A method for rearing of the medfly in the laboratory

Abstract

The application discloses an indoor breeding method of Dacus cucurbitae, and relates to the technical field of Dacus cucurbitae breeding. The specific steps are as follows: (1) collecting and hatching eggs: after fresh zucchini slices coated with an egg promoting agent are put into a gauze bag, the gauze bag is moved into a rearing cage containing mature Dacus cucurbitae to receive eggs; after the eggs are received, the gauze bag is fixed in a second container for hatching; (2) rearing larvae: after Dacus cucurbitae larvae are hatched, the larvae are reared by adding zucchini slices, and the sewage in the first container is poured out in time; when the larvae turn into light yellow and have the jumping tendency or are in the jumping state, the larvae are poured into a container filled with clean water, and the larvae sinking at the bottom of the water are taken out; (3) pupating: the larvae are put into a container filled with sand to pupate; (4) eclosion: after the pupae eclosion, the pupae are reared to complete maturity for receiving eggs again, and the next round of breeding is carried out. The application can increase the number of egg grains, improve the hatching rate, the pupation rate and the eclosion rate, and is simple to operate, and realizes the indoor large-scale breeding of Dacus cucurbitae.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of Bactrocera cucurbitae (Coquillett) breeding, in particular to an indoor breeding method of Bactrocera cucurbitae (Coquillett). BACKGROUND

[0002] Bactrocera cucurbitae (Coquillett) belongs to Diptera, Tephritidae, is commonly known as needle bee, melon maggot, is an entry quarantine harmful organism in China, and has been listed in the first batch of national key management alien invasive species list. Bactrocera cucurbitae has strong reproductive capacity, and mainly harms Cucurbitaceae crops such as cucumber, bitter gourd, luffa, zucchini and pumpkin. Bactrocera cucurbitae mainly harms by female adults laying eggs in the fruit of the melon crop, and Bactrocera cucurbitae likes to lay eggs in fresh melon or the damaged place of the skin. After the eggs hatch, the larvae feed in the fruit to develop into mature larvae, which causes the cuticle of the fruit to become hard, the taste of the fruit to become sour, and the fruit to emit a rotten odor, thereby seriously affecting the yield and quality of melon.

[0003] According to statistics, the loss degree of melon and fruit damaged by Bactrocera cucurbitae is generally between 30% and 100%, and the damage to bitter gourd is the most serious, with an average yield loss of 50%, and the yield loss can reach 100% in severe cases. Therefore, the damage of Bactrocera cucurbitae to melon plants has become a major limiting factor for obtaining high-quality melon and high yield. At present, the methods for preventing and controlling Bactrocera cucurbitae include chemical control, biological control, agricultural control and the like. The development of indoor breeding technology of Bactrocera cucurbitae can promote the research of Bactrocera cucurbitae and effectively promote the progress of Bactrocera cucurbitae control means.

[0004] At present, there are still some problems in indoor breeding of Bactrocera cucurbitae, such as incomplete collection of egg masses, poor aeration of larva hatching, and easy drowning of larvae in sewage. Patent application No. 201310045320.4 discloses a Bactrocera cucurbitae breeding method, which induces Bactrocera cucurbitae to lay eggs on a porous film and collects egg masses, and uses a pupa collection device to collect pupae and perform eclosion, and has good collection effect. However, the eggs and melons are directly placed on the sand for hatching and breeding larvae, and the sewage generated during breeding cannot be discharged in time, resulting in low hatching rate and low survival rate of larvae. In addition, it is not convenient to replace the sand. Therefore, in order to improve the breeding method of Bactrocera cucurbitae, a simple and efficient breeding method needs to be invented. SUMMARY

[0005] In view of the above problems, the present application provides an indoor breeding method of Bactrocera cucurbitae, which can not only promote egg laying, but also improve the hatching rate, pupation rate and eclosion rate, and is simple to operate, and realizes large-scale indoor breeding. The specific technical scheme is as follows:

[0006] An indoor breeding method of Bactrocera cucurbitae is adopted, and a breeding device is used;

[0007] The breeding device comprises a first container, a second container detachably clamped in the first container, the top of the first container and the top of the second container are both provided with an opening, the bottom of the second container is provided with a through hole, and there is a gap between the bottom of the second container and the bottom of the first container, and a detachable gauze bag is arranged in the second container.

