CN110754441A - Method for storing and raising small sterculia bigelovii larvae - Google Patents
Method for storing and raising small sterculia bigelovii larvae Download PDFInfo
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Abstract
The invention discloses a method for storing and feeding small sterculia malabarica larvae, which comprises the following steps: (1) sealing the apple branches and larvae of the hatched small Martin larvae with preservative films and then storing in a constant temperature and humidity box; (2) filling a small container with feed for feeding the small Martin larvae under an aseptic condition, covering with a preservative film, and sealing the container with a cover to obtain an artificial feed box; (3) taking out the larvae from the apple branches, and then sterilizing; (4) digging a small pit for accommodating larvae on the feed surface of the artificial feed box, placing the larvae into the small pit, and covering a preservative film on the feed surface; and (4) pricking vent holes on the preservative film by using a sterile insect needle, sealing the box cover, and storing in a constant temperature and humidity box. The method effectively prolongs the storage period and pupal stage of the larvae of the small Martin apples, obviously improves the eclosion rate of the larvae, and lays a foundation for completing the complete breeding of the small Martin apples from eggs, larvae, pupae, adults and eggs.
Description
Technical Field
The invention relates to a method for preserving and breeding pest larvae, in particular to a method for preserving and breeding small sterculia mala larvae, and belongs to the field of preserving and breeding of small sterculia mala larvae.
Background
The apple small Jiding Agrilus mali Matsumura is an important pest which harms branches and stems of apples in wild fruit forest in Xinjiang and in northern apple producing areas in China, and the pest is seriously harmed especially in apple orchards in management and aging periods. Since the discovery that the apple-shaped small jiding is distributed in China in 1956, previous researches report the biology, harm and transmission mode and control method of the pest. In 1995, the pest endangered in Yili apple orchard in Xinjiang, and in 2004, it was found that the damage of wild fruit forest in Xinyuan and Jiancheng county was serious and caused the death of wild apples and the like in large area.
According to the current research report, the pests are distributed in most of apple, crabapple, Chinese Shaguo and other production areas in China, and the method of cutting down the damaged trees, cutting off the damaged branches and stems and removing the pest source is commonly adopted to prevent and control serious damaged forests, so that the damage of the pests is difficult to be fundamentally solved by removing the pest source. Therefore, a large amount of test insects need to be collected from the damaged branches of the small Martin apple trees, and researches such as behaviors, host chemical information, molecular biology and indoor medicament screening are carried out so as to explore a new prevention and control technology; in order to ensure and meet the needs of the research on insect testing, the technical problem of artificial breeding of the small Marigold is necessary to be solved.
The existing artificial feed for the larvae of the apple midge can be used for feeding the larvae collected from the branches of the damaged apples to pupae and obtaining adults (Lebang, Zhang Zheng Qing, everage, biological characteristics of the adult apple midge are complemented and larva is fed, proceedings of northwest forest academy, 2017, (6): 214-.
Disclosure of Invention
The invention mainly aims to provide a method for preserving and breeding the larvae of Marigold;
the above object of the present invention is achieved by the following technical solutions:
a method for preserving and rearing of Pingting larvae comprises:
(1) cutting micro wounds on the apple branches, and inoculating small apple gerbil eggs to the micro wounds for hatching; sealing the apple branches and larvae of the hatched small Martin larvae with preservative films and then storing in a constant temperature and humidity box;
(2) filling a small container with feed for feeding the larvae of the Pingting midge under an aseptic condition, covering with a preservative film, sealing the container with a cover to obtain an artificial feed box, and storing the artificial feed box at a low temperature;
(3) taking out the larvae from the apple branches, and then sterilizing;
(4) digging a small pit for accommodating larvae on the feed surface of the artificial feed box, placing the larvae into the small pit, and covering a preservative film on the feed surface; and (4) pricking vent holes on the preservative film by using a sterile insect needle, sealing the box cover, and storing in a constant temperature and humidity box.
As a preferred embodiment of the present invention, the conditions of the constant temperature and humidity chamber in the step (1) are that the temperature is 5 ℃ and the relative humidity is 70%; experiments show that the larvae in the damaged apple branches stored in a constant temperature and humidity box with the temperature of 5 ℃ and the relative humidity of 70 percent are dissected in 3 months after storage, the larvae still do not grow into pupae and imagoes, and only a few of the original pupae in the branches grow into the imagoes; anatomical examination after 6 months of storage showed that the method preserved larvae and pupae still alive as long as the shoots did not dehydrate and dry.
