CN106689062B - Method for evaluating toxicity of pesticide on bee larvae by utilizing artificial feeding of bee larvae in laboratory - Google Patents
Method for evaluating toxicity of pesticide on bee larvae by utilizing artificial feeding of bee larvae in laboratory Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
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Abstract
The invention discloses a method for evaluating toxicity of pesticide on bee larvae by utilizing artificial feeding of the bee larvae in a laboratory, which comprises the steps of preparation of the bee larvae, laboratory feeding of the bee larvae, preparation of tested pesticide, addition of the tested pesticide and the like, and finally, the processes of defecation, silking, pupation and eclosion of the larvae in a hole are observed. The method can be used for directly researching the toxicity of the pesticide on the bee larvae in a laboratory without depending on bee swarms by breeding the bee larvae in the laboratory. The method has high survival rate of bee larva and basically consistent growth condition. The method is labor-saving and effective, can obtain the bee larvae with basically consistent growth states in batches in a short time, can visually, efficiently and accurately evaluate the influence of the pesticide on the bee larvae, can develop a plurality of in-vivo experiments taking the bee larvae as objects, has stronger practicability and expansibility, and provides better materials and methods for related research and practice work.
Description
Technical Field
The invention belongs to the field of ecotoxicity tests, relates to a method for evaluating toxicity of pesticide to bee larvae, and particularly relates to a method for evaluating toxicity of pesticide to bee larvae by utilizing artificial feeding of bee larvae in a laboratory.
Background
The bees play an important role in pollination of many crops by virtue of the advantages of high pollination efficiency, collection specificity, population sociality and the like, and are the groups with the largest variety and the largest quantity in naturally pollinating insects. However, not only the collection behavior and health of the collected bees are seriously affected by the abuse of pesticide, but the honey, pollen and pesticide accumulated in the honeycomb polluted by pesticide also have an influence on the growth and development of the bee larvae, and when the bee larvae are seriously damaged, the bee larvae die greatly. The less the number of larvae in the swarm, the less the possibility of developing strong swarm, the poorer the collecting capability, the inability to produce bee products and pollinate, and the heavy strike on the bee-keeping industry and agriculture.
At present, in order to evaluate the toxicity of pesticide on bee larvae, scientific workers develop research on the influence of pesticide in a honeycomb on the bee larvae, however, bees are typical real social insects and have definite and detailed labor division, larva food in bee colonies is provided by professional feeding bees, the larvae are usually abandoned by worker bees, pesticides added into natural bee colonies are easy to store on the honeycomb to influence the breeding of the bee colonies, and meanwhile, the pesticide dosage is diluted by an external honey source, so that the defects of poor repeatability, low persuasion of test results and the like exist. If the larvae can be raised in a laboratory for research on acute toxicity of pesticides on bees, the food components of the larvae can be artificially controlled, and the influence of feeding bees and external honey sources can be eliminated. Therefore, the research effort needs to be increased, feasible technologies need to be explored, and an effective, simple and high-repeatability indoor method needs to be established.
In order to establish a method for evaluating toxicity of indoor pesticides to bee larvae, a technology for artificially feeding the bee larvae is an indispensable project. In the past beekeeping and research practice, several breeding techniques have been developed. However, an operator needs to have good operation experience, larva transfer is needed every day, time and labor are wasted in the process, if batch honeybee larvae are needed for test development, the test cost is greatly increased, the test process is slowed down, meanwhile, certain damage can be caused to tested larvae, and the accuracy of later-stage test results is affected.
Disclosure of Invention
In order to solve the problems, the invention provides a method for evaluating the toxicity of pesticide to bee larvae by utilizing artificial feeding of the bee larvae in a laboratory, and the method has important scientific significance.
