle during the experiment (4 h).

[0053] The number of offspring female bees refers to the total number of offspring female bees produced by a single long-bodied braconid wasp during the experiment (4 hours).

[0054] The number of compound cocoon progeny refers to the number of progeny that emerge from a single host.

[0055] SPSS v.27.0 was used for data analysis, and GraphPad Prism v8.0 software was used for graphics.

[0056] 2. Results and Analysis

[0057] Depend on Figure 1 It can be seen that the parasitism rate of the third-instar larvae of Asian corn borer increased significantly with the extension of the development time of the adult bees of the belt-shaped long-bodied braconid. The parasitism rate of the belt-shaped long-bodied braconid at the age of 5, 6 and 7 days was significantly higher than that of the belt-shaped long-bodied braconid at the age of 1 to 4 days.

[0058] Depend on Figure 2 It can be seen that the number of offspring produced by the different ages of the belt-shaped long-bodied braconid wasps is consistent with the trend of the parasitism rate. As the age of the belt-shaped long-bodied braconid wasps increases, the number of offspring emergence increases significantly. However, the number of offspring female bees emerging is not related to the age of the belt-shaped long-bodied braconid wasps. The 5-day-old, 6-day-old and 7-day-old belt-shaped long-bodied braconid wasps with no egg-laying experience parasitized the 3rd instar Asian corn borer larvae and produced an average of 197.10, 213.17 and 254.19 offspring in 4 hours, of which 55.95, 41.83 and 62.31 were female bees, respectively. The number of offspring produced was significantly higher than the number of offspring produced by the 1-day-old to 4-day-old belt-shaped long-bodied braconid wasps.

[0059] Example 2

[0060] The influence of different ages of Asian corn borer larvae on the parasitism rate and number of offspring of the belt-shaped braconid wasp

[0061] 1. Materials and Methods

[0062] 1.1 Test insects

[0063] The feeding method of Ostrinia furnacalis and Long-bodied Braconidae is the same as that of Example 1

[0064] 1.2 Experimental methods

[0065] (a) Based on the results of Example 1, a pair (1♀:1♂) of 5-day-old, 6-day-old and 7-day-old inexperienced long-bodied braconid wasps were selected and placed in a plastic box (9.8 cm in diameter and 9.8 cm in height). Cotton balls dipped in 20% honey water were used as food, which was changed daily.

[0066] (b) Fifteen larvae of Ostrinia furnacalis at the end of the third instar (3L), the beginning of the fourth instar (4E) and the end of the fourth instar (4L) were picked and placed in a petri dish (9 cm in diameter) containing corn leaf fragments and then transferred to a plastic box containing the cercis cercis cercis.

[0067] (c) After 4 hours of parasitism, the larvae of the Asian corn borer were placed individually in a plastic tube (2.4 cm in diameter and 9.5 cm in height) filled with sufficient artificial diet for further feeding. The parasitized larvae of the Asian corn borer were cultured in an incubator at a temperature of 26±1°C, a relative humidity of 70±5%, and a photoperiod of 16L:8D.

[0068] (d) After 10 days of parasitism, check whether the Asian corn borer has successfully parasitized. Wrap the successfully parasitized Asian corn borer larvae with toilet paper to facilitate the cocooning of the larvae of the long-bodied braconid wasp.

[0069] (e) The calculation and statistical plotting methods of parasitism rate and progeny number are the same as those in Example 1.

[0070] 2. Results and Analysis

[0071] Depend on Figure 3 It can be seen that the parasitism rate of 5-day-old, 6-day-old and 7-day-old belt-shaped braconids parasitizing the Asian corn borer larvae at the end of the 3rd instar was significantly higher than that of parasitizing the early and late 4th instar. When the 5-day-old belt-shaped braconids parasitized the Asian corn borer larvae at the end of the 3rd instar, the average parasitism rate reached 49.39%, which was about twice that of parasitizing the early and late 4th instar.

[0072] Depend on Figure 4 It can be seen that the number of offspring produced by 5-day-old, 6-day-old and 7-day-old adult bees of the belt-belted long-bodied braconid parasitizing the late 3rd instar Asian corn borer larvae is significantly higher than that of the host parasitizing the early and late 4th instars. The number of offspring that emerged from the late 3rd instar was higher than 190, while the average number of offspring that emerged from the early and late 4th instars was the highest, with 139. The number of offspring female bees produced by 5-day-old and 7-day-old adult bees parasitizing the late 3rd instar Asian corn borer larvae was significantly higher than that of the early and late 4th instars. Although there was no significant difference in the number of offspring female bees produced by the 6-day-old belt-belted long-bodied braconid, it could produce 41.83 female bees when parasitizing the late 3rd instar, while it could only produce 29.33 and 22.00 female bees on average when parasitizing the early and late 4th instars.

[0073] Depend on Figure 5It can be seen that the number of offspring that emerged from the composite cocoons of 3rd instar Asian corn borer larvae parasitized by 5-day-old, 6-day-old and 7-day-old Longibacterium gracile wasps was significantly higher than the number of offspring that emerged from the composite cocoons of 4th instar larvae parasitized by 5th-day-old, 6th-day-old and 7th-day-old Longibacterium

[0074] It can be seen that the 5-day-old, 6-day-old and 7-day-old belt-shaped braconid wasps with no egg-laying experience had the highest parasitism rate on the third-instar larvae of Asian corn borer, and the number of offspring, female bees and composite cocoon eclosion offspring produced were the highest. The third-instar larvae of Asian corn borer were more suitable for the development and reproduction of belt-shaped braconid wasps.

