A method for artificial rearing of the whole life cycle of the eucalyptus longicorn beetle

Abstract

The present application relates to a kind of artificial feeding method of eucalyptus moth whole life cycle, belong to biological control technical field, egg mass is concentrated room temperature to hatch;Larva feeding environment is dark, temperature 23℃±2℃, humidity 70%±10%, 1~2 age larva group is fed with shiitake, 3~12 age larva is fed with fresh eucalyptus stem dry or branch dry;Pupa pre-feeding environment is same with larva, after body color changes into black brown, pupa is moved into emergence cage, keeps humidity 80%±10%, temperature 25℃±2℃, female pupa is 2℃~3℃ higher than male pupa, dark;Emergence adult is placed into net cage with length width height 9.6m×4m×8m and no light source in periphery, keeps female and male sex ratio in net ≤1:3 so that female and male moth copulation and lay eggs.The present application can make eucalyptus moth complete whole generation, its environmental condition is easy to control, feed is easy to obtain, simple operation, workload is small, especially solve the problem that adult mating is difficult and difficult to breed offspring.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological control, and particularly relates to an artificial rearing method for the whole life cycle of Endoclita signifer Walker. BACKGROUND

[0002] Endoclita signifer Walker belongs to Lepidoptera Hepialidae. Before the 3rd instar, the larvae of E. signifer live in the ground and may feed on underground dry branches, fungi or humus; the 3rd-5th instar larvae select 1-3 year old eucalyptus saplings, bore into the trunks, and then live in the trunks and feed on the callus around the bore holes until the end of the 12th instar when they pupate in the bore holes (Yang Xiuhao et al., 2012). The growth and development of eucalyptus trees damaged by E. signifer are slowed down, the volume and quality of the wood are reduced, and the trees are prone to wind breakage and death (Yu Yonghui, 2012). In recent years, with the large-scale planting of fast-growing eucalyptus plantations in southern China, the damage range of E. signifer has gradually expanded, and it has become one of the main boring pests affecting the development of eucalyptus industry (Yang Xiuhao et al., 2021). Establishing a laboratory population of E. signifer at a certain scale is conducive to the research of E. signifer in behavior ecology, molecular biology and biological control, etc. However, so far, there have been few studies on the artificial rearing of E. signifer. It is known that newly hatched larvae of E. signifer can grow on straw Volvariella volvacea medium to the 3rd instar, but the survival rate is very low, and it is found that E. signifer adults cannot successfully mate even under indoor or artificially assisted conditions, and there is a phenomenon of mating difficulty (Yang Xiuhao, 2017). At present, there is no artificial rearing method for the whole life cycle of E. signifer, including the egg stage, larval stage, pupal stage and adult stage. Experimental materials are obtained only by relying on the low-efficiency method of field artificial collection, which is time-consuming and laborious, and there is a problem of difficulty in breeding offspring due to mating difficulty of female and male adults. Based on this background, the present application develops an artificial rearing method for the whole life cycle of E. signifer. SUMMARY

[0003] The purpose of the present application is to provide an artificial rearing method for the whole life cycle of E. signifer in order to solve the problems of low survival rate of newly hatched larvae of E. signifer, mating difficulty of adults, and lack of an artificial rearing method covering the whole life cycle of E. signifer including the egg stage, larval stage, pupal stage and adult stage.

[0004] An artificial rearing method for the whole life cycle of E. signifer comprises the following steps:

[0005] 1) Open a small hole with a diameter of 2mm on the plastic box cover for ventilation and air permeability, sterilize the plastic box, soak the sterile filter paper with sterile water, and make sure that no water droplets and water stains are formed on the filter paper, then place the sterile filter paper in the transparent plastic box; after the adult lays eggs, place the eucalyptus moth eggs in the plastic box with the bottom covered with sterile filter paper, 50 eggs in each box, and try to disperse the eggs without clustering, then spray sterile water every 3 days, place at room temperature, and wait for the eggs to hatch.

[0006] 2) The newly hatched larvae are group-raised in the plastic box of step 1), and the pleurotus ostreatus sticks are used for the larvae to feed and hide, and sterile water is sprayed daily to keep the surface moist; according to the head capsule width of 3rd instar larvae being 1.6-2.0mm, the instar is determined, and after the 3rd instar larvae, single head feeding is carried out in a sterile glass culture dish with a diameter x height of 90mm x 15mm, and fresh and surface-sterilized 1-3 year old eucalyptus stems or branches are placed for the 3rd instar larvae to feed, and the stems or branches are replaced every 7 days until pupation; the whole process is kept in the dark, the temperature is 23℃±2℃, and the relative humidity is 70%±10%.

[0007] Beneficial effects: The host transfer of newly hatched larvae to 3rd instar larvae is the key to larval survival, and the present application can improve the survival rate of larvae by feeding 3rd instar and above larvae with fresh eucalyptus wood sections.