The object of the invention can be achieved by the following measures:
the invention adopts one of the technical schemes: a method for evaluating toxicity of pesticide on bee larva by using laboratory artificial feeding bee larva comprises the following steps:
(1) preparation of bee larvae: inserting an empty honeycomb into a beehive containing a bee colony, fixing a queen bee in a spawning period on the empty honeycomb for spawning, discharging the queen bee out of the honeycomb containing bee eggs after 20-28 hours, and continuously keeping the honeycomb containing the bee eggs in the beehive until the bee eggs in the honeycomb hatch into bee larvae;
(2) laboratory rearing of bee larvae: taking out the inserted bee spleen from the beehive, picking the bee larva, carrying out adaptive breeding for 3 days by using basic ration, carrying out health examination on the 4 th day, and selecting the healthy larva;
(3) addition of the tested pesticide: dividing the healthy larvae selected in the step (2) into control group larvae and treatment group larvae and continuously feeding for 3 days, wherein the treatment group larvae are fed with a tested feed, and the tested feed contains tested pesticide with various concentrations and basic ration;
(4) pupating and observing: transferring the larvae which are bred for 3 days in the step (3) and are about to enter a pupation stage into plate holes of a pupation plate to prepare for pupation, observing the processes of defecation, silking, pupation and eclosion of the larvae in the plate holes, and counting the survival, pupation or/and eclosion conditions of the larvae.
The second technical scheme of the invention is as follows: a method for artificially feeding bee larvae in a laboratory comprises the following steps:
(1) preparation of bee larvae: inserting an empty honeycomb into a beehive containing a bee colony, fixing a queen bee in a spawning period on the empty honeycomb for spawning, discharging the queen bee out of the honeycomb containing bee eggs after 20-28 hours, and continuously keeping the honeycomb containing the bee eggs in the beehive until the bee eggs in the honeycomb hatch into bee larvae;
(2) laboratory rearing of bee larvae: taking out the inserted bee spleen from the beehive, picking the bee larva, carrying out adaptive breeding for 3 days by using basic ration, carrying out health examination on the 4 th day, and selecting the healthy larva to continue breeding for 3 days;
(3) pupating and observing: transferring the larvae which are about to enter the pupation stage after continuously breeding for 3 days in the step (2) to plate holes of a pupation plate for preparing pupation, observing the processes of defecation, silking, pupation and eclosion of the larvae in the plate holes, and counting the survival, pupation or/and eclosion conditions of the larvae.
The third technical scheme of the invention is as follows: the application of the method for artificially feeding the bee larvae in the laboratory in evaluating the toxicity of the pesticide on the bee larvae is disclosed.
The invention also discloses a specific scheme, a method for evaluating toxicity of pesticide to bee larvae by utilizing artificial feeding of the bee larvae in a laboratory, which comprises the following steps:
(1) preparation of bee larvae: inserting 1 empty honeycomb into a beehive containing bee colonies, fixing a queen bee in a spawning period on the empty honeycomb by using a queen bee partition plate to spawn, removing the queen bee partition plate after 20-28 hours to discharge the queen bee out of the inserted honeycomb, and continuously keeping the queen bee containing bee eggs in the beehive until the bee eggs in the queen bee hatching into bee larvae;
(2) laboratory rearing of bee larvae: taking out the inserted bee spleen from the beehive, picking the bee larva, placing the bee larva on a pore plate filled with basic ration of the bee larva, placing the bee larva in an incubator for breeding, transferring the bee larva to a pupation plate to prepare for pupation when the bee larva is about to enter a pupation stage, and then moving the bee larva no longer until the bee larva is completely developed. The tested pesticide can be further added in the process;
(3) preparation of the tested pesticide: dissolving tested pesticide with distilled water or/and organic solvent without toxicity to bee larva, preparing into one or more treatment groups with different concentrations, and preparing corresponding solvent control group;
(4) addition of the tested pesticide: and (3) feeding the larvae in the step (2) for 3 days, carrying out health examination on the 4 th day, removing abnormal, sick and injured larvae, and distributing the larvae to each treatment group. And (2) when the larvae are about to enter the pupation stage, transferring the larvae into plate holes of a pupation plate to prepare for pupation, observing the processes of defecation, silking, pupation and eclosion of the larvae in the holes, and counting the survival, pupation and eclosion conditions of the larvae.
Preferably, wherein the step of bee larva preparation comprises: the bee colony is a healthy bee colony which is not used with medicine and has a known source and physiological state in four weeks; the time for the queen bee to lay eggs on the empty spleen in the oviposition period is preferably 24 hours.
Preferably, the bee egg hatching period in the preparation step of the bee larvae is 2-4 days, generally 3 days.