[0075] Example 3

[0076] Comparison of two different reproduction methods

[0077] 1. Materials and Methods

[0078] 1.1 Test insects

[0079] The feeding method of Ostrinia furnacalis and Longibotium waistband wasp is the same as that in Example 1.

[0080] 1.2 Experimental methods

[0081] The optimal conditions for the reproduction of the belt-shaped long-bodied braconid was determined according to Examples 1 and 2, i.e., 5-day-old, 6-day-old and 7-day-old belt-shaped long-bodied braconid wasps without egg-laying experience and 3-instar larvae of Asian corn borer were compared with 2-day-old belt-shaped long-bodied braconid wasps and 4-instar larvae of Asian corn borer used for subculture in the laboratory.

[0082] (a) A pair of 5-day-old long-bodied braconid wasps (1♀:1♂) without egg-laying experience were selected and placed in a plastic box (9.8 cm in diameter and 9.8 cm in height) and fed with 20% honey water. They were then parasitized with 15 3rd-instar larvae of Ostrinia furnacalis.

[0083] (b) A pair of (1♀:1♂) 2-day-old long-bodied braconid wasps without egg-laying experience were selected and placed in a plastic box (9.8 cm in diameter and 9.8 cm in height) and fed with 20% honey water. They were then parasitized with 15 4th-instar larvae of Asian corn borer.

[0084] (c) After 4 hours of parasitism, the larvae of the Asian corn borer were placed individually in a plastic tube (2.4 cm in diameter and 9.5 cm in height) filled with sufficient artificial diet for further feeding. The parasitized larvae of the Asian corn borer were cultured in an incubator at a temperature of 26±1°C, a relative humidity of 70±5%, and a photoperiod of 16L:8D.

[0085] (d) After 10 days of parasitism, check whether the Asian corn borer has successfully parasitized. Wrap the successfully parasitized Asian corn borer larvae with toilet paper to facilitate the cocooning of the larvae of the long-bodied braconid wasp.

[0086] (e) The calculation and statistical analysis methods of parasitism rate and progeny number are the same as those in Example 1.

[0087] 2. Results and Analysis

[0088] Depend on Figure 6 It can be seen that the average parasitism rate of 5-day-old long-bodied braconid wasps parasitizing the 3rd instar Asian corn borer larvae is 49.39%, and the average parasitism rate of 2-day-old long-bodied braconid wasps parasitizing the 4th instar Asian corn borer larvae is 8.01%. The parasitism rate obtained by the method of the present invention is 6 times that of the conventional laboratory breeding method.

[0089] Depend on Figure 7 It can be seen that the total number of offspring and the number of female bees produced by the method of the present invention are significantly higher than the current laboratory breeding method. Using 5-day-old 5-day-old 5-day-old parasitizing Asian corn borer at the end of the 3rd instar for 4 hours can produce 197.10 offspring, of which 55.95 are female bees. The number of offspring produced is about 2.5 times that of the conventional laboratory breeding method, and the number of female bees is 4 times that of the conventional laboratory breeding method.

[0090] Figure 8 It can be seen that when the method of the present invention is used to breed the belt-shaped long-bodied braconid, the number of offspring and the number of female bees that have emerged from the composite cocoons are greater than those of the laboratory breeding method, although the difference is not significant.

[0091] For matters not mentioned above, the prior art applies.

[0092] Although some specific embodiments of the present invention have been described in detail through examples, those skilled in the art should understand that the above examples are for illustration only and are not intended to limit the scope of the present invention. Those skilled in the art to which the present invention belongs may make modifications or supplements to the described specific embodiments or replace them in a similar manner, but they will not deviate from the direction of the present invention or exceed the scope defined by the attached claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc. made to the above implementations based on the technical essence of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for improving the reproductive efficiency of the belt-shaped long-bodied braconid wasp, characterized in that: The following steps are involved: S1. Select the third-instar larvae of Asian corn borer and place them in a petri dish containing corn leaf fragments as the test host; S2, select the fully mated but inexperienced Long-bodied Braconid wasps of the waistline at 5, 6 and 7 days after emergence as parasitic wasps; S3, move the pair of long-bodied braconid wasps in step S2, with the ratio of male to female being 1:1, into a container, and feed them with honey water every day; S4, moving the Asian corn borer larvae of step S1 into the container of step S3, and after parasitizing for a preset time, taking out the Asian corn borer larvae for single head feeding.

2. The method according to claim 1, characterized in that In step S1, the instars of the Asian corn borer larvae are distinguished by observing the separation of the head shell and the pronotum and the individual size to determine the end of the third instar.

3. The method according to claim 2, characterized in that In step S2, the long-bodied braconid wasps used are individuals of similar size, fully mated and without egg-laying experience.

4. The method according to claim 3, characterized in that In step S3, the size of the container is 9.8 cm in diameter and 9.8 cm in height.

5. The method according to claim 4, characterized in that In step S3, the mass concentration of the honey water is 20% to 25%.

6. The method according to claim 4, characterized in that In step S4, the parasitic time is 4 hours to 4.5 hours.

7. The method according to claim 6, characterized in that In step S4, the parasitized Asian corn borer larvae were raised individually in a plastic tube containing sufficient artificial feed, with a diameter of 2.5 cm and a height of 9.5 cm.

8. The method according to claim 7, characterized in that In step S4, the temperature of the feeding is 26±1°C.

9. The method according to claim 8, characterized in that In step S4, the light cycle of the culture is 16L:8D.

10. The method according to claim 9, characterized in that In step S4, the relative humidity of the breeding environment is 70±5%.

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