[0008] The breeding method comprises the following steps:

[0009] (1) Collection and hatching of eggs: first, put fresh zucchini slices smeared with an oviposition stimulant into the gauze bag, fix the gauze bag in a culture dish, and then move it into a rearing cage containing mature fruit flies to collect eggs, after 6-8 hours, take out the gauze bag and fix it in the second container for hatching;

[0010] (2) Rearing of larvae: after the fruit fly larvae are hatched, add zucchini slices every half day, and discard the sewage in the first container before adding zucchini slices each time; when the larvae are yellowish in color and have the tendency to jump or are in the jumping state, take out the gauze bag, pour the larvae in the gauze bag into a container filled with water, remove the floating debris after 1-2 minutes, and take the mature larvae;

[0011] (3) Pupation: put the mature larvae into a container filled with sand for pupation;

[0012] (4) Emergence: after the pupae emerge into fruit flies, part of them are put into a rearing cage for feeding, and the other part is fed to complete maturity for collecting eggs again for the next round of breeding.

[0013] Preferably, the breeding device is further provided with a cover, the cover is matched with the opening of the first container, and the cover is black gauze.

[0014] Preferably, the mesh number of the gauze bag is 30-40 mesh, and the opening of the gauze bag is provided with a elastic rope.

[0015] Preferably, the oviposition stimulant in step (1) is prepared by mixing pumpkin extract and tomato juice at a weight ratio of 1:2-8; the preparation method of the pumpkin extract is as follows: freeze-drying mature pumpkin fruits to obtain pumpkin powder, adding petroleum ether to the pumpkin powder until it is completely immersed, sealing, taking the filtrate every 10-12 hours at room temperature, and continuously extracting 6-8 times, combining the filtrate, evaporating and concentrating at 60-70°C to half of the original volume, adding acetone to the concentrated solution to obtain an extract, and then reducing and concentrating the extract to obtain the pumpkin extract.

[0016] Preferably, the mature C. pipiens in step (1) refers to C. pipiens after 15 days of emergence.

[0017] Preferably, the indoor environment temperature of the egg receiving room in step (1) is 27-31℃.

[0018] Preferably, the thickness of the sand in step (3) is 3-4cm.

[0019] Preferably, the feed used in the feeding of C. pipiens in step (4) is yeast and sugar mixed in a weight ratio of 1:2-4.

[0020] Preferably, the light cycle of the feeding in step (4) is L:D=14:10.

[0021] Preferably, the thickness of the zucchini slices is 1-4mm.

[0022] Compared with the prior art, the present application has the following beneficial effects:

[0023] 1. The breeding method of the present application is simple in operation, low in cost and high in survival rate, can be used for large-scale indoor breeding, can meet the experimental demand, and can provide a test basis for the research and prevention of C. pipiens.

[0024] 2. The egg promoting agent of the present application can promote the oviposition of C. pipiens females and increase the number of egg grains. The pumpkin extract mainly contains ethyl acetate, benzoic acid and octyl acetate and the like, which, in combination with tomato juice, stimulates and induces the oviposition of females, and effectively increases the number of egg grains.

[0025] 3. The gauze bag in the breeding device of the present application can completely collect the eggs, and the feeding sewage can be discharged into the first container through the through hole at the bottom of the second container, so as to timely separate the sewage from the larvae, avoid the death of the larvae due to drowning, and improve the hatching rate and emergence rate. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced as follows.

[0027] Figure 1 It is a front view of the breeding device of the present application.

[0028] 1-cover, 2-gauze bag, 3-second container, 4-first container. DETAILED DESCRIPTION

[0029] The specific embodiments of the present application will be described in detail below, but it should be understood that the protection scope of the present application is not limited by the specific embodiments.