Preferably, wherein in the step of laboratory rearing of the larvae of bees: the bee larva is 1 day old later stage larva, and its body color is white. Compared with larvae of other ages in days, the larvae of later stage of 1 day can grow well under the condition of artificial feeding in a laboratory, the survival rate of the larvae is improved to more than 90%, and more larvae can successfully enter the pupation stage.
Preferably, the basic ration comprises the following components in percentage by mass: 50 plus or minus 5 percent of fresh royal jelly, 37 plus or minus 2 percent of sterile water, 6 plus or minus 1 percent of glucose, 6 plus or minus 1 percent of fructose and 1 plus or minus 0.5 percent of yeast extract; the preferable quality of the basic ration comprises the following components: 50% of fresh royal jelly, 37% of sterile water, 6% of glucose, 6% of fructose and 1% of yeast extract. More preferably, the basal ration is ready for use and is preheated in an incubator before feeding. For example, the culture medium is preheated in an incubator for 30 minutes before feeding. Compared with other bee larva feeds, the basic ration provided by the invention can enable the larva to grow well, and greatly improve the survival rate of the larva.
Preferably, wherein in the step of laboratory rearing of the larvae of bees: the adaptive feeding is carried out by adopting a pore plate filled with basic ration; one bee larva is distributed in each hole of the pore plate, the feed amount of the basic ration of the bee larva in each hole is 40-60 mu L, and the basic ration in the pore plate is replaced every 24 hours. The preferred orifice plate may be a 48-orifice plate or other orifice plate.
Preferably, the environmental conditions in the laboratory rearing of the bee larvae and the step of adding the pesticide under test are both: the temperature is 34 +/-2 ℃, the humidity is 95 +/-1 percent, and the preferred temperature is 34 +/-1 ℃ and the preferred humidity is 95 percent; more preferably, the well plate is placed in a closed box filled with glycerol-water mixture at the bottom of the box during feeding, and then is placed in an incubator at 34 + -1 deg.C for culturing.
Preferably, wherein in the step of adding the pesticide to be tested, the larvae of the control group are divided into larvae of a blank control group or/and larvae of a solvent control group; feeding the larvae of the blank control group by using basic ration, feeding the larvae of the solvent control group by using the basic ration added with a solvent as feed, wherein each 41-61 mu L of the feed contains 0.5-5 mu L of the solvent; the treatment group larvae are divided into a plurality of treatment groups, preferably into at least 5 treatment groups, each treatment group being fed with a test feed containing a different concentration of the test pesticide.
Preferably, in the step of adding the tested pesticide, the preparation method of the tested feed comprises the following steps: dissolving tested pesticide by using distilled water or/and an organic solvent which is nontoxic to bee larvae, preparing one or more tested pesticide solutions with different concentrations, and adding the solutions into basic daily ration respectively to prepare tested feed, wherein each 41-61 mu L of the tested feed contains 0.5-5 mu L, preferably 1 mu L of the tested pesticide solution;
preferably, the feed amount of each bee larva in the continuous feeding process in the step of adding the tested pesticide is 41-61 mu L, and the feed is replaced every 24 hours. More preferably, the amount of each concentration treatment group and solvent control group in the test feed does not exceed 5% of the volume of the basal ration.
Preferably, in the step of adding the test pesticide, the test pesticide is added to the test pesticide, and the test subject is a bee larva after 4 days old.
Preferably, the tested feed is replaced at regular time (such as 24h) during the continuous feeding process, wherein the basic ration is prepared immediately and needs to be preheated in the incubator before feeding.
Preferably, in the pupation and observation step, the pupation plate holes are padded with dry sterilized paper, such as dry sterile lens wiping paper; preferably, the larva entering the pupation stage is sucked to dry the feed on the body surface of the larva before being transferred to a pupation plate; more preferably, the larvae transferred into the pupation plates do not move any more until the bee larvae have developed completely. Experiments show that the eclosion rate from pupation to bee formation can be improved by more than 50% by filling the dry sterilizing paper in the pupation plate hole.
Preferably, in the pupation and observation step, the pupation plate can adopt a 24-pore plate or other pore plates.