[0008] 3) The female and male pupae of step 2) are individually moved into glass test tubes with a diameter x height of 20mm x 153mm, 20-50 heads are placed in one sterilized plastic box with a length x width x height of 33.9cm x 22.9cm x 15.7cm, a small hole is punched on the top of the box to ensure ventilation and air permeability; the plastic box is placed in an artificial climate incubator, the relative humidity is set to 70%±10%, the temperature is 22℃±2℃, and it is kept in the dark; the temperature of the artificial climate incubator where the female pupae are placed is 2-3℃ higher than that of the male pupae, so that the adult emergence is more uniform; the dead pupae are cleaned up in time, the plastic box and the incubator are disinfected every week; after the body color of the pupae turns black brown, the relative humidity is kept at 80%±10%, the temperature is 25℃±2℃, the pupae are taken out from the glass test tubes and moved into the emergence cage made of a mesh insect rearing cage with a length x width x height of 60cm x 40cm x 40cm, 20-50 female or male pupae are placed in each cage, and a 50-cell long x wide x high 84mm x 16mm x 15mm suction tray is placed in the cage as a pupal emergence carrier, and a layer of loose mesh is fixed on the upper surface of the tray along the four edges with hot melt adhesive to provide a climbing surface for adult emergence.

[0009] Beneficial effects: Eucalyptus moth is a single worm single tunnel damage to eucalyptus, and the female and male pupae are individually moved into glass test tubes to simulate the single worm single tunnel environment in the wild; in addition, under the same temperature conditions, the emergence time of female moths is 2-3 days later than that of male moths, in order to ensure the synchronous emergence of female and male moths and promote copulation, the cultivation temperature of female pupae is 2-3℃ higher than that of male pupae.

[0010] 4) Put the step 3) initial emergence of unmated female, male moth into the net cage with length x width x height of 9.6m x 4m x 8m for adult climbing, mating, copulation, and the net cage periphery has no light source; keep the female to male ratio in the net less than or equal to 1:3 to promote adult copulation; spray clean water on the ground to make it wet, which can prolong the adult life and improve the activity; after the adult mating is stable, move the paired adults into the net cage with length x width x height of 60cm x 40cm x 40cm, and the bottom of the cage is placed with a kraft paper slightly larger than the bottom surface for collecting the eggs laid by female moths.

[0011] Beneficial effects: Eucalyptus moth copulation has always been a problem in the industry, and through experiments, the application finds that space, light source and sex ratio are the key factors interfering with adult copulation, and provides a net cage with length x width x height of 9.6m x 4m x 8m and no light source around the periphery for the adults, and the female to male ratio is less than or equal to 1:3. The conditions can guarantee successful copulation of the adults.

[0012] Beneficial effects of the application:

[0013] (1) The application can improve the survival rate of larvae by feeding 3rd instar and above larvae with fresh eucalyptus wood segments.

[0014] (2) The application can improve the survival rate of pupae and guarantee the synchronous emergence of female and male moths by moving female and male pupae into glass test tubes separately, and the cultivation temperature of female pupae is 2-3℃ higher than that of male pupae.

[0015] (3) The application can guarantee successful copulation of the adults by providing a net cage with length x width x height of 9.6m x 4m x 8m and no light source around the periphery, and the female to male ratio is less than or equal to 1:3.

[0016] (4) The application has simple requirements for the feeding environment conditions, and the light, temperature and humidity are easy to control. The raw materials used are easy to obtain, the operation steps are simple, the workload in the feeding process is small, and the method is simple and easy to implement. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 It is a process diagram for artificial feeding of eucalyptus moth.

[0018] Figure 2 It is a process diagram for artificial feeding of eucalyptus moth larvae. DETAILED DESCRIPTION

[0019] Now, a variety of exemplary embodiments of the application will be described in detail, which should not be considered as limiting the application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the application.

[0020] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. In addition, where a range of values is provided, it is understood that each intervening value, to the upper and lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed. The upper and lower limits of these smaller ranges can independently be included or excluded in the range, and are also encompassed by the application, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included.

[0021] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in detail the methods and / or materials useful in connection to the documents. In the event of conflict between the content of the specification and that of any document incorporated herein by reference, the content of the specification controls.

[0022] Many modifications and variations of the present application described in the specification are possible without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The specification and examples are illustrative only.

[0023] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", and the like are open-ended terms that are intended to mean including, but not limited to.

[0024] As shown in Figure 1 , the embodiment of the present application provides a method for artificial feeding of the whole life cycle of the Eucosia eucyrtia:

[0025] 1) The Eucosia eucyrtia eggs are placed in a plastic box with sterile filter paper on the bottom, 50 eggs per box, and then sterile water is sprayed every 3 days, and the plastic box is placed at room temperature, with a 2mm diameter hole on the cover for ventilation, and the eggs are allowed to hatch.

[0026] 2) As shown in Figure 2 , the newly hatched larvae are fed and hidden with Pleurotus ostreatus sticks, and after the 3rd instar, the larvae are individually fed in a glass culture dish with fresh and surface-sterilized eucalyptus stems or branches, which are replaced every 7 days until pupation; the feeding process is kept in complete darkness, with a temperature of 23°C ± 2°C and a relative humidity of 70% ± 10%.