As a preferred embodiment of the present invention, the length of the micro wound in step (1) is less than 4 mm.
As a preferred embodiment of the present invention, the small container in step (2) is preferably a plastic dish with a cover; more preferably, the vessel is a circular plastic vessel with a lid, and has a diameter of 1.5cm and a height of 1 cm.
In a preferred embodiment of the present invention, the low temperature in step (2) is preferably a temperature of 0 to 5 ℃, more preferably 5 ℃.
In a preferred embodiment of the present invention, the step (3) of sterilizing the larvae is performed by sterilizing the larvae for 30 seconds under an ultraviolet lamp.
As a preferred embodiment of the present invention, only 1 larva is placed in each artificial feed box in the step (4); and (4) pricking 3-5 more air holes on the preservative film by using a sterile insect needle.
In a preferred embodiment of the present invention, in the step (4), the temperature of the constant temperature and humidity chamber is 5 ℃ and the relative humidity is 70%.
In addition, if the larvae need to be bred into pupae or imagoes, the larvae stored in the constant-temperature and constant-humidity box are taken out together with the feed box, and are bred in a dark environment and in a moisture-preserving manner in an artificial climate box at the temperature of 25 ℃ until all the larvae emerge or die.
The eclosion observation is carried out on 500 apple midge pupae which are stored at 5 ℃ for 15 days and fed in an artificial feed box, and 1 batch of the apple midge pupae is fed in each 100 batches. The 5 batches of eclosion shows that the eclosion rate is 50.0-70.0 percent, the average is 59.0 percent, the pupal stage is 10-13 days, the average is 11.9 days, and the male-female ratio is about 1: 1.
The apple branches of the hatched small sterling larvae of the apples and the larvae are sealed by a preservative film and then stored in a constant-temperature and constant-humidity box with the temperature of 5 ℃ and the relative humidity of 70 percent, the branches are dissected in 3 months after storage, the larvae are not developed into pupae or imagoes, and only a few of the original pupae in the branches are developed into the imagoes; anatomical examination after 6 months of storage showed that the method preserved larvae and pupae still alive; in addition, the invention adopts an artificial feed box to feed the larvae,
the method effectively prolongs the storage period and pupal stage of the larvae of the small Martin apples, obviously improves the eclosion rate of the larvae, and lays a foundation for completing the complete breeding of the small Martin apples from eggs, larvae, pupae, adults and eggs.
Drawings
FIG. 1 is a schematic view of an egg collection cuvette.
Fig. 2 apple-small gerbil eggs on leaves.
FIG. 3 shows the trend of the total egg laying amount of the adults in the treatment.
FIG. 4 is the micro wound of the egg on the branch of the egg incubator and the micelle and the wormhole generated after the larva hatched into the cortex.
Detailed Description
The invention is further described below in conjunction with specific embodiments, the advantages and features of which will become apparent from the description. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.
Test example 1 Collection, preservation and hatching of apple-Proctedinium parvum eggs and preservation and rearing of larvae test 1 materials and methods
1.1 insect sources and treatments
Apple branches endangered by apple Xiaojiding are collected from Yili wild fruit forest in Xinjiang in 2017 and 2018 in 6 months, cut into branch sections with the length of about 30-40 cm and 1 bundle of each 8-10 branch sections, completely wrapped by a preservative film to keep moisture, and then pricked with 20-30 air holes by using insect needles on the preservative film, and then placed in an RXZ type artificial climate box at the temperature of 4-5 ℃ and the relative humidity of 70-80% for later use. And carefully dissecting the preserved damaged apple branches in a super-clean workbench in 7 months, wherein the dissected larvae and pupae are used for storage and continuous feeding research, and adults are used for spawning research and the like.
1.2 adult oviposition and egg collection
1) The method comprises the steps of manufacturing an egg collector, namely ① cutting an apple branch which is about 15cm high and about 1cm in diameter and carries 2-3 leaves from apple trees in a tree garden in the southern school of northwest agriculture and forestry science and technology university, meanwhile, filling sterilized sand in a plastic water cup which is 8cm in diameter and 15cm in height to 2/3 of the height of the water cup, filling sterile water in the sand, inserting the cut apple branch carrying the leaves into the sterilized sand, then, inversely buckling and sealing the apple branch with the diameter of 9cm and the height of 15cm, and using an insect to prick 20-30 air holes on the inversely buckled plastic water cup, ② the large egg collector, manufacturing the large egg collector with a plastic barrel with the diameter of 23cm and the height of 25cm, manufacturing the large egg collector with the thickness of 15cm of sandy soil, and inserting the apple branch with the height of about 50cm and the diameter of 1.5-2 cm in diameter and the diameter of 2-3 leaves.