A specific method for breeding in a bee larva laboratory comprises the following steps: the method comprises the following steps of moving bee larvae at the later stage of 1 day age from a bee spleen to a 48-hole plate containing basic ration of the bee larvae, placing one larva in each hole, placing the 48-hole plate in a closed small tidying box with 1L of sterilized glycerol-water mixed liquid (15.5% of glycerol and 84.5% of water) at the bottom of the box, placing the box in an incubator at the temperature of 34 +/-1 ℃ for cultivation, replacing feed in the hole plate every 24 hours later, when the larvae are about to enter a pupation stage, sucking the feed on the body surfaces of the larvae with mirror wiping paper, transferring the larvae into a 24-hole plate padded with sterilized paper for pupation, and then moving the larvae until the bee larvae are completely developed. During the process, pesticide or other reagent to be tested can be further added, so that a plurality of living experiments taking bee larvae as objects can be further carried out.
The invention has the following remarkable advantages:
1. is quick and simple. By optimizing the basic ration, the larva age and the pupation plate of the bee larva, the simple and easily-mastered indoor feeding technology is obtained, and an operator can master the technology in a short time.
2. Is visual and accurate. The method for feeding the bee larvae in the laboratory can help us to develop the toxicity research of the pesticide on the bee larvae in the laboratory without depending on bee colonies, avoid the restriction of various uncontrollable factors in the bee colonies, and intuitively, efficiently and accurately evaluate the influence of the pesticide on the bee larvae.
3. And stronger practicability and expansibility. The method for feeding the bee larvae in the laboratory can develop a plurality of living experiments taking the bee larvae as objects on the basis, so the method is suitable for other researches except pesticides and has wider application range.
Drawings
FIG. 1 shows bee larvae of different developmental stages raised in the laboratory using 48-well plates, wherein A, B, C, D, E, F represent day-old larvae at 1, 2, 3, 4, 5, and 6 days old, respectively.
Fig. 2 shows bee larvae pupated and emerged in the laboratory using a 24-well plate, wherein A, B, C represents newly pupated bee larvae, mature pupae bodies, and emerged brood, respectively.
Detailed Description
Example 1: the invention relates to a method for artificially feeding bee larvae in a laboratory, which comprises the following steps:
(1) preparation of bee larvae
During the queen bee oviposition period, three cases of healthy bee colonies of known origin and physiological state are selected. Opening the beehive cover, inserting 1 empty spleen respectively, fixing the queen bee on the empty spleen by a queen bee separation plate for laying eggs, removing the queen bee separation plate after 24 hours, releasing the queen bee, and checking the new egg laying condition. According to the fertility of queen bees, the isolation time can be shortened appropriately to reduce the size and age difference between larvae. The hive containing the bee eggs is kept until hatching (the hatching period of the bee eggs is generally 3 days).
(2) Laboratory rearing of bee larvae
And taking out the inserted bee spleen from the beehive, picking the bee larvae, placing the bee larvae on a pore plate filled with basic ration of the bee larvae, and placing the bee larvae into an incubator for breeding.
In the laboratory, 48-hole plates are used for feeding the bee larvae.
The basic ration formula of the larva comprises fresh royal jelly 50%, sterile water 37%, glucose 6%, fructose 6% and yeast extract 1%. The specific operation method comprises the following steps: preparing larva basic ration, placing in an incubator, preheating for 30 minutes, and adding 50 μ L of larva ration into each well to 48-well plate.
The bee larvae at the later stage of 1 day are transferred from the bee spleens to a 48-hole plate containing basic ration of the bee larvae, one larva is placed in each hole, the 48-hole plate is placed in a small sealed storage box, the bottom of the box is filled with 1L of sterilized glycerol-water mixed liquid (15.5% of glycerol and 84.5% of water), the small sealed storage box is placed in an incubator at the temperature of 34 +/-1 ℃ for culture, and then the basic ration of the larvae in the hole plate is replaced every 24 hours.
When the larva is about to enter a pupation stage, sucking feed on the body surface of the larva by using a lens wiping paper, transferring the larva into a 24-hole plate filled with a sterilizing paper to prepare for pupation, and then, not moving the larva until the larva grows completely, wherein the specifically cultured larva of the bee in each day age can grow normally and pupate as shown in figure 1.