[0030] Example 1

[0031] The indoor breeding method of the fruit fly comprises the following steps:

[0032] (1) Collection and hatching of eggs: first, put fresh zucchini slices with a thickness of 4 mm smeared with an egg-promoting agent into a gauze bag, and fix the gauze bag in a culture dish, open the gauze bag opening, expose the zucchini slices, and then move into a rearing cage containing mature fruit flies, i.e. fruit flies that have hatched for 18 days, to receive eggs, 8 hours after receiving eggs, take out the gauze bag and fix it in a second container for hatching, and control the hatching temperature to be 31℃.

[0033] The egg-promoting agent is prepared by mixing pumpkin extract and tomato juice at a weight ratio of 1:2; the preparation method of the pumpkin extract is as follows: after the mature pumpkin fruit is frozen at low temperature and crushed into pumpkin powder, petroleum ether is added until it is completely immersed, sealed, and taken out every 10 hours at room temperature, and petroleum ether is continuously added, and the filtrate is continuously extracted for 6 times, the filtrate is combined, and evaporated and concentrated at 60℃ to half of the original volume to obtain a concentrated solution, and then acetone is added to extract to obtain an extract solution, and the extract solution is reduced pressure concentrated to obtain the pumpkin extract.

[0034] (2) Rearing of larvae: after the fruit fly larvae are hatched, 4 mm thick zucchini slices are added every half day, and the sewage in the first container is discarded before adding the zucchini slices each time; when the larvae are reared to have a light yellow body surface and have a jumping tendency or are in a jumping state, the gauze bag is taken out, the larvae in the gauze bag are poured into a container filled with clean water, 2 minutes later, the sundries floating on the water surface are removed, and the larvae sinking at the bottom of the water are taken to obtain mature larvae.

[0035] (3) Pupation: the mature larvae are put into a container filled with 4 cm thick sand for pupation, and the sand humidity is controlled to be 70%.

[0036] (4) Emergence: after the pupae emerge into fruit flies, a part is put into a rearing cage for feeding for experimental needs, and the other part is fed to complete sexual maturity for putting into a rearing cage again to receive eggs for the next round of breeding, wherein the feed used for feeding is prepared by mixing yeast and sugar at a weight ratio of 1:4, and the light cycle for feeding is L:D=14:10.

[0037] Example 2

[0038] The indoor breeding method of the fruit fly comprises the following steps:

[0039] (1) Collection and hatching of eggs: first, put fresh zucchini slices with a thickness of 4 mm smeared with an egg-promoting agent into a gauze bag, and fix the gauze bag in a culture dish, open the gauze bag opening, expose the zucchini slices, and then move into a rearing cage containing mature fruit flies, i.e. fruit flies that have hatched for 18 days, to receive eggs, 8 hours after receiving eggs, take out the gauze bag and fix it in a second container for hatching, and control the hatching temperature to be 31℃.

[0040] The promoting ovum agent is prepared by mixing pumpkin extract and tomato juice at a weight ratio of 1:8; the pumpkin extract is prepared as follows: the mature pumpkin fruits are freeze-dried at low temperature and then crushed into pumpkin powder, petroleum ether is added to the pumpkin powder until it is completely immersed, sealed, and then the filtrate is taken out every 12 hours at room temperature, and petroleum ether is continuously added, the extraction is carried out for 7 times, the filtrate is combined, and then concentrated at 70°C until the volume is reduced to half of the original volume to obtain a concentrated solution, acetone is added to the concentrated solution for extraction to obtain an extracted solution, and then the extracted solution is concentrated under reduced pressure to obtain the pumpkin extract.

[0041] (2) The larvae are fed: after the real fly larvae are hatched, 1mm-thick zucchini slices are added every half day, and the sewage in the first container is poured out before adding the zucchini slices each time; the larvae are fed until the body surface becomes light yellow, and the larvae are jumping or in the jumping state, then the larvae in the gauze bag are poured into a container filled with clean water, the sundries floating on the water surface are removed after 1 minute, and the mature larvae are obtained by taking the larvae sinking at the bottom of the water.

[0042] (3) Pupation: the mature larvae are put into a container filled with 3cm-thick sand for pupation, and the sand humidity is controlled to be 60%.