2) Collection and preservation of eggs: placing 5 pairs of male and female adults collected in the step 1.1 into a small container for laying eggs and collecting eggs, placing an egg collecting device indoors, replacing apple branches every 2 days, and collecting small apple gerbil eggs. Putting clean filter paper into a sterilized culture dish, and spraying the filter paper with sterile water; placing at most 3 pieces of the leaves with ovum above the filter paper, then placing in a constant temperature and humidity box with 5 deg.C, 10 deg.C, 15 deg.C for storage, periodically checking and supplementing water.
1.3 egg hatching method
1) Manufacturing an incubation box: cutting small apple branches with the diameter of 1.0-1.5 cm and the length of 15-20 cm, disinfecting the surfaces of the branches by using 95% ethanol, and sealing the cut openings at the two ends of the branches by using preservative films; then selecting a transparent plastic box with the length of 18cm, the width of 12cm and the height of 10cm, adding the sterilized sand to 1/5 of the height of the plastic box, and adding sterile water into the sand without overflowing; and horizontally placing the treated branches on wet sand in a plastic box, and placing 4 branches in each box. The diameter of a large branch for hatching eggs is 1.5-2.0 cm, the length of the large branch is 35-50 cm, the small branch is treated in the same way, and then the large branch is placed into the 1.2 large egg collector for hatching.
2) Egg hatching test: pricking 5 micro wounds with the lengths of less than 4mm on the bark of branches in an incubation box at equal intervals by using tool tips, putting 1 egg of apple and gerbil into each wound, and putting 20 eggs on 4 branches. And then sealing the opening of the plastic box by using a preservative film, pricking 15-30 air holes on the preservative film by using a sterile insect needle, and culturing at room temperature to observe and record the hatching condition of the eggs. If the branches are infected by mixed bacteria, spraying 0.5 percent carbendazim on the branches for sterilization.
Judging the hatching of apple small gerbil eggs: after the 1 st larva of the apple midge Jiding invades the bark of the branch, the epidermis of the invaded part is changed from grey brown to red brown, and red brown liquid with the diameter of about 1 mm-2 mm is exuded. Therefore, the judgment standard of whether the eggs in the branches hatch is determined according to whether the surfaces of the branches where the eggs are placed turn red or have reddish brown colloidal liquid grains.
1.4 preservation and rearing of larvae
1) Preparing a feed: the formulation is prepared according to the literature report, sealed in a beaker by a preservative film and stored in a refrigerator at the temperature of-5 ℃ (Limeng, Zhangqing, everage, apple Xiaojiding adult biological characteristic supplement and larva feeding, academy of northwest Linn academy, 2017, (6) 214-.
TABLE 1 Artificial feed formula for Martin larvae
A2, improved feed formulation. And D, feed formula reported in literature.
2) Manufacturing an artificial feed box: selecting a round plastic dish with diameter of 1.5cm and height of 1cm and a box cover, scrubbing with 75% ethanol, and sterilizing. Taking the preserved feed under aseptic condition, heating the feed to room temperature, filling the feed in a circular plastic dish, covering the feed with a preservative film, sealing a box cover, and preserving in a refrigerator at 5 ℃.
3) And (3) storing the larvae in the damaged apple branches: according to the method in the 1.1 insect source and treatment, the branches are sealed by preservative films and stored in a constant temperature and humidity box.
4) Preservation of larvae dissected from shoots: taking the branches in the '1.1 insect source and treatment', dissecting larvae from the branches, and sterilizing for 30 seconds under an ultraviolet lamp; the method comprises the steps of opening a stored artificial feed box, digging a smooth pit capable of containing worm bodies on the surface of feed, carefully placing larvae into the pit, only placing 1 head of each box, covering a preservative film, pricking 3-5 air holes on the preservative film by using sterile insect needles, and sealing a box cover and then storing in a constant-temperature constant-humidity box at 5 ℃ for later use.
If the larvae need to be bred into pupae or adults, the larvae stored in the constant-temperature and constant-humidity box are taken out together with the feed box, bred in a dark and moisture-preserving manner in a 25 ℃ artificial climate box, and observed and recorded the development phenomenon of the larvae every day until all the larvae emerge or die.
1.5 preservation and rearing of pupae
Dissecting pupa from the damaged branch, placing into plastic culture dish, placing 1 pupa per dish, placing 1 piece of filter paper under pupa body, moistening with water, opening 1 air vent on the culture dish cover, covering with culture dish cover, and storing in 5 deg.C constant temperature and humidity box for use.