The bee larva cultured by the method can normally develop, and the development condition of the bee larva is basically consistent with that of the bee larva fed in the beehive. On the basis, a plurality of living experiments taking the bee larvae as objects can be developed, and the restriction of a plurality of uncontrollable factors in bee colonies is avoided.
Comparative example 1: influence of daily ration formula on growth and development of bee larvae
In order to obtain the optimal ratio of the bee larva daily ration, according to the nutrition requirement of the bee larva, the basic daily ration of the bee larva is prepared by the invention: fresh royal jelly accounts for 50%, sterile water accounts for 37%, glucose accounts for 6%, fructose accounts for 6%, and yeast extract accounts for 1%.
Three diets were formulated simultaneously at different stages given in the OECD237 guidelines:
diet a (day 1): 50% of fresh royal jelly and 50% of aqueous solution containing 2% of yeast extract, 12% of glucose and 12% of fructose;
diet B (day 3): 50% of fresh royal jelly and 50% of aqueous solution containing 3% of yeast extract, 15% of glucose and 15% of fructose;
diet C (day 4 to day 6): 50% of fresh royal jelly and 50% of aqueous solution containing 4% of yeast extract, 18% of glucose and 18% of fructose.
The experiment is divided into 2 treatments according to daily ration, each treatment is carried out 3 culture plates (with 48 holes), namely 3 treatments are repeated, and the bee larvae with different ages of days are selected for feeding, so that the larvae of the fundamental daily ration treatment group of the larvae grow well and can be defecated to enter a pupation period. The OECD237 treated group larvae grew poorly, and the larvae died after being transplanted to the plates for about 3 days, and individual larvae stopped developing after they developed to the pupal stage and then died.
The basic daily ration formula of the bee larvae is superior to the OECD237 criterion, mainly because the bee larvae develop slowly and have less sugar demand, but the bee larvae undergo a series of complex changes, the decomposition and synthesis of substances are continuously carried out in vivo, water is a main medium of physiological metabolism and biochemical reaction and has large water demand, and the OECD237 criterion increases the sugar content and reduces the water content, so the larvae grow badly.
Comparative example 2: effect of insect age on larva survival Rate
The experimental diets were unified into the best feeding formula of example 1, two treatment groups of larvae at the early stage of 1 day (egg cyan) and larvae at the later stage of 1 day (white), each treatment consisting of 3 plates (48 wells) i.e. 3 replicates. During feeding, the larvae at the earlier stage of 1 day are relatively good in growth vigor 3 days before being transplanted to a culture plate, more than 50% of the larvae on the fourth day have weakened body elasticity, gradually stop developing and die. The larvae grow well at the later stage of 1 day, more than 90% of the larvae survive, and the defecate enters the pupation stage.
Comparative example 3: influence of pupation plates on larval development
The experiment designed 2 treatment groups, one with sterile dry lens wiping paper padded in the wells of a 24-well plate, and the other with a blank 24-well plate. The experimental daily ration is unified into the daily ration formula with the best feeding effect in the example 1, the larva with the best feeding effect in the example 2 is selected for feeding, and then the larva to be pupated (prepupa) in the defecation period is moved into the pupation plate holes by using the larva moving needle, wherein 1 larva is placed in each hole. Pupation patterns of the blank pupation plate only achieved an emergence rate of 44.44% from pre-pupation to bee formation, compared to a pupation plate of pad-dried sterile mirror paper, which achieved a survival rate of up to 95.83%.
Example 2: the invention relates to a method for evaluating toxicity of pesticide to bee larvae by utilizing artificial feeding of the bee larvae in a laboratory, which comprises the following steps:
(1) preparation of bee larvae
During the queen bee oviposition period, three cases of healthy bee colonies of known origin and physiological state are selected. Opening the beehive cover, inserting 1 empty spleen respectively, fixing the queen bee on the empty spleen by a queen bee separation plate for laying eggs, removing the queen bee separation plate after 24 hours, releasing the queen bee, and checking the new egg laying condition. According to the fertility of queen bees, the isolation time can be shortened appropriately to reduce the size and age difference between larvae. The hive containing the bee eggs is kept until hatching (the hatching period of the bee eggs is generally 3 days).