[0043] (4) Emergence: after the pupae emerge into cucurbit fruit flies, part of the cucurbit fruit flies are put into a rearing cage for feeding for experimental needs, and the other part of the cucurbit fruit flies are fed to complete sexual maturity for placing zucchini again to receive eggs for the next round of reproduction, wherein the feed used for feeding is prepared by mixing yeast and sugar at a weight ratio of 1:2, and the light cycle for feeding is L:D=14:10.

[0044] Example 3

[0045] The indoor breeding method of the cucurbit fruit fly comprises the following steps:

[0046] (1) Collection and hatching of eggs: fresh 2mm-thick zucchini slices coated with the promoting ovum agent are put into a gauze bag, and the gauze bag is fixed in a culture dish, the opening of the gauze bag is opened, the zucchini slices are exposed, and then the gauze bag is moved into a rearing cage containing mature cucurbit fruit flies, i.e. cucurbit fruit flies that have emerged for 15 days, to receive eggs, the gauze bag is taken out and fixed in a second container to hatch the eggs after 7 hours of egg receiving, and the hatching temperature is controlled to be 28°C.

[0047] The promoting ovum agent is prepared by mixing pumpkin extract and tomato juice at a weight ratio of 1:5; the pumpkin extract is prepared as follows: the mature pumpkin fruits are freeze-dried at low temperature and then crushed into pumpkin powder, petroleum ether is added to the pumpkin powder until it is completely immersed, sealed, and then the filtrate is taken out every 11 hours at room temperature, and petroleum ether is continuously added, the extraction is carried out for 8 times, the filtrate is combined, and then concentrated at 65°C until the volume is reduced to half of the original volume to obtain a concentrated solution, acetone is added to the concentrated solution for extraction to obtain an extracted solution, and then the extracted solution is concentrated under reduced pressure to obtain the pumpkin extract.

[0048] (2) Rearing of larvae: after the emergence of the fly larvae, add 2mm thick zucchini slices every half day, and pour out the sewage in the first container before adding zucchini slices each time; when the larvae are reared to have a light yellow body surface and have the tendency to jump or are in the jumping state, take out the mesh bag, pour the larvae in the mesh bag into a container filled with clean water, remove the sundries floating on the water surface after 2 minutes, and take the mature larvae sinking at the bottom of the water.

[0049] (3) Pupation: put the mature larvae into a container filled with 3.5cm thick sand for pupation, and control the sand humidity to be 65%.

[0050] (4) Emergence: after the emergence of the pupae into the fruit fly, part of them are put into the insect cage for feeding for the experimental needs, and the other part is fed to complete maturity for placing zucchini for egg laying again to carry out the next round of breeding, wherein the feed used for feeding is yeast and sugar mixed in a weight ratio of 1:3, and the light cycle for feeding is L:D=14:10.

[0051] The breeding device of the above embodiment comprises: a first container 4, a second container 3 detachably clamped in the first container 4; the top of the first container 4 and the second container 3 is provided with an opening, the bottom of the second container 3 is provided with a through hole, and there is a gap between the bottom of the second container 3 and the bottom of the first container 4, and a detachable mesh bag 2 is arranged in the second container 3. The breeding device is also provided with a cover 1 which matches the opening of the first container 4, and the cover 1 is black gauze.

[0052] The mesh bag 2 is taken out and put into the insect cage to collect the egg grains, and after the egg laying, the mesh bag 2 is moved into the second container 3 and fixed in the second container 3 by the elastic rope, and the cover 1 is covered to carry out egg incubation and larva breeding. The breeding wastewater flows into the first container 4 through the through hole at the bottom of the second container 3, the second container 3 is taken down every time the larvae are fed with zucchini, and the breeding sewage in the first container 4 is poured out to keep the breeding environment clean. The cover 1 is black gauze, which can make the larvae breed better in the dark environment.

[0053] Comparative Example 1

[0054] Step (1) does not use a mesh bag to lay eggs, but directly places zucchini slices smeared with an oviposition stimulant in a culture dish for egg laying. The number of zucchini slices is the same as that of Example 3, which is 1 piece. The culture dish containing the zucchini slices is directly placed in the insect cage for egg laying, and the average number of egg grains collected after 1h, 2h and 3h of egg laying in Example 3 and Comparative Example 1 is counted respectively, with 5 repetitions. The results are shown in Table 1:

[0055] Table 1 Comparison of egg grain numbers at different times

[0056]

[0057] As shown in Table 1, the number of eggs collected using the mesh bag of the present invention is higher than that collected using conventional methods on petri dishes or trays.