When feeding pupa, taking out the preserved pupa together with culture dish, placing into 25 deg.C constant temperature and humidity chamber for conservation, checking pupa growth and eclosion every 2d, and adding water for keeping moisture.
2 results and analysis
2.1 Collection and preservation of apple-Proctens
Adult apple midge was housed in an egg collection container, setting A, B, C3 treatments:
a small branch egg collector, scattering light at 25 ℃ in a room.
And B, small branch egg collector and outdoor illumination.
C, large branch egg collector, and outdoor illumination.
In the experiment, apple-small gerbil eggs mostly lay on the main veins and branch veins of the leaves, and a few lay eggs on plastic cups (fig. 1 and 2). Of A, B, C three kinds of induced imagoes, the average life of the imagoes treated by B is longer, the total egg production A is more than B and more than C, the egg laying period A is 7d, C is 6d, and B is 11d (Table 2). Therefore, the small egg collecting container can attract the adult to lay eggs on the apple leaves and collect the eggs laid by the adult, and the effect of collecting the eggs by the A treatment is better.
TABLE 2 egg laying in egg collecting Small containers
1) emergence in 6/14 th in 2018, treatment started in 6/16 th in 2018, and 2 females and males per egg collector. 2) Test treatments and symbols in the table: a, a small branch egg collector, scattering light at 25 ℃ in a room. And B, a small branch egg collector and outdoor illumination. And C, large branch egg collector and outdoor illumination. S means death of the adult.
A. B, C, the variation trend of the total egg production of the adult midge apple in the three treatments shows that the variation trends of the egg production of the three treated adults are basically similar, the adults start to lay eggs at the 8 th day after eclosion and stop laying eggs at the 26 th day after eclosion, and the egg laying period lasts 28.6 to 30.0 days; however, the amount of eggs laid was small at the first and large at 10 th to 16 th days, and the number of eggs laid was gradually reduced thereafter (FIG. 3).
2.2 incubation of apple-small Geding eggs
Collecting small Germin ova of apple by using a small egg collector from 19 days to 22 days in 7 months in 2017, carrying out no low-temperature preservation treatment on the ova after every 20 ova are collected, and transferring the ova into branch cortex after the ova are placed for 3 days at room temperature for carrying out an incubation test (figure 4). In an incubation test of 19 days in 7 months and 7 months in 2017, the total incubation rate of 440 eggs is 13.86 percent, the egg period is 13-20 days, and the average time is 17.09 days; however, the egg phase tended to gradually increase with the delay of the incubation test date (Table 3). The reason that the eggs transferred and connected to the branch micro-wounds of the hatching box can be hatched is that the branches in the hatching box are all mildewed, dehydrated and dried, and larvae are difficult to invade the cortex of the branches to die.
160 and 100 eggs of small apple gerbil stored in a refrigerator at 5 ℃ for 14d and 24d were taken in 30 and 10 in 2017 and 8 and 10 in 2017, respectively, and 2 groups of hatching experiments were carried out but no eggs were hatched. To further determine whether the eggs could be hatched after cryopreservation, the eggs were stored at 5 ℃, 10 ℃ and 15 ℃ for 6, 23 and 3 months from 2018, and then comparative hatching tests were performed for the eggs stored at low temperature and the eggs not stored at low temperature (Table 4). The results showed that eggs stored at low temperatures of 5 ℃ and 10 ℃ could not be hatched, that eggs stored for 10d and 20d were hardly hatched, and that 30 eggs stored for 10d at 15 ℃ were hatched only 3 eggs. However, the total hatchability after the primary eggs are directly transferred to the small branches is 21.67%, the total hatchability after the primary eggs are directly transferred to the large branches is 32.00%, and the reason that the hatchability of the eggs on the large branches is high is probably caused by the slow dehydration and drying rate. In addition, compared with the results of transferring and hatching after being placed at room temperature for 3 days, the egg period of the low-temperature preservation and the egg period of the non-low-temperature preservation treatment are only 11 days to 14 days, and the average is 12.5 days. The results show that the egg period is short when the eggs are hatched immediately at the beginning, the egg period is not prolonged by low-temperature preservation treatment, and the hatching rate of the eggs is lower or the eggs are difficult to hatch after the eggs are preserved at low temperature.
TABLE 3 incubation results of unstopped apple Gilles eggs
Eggs were received on twigs and incubated at 25 ℃. Minor branches of eggs. Eggs hash at 25 deg.C
TABLE 4 incubation results of treated apple-Germin eggs
Eggs were incubated at 25 ℃. Control, no preservation treatment.