[0027] 3) As shown in Figure 1The shown female and male pupae are individually moved into glass test tubes, 20-50 heads are placed in a sterilized plastic box, a small hole is punched on the top of the box to ensure ventilation; the plastic box is placed in an artificial climate incubator with relative humidity of 70%±10%, temperature of 22℃±2℃, and darkness; the temperature of female pupae is 2-3℃ higher than that of male pupae; after the body color of the pupae turns black brown, the relative humidity is kept at 80%±10% and the temperature is kept at 25℃±2℃, the pupae are taken out from the glass test tubes and moved into a pupation cage with a size of 50cm×50cm×50cm for adult emergence and climbing.

[0028] 4) The unmated female and male moths after emergence are placed in a net cage with a size of 9.6m×4m×8m and no light source around the cage, the ratio of female to male is less than or equal to 1:3, the paired adults are moved into a rearing cage with a size of 50cm×50cm×50cm, as shown in Figure 1 The bottom of the cage is placed with a kraft paper slightly larger than the bottom surface for collecting the eggs laid by female moths.

[0029] Example 1

[0030] Effect of activity space size on courtship and mating behavior

[0031] 20-30 heads of 1-3 day old adults after initial emergence and unmating are respectively placed in A, B, C, and D types of net cages. A type is a small net cage with a size of 1.5m×1.5m×1.5m, the six sides are composed of a gauze net; B type is a net cage with a size of 7.5m×7.5m×2m, the six sides are composed of an iron net, the top is a sloping roof, and eucalyptus branches and eucalyptus wood segments are placed in the center of the net and filled with clean water; C type is a large net cage with a size of 6m×6m×5m, the six sides are composed of a gauze net; D type is a greenhouse with a size of 9.6m×4m×8m, the four sides are composed of a 100-mesh iron net, and the top is a sloping roof covered with transparent glass. All experiments are carried out under natural light at the same time period, and Sony Lice 6500 cameras and a hundred-step infrared recorder are used to record the courtship and mating behavior. The experiments are repeated 3 times in each type of cage, and the total mating number is counted, and the results are shown in Table 1.

[0032] Example 2

[0033] Effect of sex ratio on courtship and mating behavior

[0034] In the D-shaped net greenhouse in Example 1, 20 1-3 day old virgin adults were put in the net at a ratio of 3:1, 1:1 and 1:3 of female to male, respectively. The process from the first adult starting to move to the last adult resting was observed at sunset every day, the number and ratio of female and male moths in the net were kept, and the observation was continued for 3 days. The courtship and mating behaviors were recorded by Sony Lice 6500 camera and Baibu Tong infrared recorder, and the results are shown in Table 1.

[0035] Table 1 Influence of activity space and sex ratio on courtship and mating behaviors of Echymipera maculosa

[0036]

[0037] Note: ♀, male moth; ♂, female moth.

[0038] As shown in Table 1, complete courtship and mating behaviors occurred only in the D activity space with a length of 9.6 m, a width of 4 m and a height of 8 m, while the moths in treatments A, B and C did not mate successfully. In the D-shaped net cage, moths mated when the female to male ratio was 1:3 and 1:1, and the former was more likely to mate successfully.

[0039] The above examples only describe the preferred modes of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A method for artificially rearing the eucalyptus bat moth throughout its entire life cycle, characterized in that, The feeding method is as follows: 1) Place the eucalyptus moth eggs in a plastic box with sterile filter paper on the bottom. Make a small hole with a diameter of 2 mm on the lid of the plastic box for ventilation. There should be 50 eggs in each box. Spray sterile water once every 3 days and place at room temperature. 2) Newly hatched larvae are provided with oyster mushroom spawn for hiding and feeding. Larvae older than 3rd instar are raised on eucalyptus stems or branches. During the rearing process, keep the area completely dark, with a temperature of 22℃±2℃ and a relative humidity of 70%±10%. 3) Transfer the male and female pupae individually into glass test tubes. Place 20 to 50 pupae into a sterilized plastic box. Make small holes in the top of the box to ensure ventilation. The environmental conditions should be the same as those for the larvae. After the pupae turn dark brown, maintain a relative humidity of 80% ± 10% and a temperature of 25℃ ± 2℃. Remove the pupae from the glass test tubes and transfer them into the rearing cage. 4) Place the emerged, unmated female and male moths into a net cage, with a piece of kraft paper slightly larger than the bottom of the cage placed at the bottom. In condition 4), the adult insects need a net cage with a length × width × height of 9.6m × 4m × 8m and no light source around it; In condition 4), the female-to-male ratio is equal to or less than 1:

3.

2. The artificial rearing method for the entire life cycle of the Eucalyptus bat moth according to claim 1, characterized in that, In condition 2), the feed for larvae in their third instar or older is fresh eucalyptus stems or branches from 1 to 3 years old, and it should be replaced every 7 days.

3. The artificial rearing method for the entire life cycle of the Eucalyptus bat moth according to claim 1, characterized in that, In condition 3), the temperature of the female pupa is 2℃~3℃ higher than that of the male pupa.

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