[0058] Comparative Example 2

[0059] The zucchini slices in step (1) were not coated with an ovulation stimulant and were designated as group A; the zucchini slices in step (1) were coated with an ovulation stimulant that did not contain pumpkin extract and were designated as group B; the zucchini slices in step (1) were coated with an ovulation stimulant that did not contain tomato juice and were designated as group C; the zucchini slices in step (1) were replaced with bitter melon slices and were designated as group D; the other conditions for groups A, B, C and D were the same as in Example 3, with 5 replicates in each group. The average number of eggs 3 hours after egg reception was counted, and the results are shown in Table 2:

[0060] Table 2 Comparison of oocyte count under different treatments

[0061]

[0062] Table 2 shows that the oviposition stimulant of this invention can promote oviposition in female melon fly larvae and increase the number of eggs. Specifically, the combined action of pumpkin extract, tomato juice, and zucchini slices stimulates and induces oviposition in females, effectively increasing the number of eggs.

[0063] Comparative Example 3

[0064] Using the technical method of this invention and an innovative device, a generation of melon flies was reared indoors. The losses during egg collection in gauze bags and petri dishes at 60 min, 120 min, and 180 min were calculated.

[0065] Cut the zucchini horizontally into 1mm thick slices. Place one slice in the middle of a gauze bag and name it A1. Place the gauze bag in the insect rearing cage (the adult insects are all sexually mature). Then take another slice of zucchini and place it directly into the insect rearing cage and name it CK1. After different times, take out A1 and CK1 and record the amount of loss.

[0066] Table 3. Loss of egg production (eggs) in melon fly at different time points.

[0067]

[0068] Comparative Example 4

[0069] The larval rearing stage does not use the breeding device of this invention, but instead uses conventional methods. Specifically, 100 eggs are placed on a slice of zucchini and then placed in a tilted petri dish for culture. Wastewater in the tilted petri dish is promptly treated. This is named CK2. Another 100 eggs are placed on a slice of zucchini and then placed in the equipment of this invention for culture. This is named A2. The number of hatched eggs is recorded after 3 days.

[0070] After the larvae mature and are able to jump to the ground, the CK2 group of larvae is poured together with the rotten melon residues into another culture dish with sand on the surface covered with newspaper, and pupation is waited for. The A2 is cultured in the inventive device to maturity, and after filtering with clean water, the larvae are placed in a plastic bottle containing sand for culture, and pupation is waited for. After pupation, the number of pupae and the pupa weight are recorded.

[0071] After the pupa weight is measured, the melon fruit fly pupae are put back to the original place, and after 10 days, the emergence of the melon fruit fly is recorded, and the number of adult melon fruit flies is recorded.

[0072] The above experiment is set with 5 repetitions, and the results are shown in Table 4. Among them, the hatching rate (%) = test 1st instar larvae / 100 x 100; the pupation rate (%) = test pupae number / test 1st instar larvae x 100; the average pupa weight (mg) = the sum of the weight of each pupa (mg) / test pupae number x 100.

[0073] Table 4 Comparison of effects of different melon fruit fly rearing methods

[0074]

[0075] Note: The data in the table are average number and standard error, and the difference is significant (P<0.05)

[0076] From Table 4, it can be seen that the hatching rate of the melon fruit fly cultured in the inventive device is higher than that in the culture dish (traditional method), the larvae can completely pupate in both culture devices, and there is no difference in the emergence rate between the melon fruit flies cultured in the inventive device and the culture dish.