(2) Laboratory rearing of bee larvae
And taking out the inserted bee spleen from the beehive, picking the bee larvae, placing the bee larvae on a pore plate filled with basic ration of the bee larvae, and placing the bee larvae into an incubator for breeding.
In the laboratory, 48-hole plates are used for feeding the bee larvae.
The basic ration formula of the larva comprises fresh royal jelly 50%, sterile water 37%, glucose 6%, fructose 6% and yeast extract 1%. The specific operation method comprises the following steps: preparing larva basic ration, placing in an incubator, preheating for 30 minutes, and adding 50 μ L of larva ration into each well to 48-well plate.
The bee larvae at the later stage of 1 day are transferred from the bee spleens to a 48-hole plate containing basic ration of the bee larvae, one larva is placed in each hole, the 48-hole plate is placed in a small sealed storage box, the bottom of the box is filled with 1L of sterilized glycerol-water mixed liquid (15.5% of glycerol and 84.5% of water), the small sealed storage box is placed in an incubator at the temperature of 34 +/-1 ℃ for culture, and then the basic ration of the larvae in the hole plate is replaced every 24 hours.
(3) Preparation of the pesticide tested
The tested pesticide is dissolved by distilled water, and for insoluble compounds, an organic solvent which is nontoxic to bee larvae is selected for assisting dissolution, wherein the amount of the solvent is not more than 5% of the diet volume, and a solvent control group is added. At least 5 concentration treatment groups were set up for the experiment.
(4) Addition of test pesticides
When tested, the bee larvae selected in the step (2) are adaptively raised for three days (the temperature is 34 +/-1 ℃, the humidity is 95 percent), health examination is carried out on the 4 th day, abnormal, sick and injured larvae are removed, and the bee larvae are distributed to each treatment group. Adding 1 μ L of pesticide to be tested into basic daily ration to obtain feed to be tested, feeding bee larva of each treatment group with the feed to be tested, changing feed in the pore plate every 24 hr, continuously feeding for 3 days, and then feeding no more in pupation stage. The larvae were observed to complete the processes of defecation, silking, pupation and eclosion in the wells.
Example 3:
using a method similar to that of example 2, using Italian bee larvae as the groundThe research on the toxicity of the pesticide on the bee larvae is carried out by taking typical neonicotinoid pesticide imidacloprid as a representative object. After the preparation of the bee larvae is completed, in the 1 st test, the larvae in the later stage of 1 day are selected from three bee colonies, the larvae are placed on a 48-hole plate, the equivalent basic ration is fed, adaptive feeding is carried out for three days (the temperature is 34 +/-1 ℃, the humidity is 95%), the health examination is carried out in the 4 th test after the test is started, abnormal, sick and injured larvae are removed, and the larvae are redistributed to each treatment group. The test was set up with 1 blank control group (C1, always fed basal diet), 1 solvent control group (C2, always fed basal diet) and 5, 50, 500, 1000, 2000 and 3000mg · L-16 concentration treatment groups (1. mu.L of each solution was added to the basal diet at 4d, 5d, and 6d after the start of the test). Feeding continuously for 3 days, then entering pupation stage, and no feeding. The larvae were observed to complete the processes of defecation, silking, pupation and eclosion in the wells.
The test result shows that the imidacloprid treatment dose is 15ng larva-1The survival rate, pupation rate and eclosion rate of the bee larvae are not influenced; when the treatment dose reaches 150ng larva-1When the bee larva emerges, the eclosion rate of the bee larva is reduced, but the survival rate and the pupation rate of the bee larva are not influenced; the half lethal dose of the larvae in 7 days is 2300ng of larvae-1。
Example 4:
using a method similar to that of example 2, using Italian bee larvae as a research object and using typical neonicotinoid pesticide dinotefuran as a representative, toxicity research of the pesticide on the bee larvae was carried out. The test was set up with 1 blank control group (C1, always fed basal diet), 1 solvent control group (C2, always fed basal diet) and 1, 5, 50, 500, 1500 and 3000mg · L-16 concentration treatment groups. Other procedures were tested in a similar manner as in example 3.