2.3 feathering results after preservation of Martin larvae
Putting larvae in the damaged apple branches stored in a constant temperature and humidity box with the temperature of 5 ℃ and the relative humidity of 70% in an experiment, dissecting the branches after the storage for 3 months to find that the larvae still do not develop into pupae and imagoes, wherein only a few of the original pupae in the branches develop into the imagoes; anatomical examination after 6 months of storage showed that the shoots did not dehydrate and dry, and the method preserved larvae and pupae to remain viable.
Larvae stored at 5 ℃ for 15 days in the A2 and D feed boxes were taken and fed to the 25 th day in the respective feed boxes at 25 ℃. The results showed that 30 larvae fed with A2 feed failed to emerge adults and that larvae fed with D feed had a 10% emergence rate, but in past studies larvae fed with A2 feed were also able to emerge adults where they had emerged[8]. Compared with the feed D, the feed A2 has the advantages that the apple peel powder is used for replacing cellulose in the feed A2, the composite vitamin is lacked, and the preservative is different from the feed D. The larva may die because the larva is not developed and feathered in the A2 feed and the larva feathering rate in the D feed is low because the larva is dissected from the injured branch in the test without care and damages the body of the larva, or the apple bark in the A2 feed is not smashed into fine and sharp wood filament structure and damages the body of the larva.
2.4 preservation and feathering of apple Gilles pupae
Separately collecting 500 pupa Bombycis of Aphancei stored at 5 deg.C for 15d, and observing eclosion at a rate of 1 batch per 100. The 5 batches of eclosion shows that the eclosion rate is 50.0-70.0%, the average is 59.0%, the pupal period is 10-13 d, the average is 11.9d, and the male-female ratio is about 1:1 (table 5), but the pupal body turns black and dies within 3-4 d in the eclosion process of the pupal, and the reason may be that the pupal body is damaged in the operation process, so that the pupal body is infected with diseases and dies.
TABLE 5 eclosion statistics of apple small gerd pupae
Claims (10)
1. A method for preserving and breeding the larvae of Martin, which is characterized by comprising the following steps:
(1) cutting micro wounds on the apple branches, and inoculating small apple gerbil eggs to the micro wounds for hatching; sealing the apple branches and larvae of the hatched small Martin larvae with preservative films and then storing in a constant temperature and humidity box;
(2) filling a small container with feed for feeding the larvae of Pingting.Martin under aseptic conditions, covering with a preservative film, sealing the container with a cover to obtain an artificial feed box, and storing the artificial feed box at low temperature;
(3) taking out the larvae from the apple branches, and then sterilizing;
(4) digging a small pit for accommodating larvae on the feed surface of the artificial feed box, placing the larvae into the small pit, covering a preservative film on the feed surface, pricking vent holes on the preservative film by using a sterile insect needle, sealing a box cover, and storing in a constant temperature and humidity box.
2. The preservation and feeding method according to claim 1, wherein the conditions of the constant temperature and humidity chamber in the step (1) are a temperature of 5 ℃ and a relative humidity of 70%.
3. The preservation and feeding method according to claim 1, wherein the length of the micro-wound in step (1) is less than 4 mm.
4. The preservation and feeding method according to claim 1, wherein the small container in the step (2) is a plastic dish with a lid.
5. The preservation and feeding method according to claim 4, wherein the plastic dish with a lid is a circular plastic dish with a lid having a diameter of 1.5cm and a height of 1 cm.
6. The preservation and feeding method according to claim 1, wherein the low temperature in the step (2) is a temperature of 0 to 5 ℃, preferably 5 ℃.
7. The preservation and feeding method according to claim 1, wherein the sterilization treatment in the step (3) is sterilizing the larvae for 30S under an ultraviolet lamp.
8. The preservation and feeding method according to claim 1, wherein only 1 larva is placed in each artificial feed box in the step (4); and (4) pricking 3-5 more air holes on the preservative film by using a sterile insect needle.
9. The preservation and feeding method according to claim 1, wherein the temperature of the constant temperature and humidity chamber in the step (4) is 5 ℃ and the relative humidity is 70%.
10. The preservation and feeding method according to claim 1, wherein if it is desired to breed the larvae into pupae or adults, the larvae stored in the constant temperature and humidity chamber are taken out together with the artificial feed box, and bred in the dark in the artificial climate chamber at 25 ℃ in a humidified state until all the larvae emerge or die.
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