[0077] Using the device and process of the present application, the growth and development conditions of the melon fruit fly at each stage can be met, the steps of separating the larvae and sewage are improved in the breeding process, the problems of poor air permeability and light transmission during larva hatching and untimely sewage treatment are solved, the situation that the larvae are easily drowned by sewage is avoided, the probability of mold growth and odor generation of the host melon is reduced, the collection amount of egg grains, the hatching rate and the emergence rate are improved, and the larva mortality rate is reduced, so that large-scale and rapid breeding of the melon fruit fly is realized. Not only is the operation simple, but also manpower and material resources are saved, and it is suitable for promotion.

[0078] The foregoing description of specific exemplary embodiments of the application is intended to be illustrative only. These descriptions are not intended to be limiting to the precise forms disclosed. Rather, these descriptions are intended to be illustrative of the specific form of the application which is described in sufficient detail to enable anyone skilled in the art to implement the application. Numerous modifications and changes can be made by persons having ordinary skill in the art without departing from the scope of the application. The scope of the application is intended to be defined by the claims and their equivalents.

Claims

1. A method for indoor breeding of melon flies, characterized in that, Breeding devices are used; The breeding device includes: a first container and a second container detachably snapped into the first container; both the first container and the second container have openings at the top, the bottom of the second container has a through hole, there is a gap between the bottom of the second container and the bottom of the first container, and a detachable mesh bag is provided inside the second container. The breeding method includes the following steps: (1) Egg collection and hatching: First, put fresh zucchini slices coated with egg-inducing agent into the mesh bag and fix the mesh bag in the petri dish. Then, transfer it into the insect cage containing mature melon flies to collect eggs. After collecting eggs for 6-8 hours, take out the mesh bag and fix it in the second container for hatching. The ovulation stimulant is prepared by mixing pumpkin extract and tomato juice in a weight ratio of 1:2-8. The preparation method of the pumpkin extract is as follows: ripe pumpkin fruit is freeze-dried at low temperature and then pulverized into pumpkin powder. Petroleum ether is added to the pumpkin powder until it is completely submerged. The mixture is sealed and extracted at room temperature. The filtrate is taken every 10-12 hours and petroleum ether is added again. The extraction is repeated 6-8 times. The filtrates are combined and evaporated and concentrated at 60-70℃ to half of the original volume to obtain a concentrate. Acetone is added to the concentrate for extraction to obtain an extract. The extract is concentrated under reduced pressure to obtain the final product. (2) Larval rearing: After the fruit fly larvae hatch, add zucchini slices once every half day and pour out the sewage in the first container; when the larvae are raised until their body surface turns light yellow and they show signs of jumping or are in the jumping state, take out the mesh bag and pour the larvae in the mesh bag into a container filled with clean water. After 1-2 minutes, remove the debris floating on the water surface and take out the larvae that have sunk to the bottom of the water to obtain mature larvae. (3) Pupation: The mature larvae are placed in a container filled with sand to pupate; (4) Emergence: After the pupae emerge into melon flies, some are placed in an insect rearing cage for experimental purposes, while others are raised until they reach full sexual maturity for the purpose of laying melons and catching eggs again for the next round of reproduction.

2. The indoor breeding method for melon flies according to claim 1, characterized in that, The breeding device is also provided with a lid that matches the opening of the first container, and the lid is made of black mesh.

3. The indoor breeding method for melon flies according to claim 1, characterized in that, The mesh bag has a mesh size of 30-40, and the opening of the mesh bag is provided with an elastic cord.

4. The indoor breeding method for melon flies according to claim 1, characterized in that, The mature melon fly mentioned in step (1) refers to a melon fly that has emerged 15 days after its emergence.

5. The indoor breeding method for melon flies according to claim 1, characterized in that, The ambient temperature inside the insect rearing cage in step (1) is 27-31℃.

6. The indoor breeding method for melon flies according to claim 1, characterized in that, The thickness of the sand in step (3) is 3-4 cm.

7. The indoor breeding method for melon flies according to claim 1, characterized in that, The feed used for raising melon flies in step (4) is a mixture of yeast and sugar in a weight ratio of 1:2-4.

8. The indoor breeding method for melon flies according to claim 1, characterized in that, The light cycle for raising animals in step (4) is L:D=14:

10.

9. A method for indoor breeding of melon flies according to claim 1, characterized in that, The thickness of the zucchini slices is 1-4 mm.

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