The test result shows that the dinotefuran treatment dose is 3ng larva-1The survival rate, pupation rate and eclosion rate of the bee larvae are not influenced; when the treatment dose reaches 15ng larva-1When the bee larva emerges, the eclosion rate of the bee larva is reduced, but the survival rate and the pupation rate of the bee larva are not influenced; the half lethal dose of 7 days on larvae is 577ng larvae-1。
Claims (21)
1. A method for evaluating toxicity of pesticide to bee larvae by utilizing artificial feeding of the bee larvae in a laboratory is characterized by comprising the following steps:
(1) preparation of bee larvae: inserting an empty honeycomb into a beehive containing a bee colony, fixing a queen bee in a spawning period on the empty honeycomb for spawning, discharging the queen bee out of the honeycomb containing bee eggs after 20-28 hours, and continuously keeping the honeycomb containing the bee eggs in the beehive until the bee eggs in the honeycomb hatch into bee larvae;
(2) laboratory rearing of bee larvae: taking out the inserted bee spleen from the beehive, picking the bee larva, carrying out adaptive breeding for 3 days by using basic ration, carrying out health examination on the 4 th day, and selecting the healthy larva;
(3) addition of the tested pesticide: dividing the healthy larvae selected in the step (2) into control group larvae and treatment group larvae and continuously feeding for 3 days, wherein the treatment group larvae are fed with a tested feed, and the tested feed contains tested pesticide with various concentrations and basic ration;
(4) pupating and observing: transferring the larvae which are bred for 3 days in the step (3) and are about to enter a pupation stage into plate holes of a pupation plate to prepare for pupation, observing the processes of defecation, silking, pupation and eclosion of the larvae in the plate holes, and counting the survival, pupation or/and eclosion conditions of the larvae.
2. The method according to claim 1, wherein in step (1), the bee colony is a healthy bee colony of known origin and physiological status that is not dosed for four weeks; in the step (2), the selected bee larvae are 1 day old later stage larvae, and the body color is white.
3. The method according to claim 1, wherein in step (1), the queen bee in the oviposition period performs oviposition on the empty spleen for 24 hours.
4. The method of claim 1, wherein the basal ration comprises, by mass: 50 plus or minus 5 percent of fresh royal jelly, 37 plus or minus 2 percent of sterile water, 6 plus or minus 1 percent of glucose, 6 plus or minus 1 percent of fructose and 1 plus or minus 0.5 percent of yeast extract.
5. The method of claim 4, wherein the basal ration comprises, by mass: 50% of fresh royal jelly, 37% of sterile water, 6% of glucose, 6% of fructose and 1% of yeast extract.
6. The method according to claim 1, wherein in the step (2), the adaptive feeding is performed using a well plate filled with a basal diet; one bee larva is distributed in each hole of the pore plate, the feed amount of the basic ration of the bee larva in each hole is 40-60 mu L, and the basic ration in the pore plate is replaced every 24 hours.
7. The method according to claim 6, wherein the environmental conditions of the adaptive feeding in step (2) and the feeding in step (3) are both: the temperature is 34 +/-2 ℃, and the humidity is 95 +/-1%.
8. The method according to claim 7, wherein the environmental conditions of the adaptive feeding in step (2) and the feeding in step (3) are both: the temperature is 34 +/-1 ℃ and the humidity is 95 percent.
9. The method according to claim 8, wherein the environmental conditions of the adaptive feeding in step (2) and the feeding in step (3) are both: during breeding, the pore plate is placed in a closed box filled with glycerol-water mixed liquid at the bottom of the box, and then is placed in an incubator at the temperature of 34 +/-1 ℃ for culture.
10. The method according to claim 1, wherein in step (3), the control larvae are either blank control larvae or/and solvent control larvae; feeding the larvae of the blank control group by using basic ration, feeding the larvae of the solvent control group by using the basic ration added with a solvent as feed, wherein each 41-61 mu L of the feed contains 0.5-5 mu L of the solvent; the treatment group larvae were divided into multiple treatment groups, each treatment group being fed with test feed containing different concentrations of test pesticide.
11. The method of claim 10, wherein in step (3), the treatment group larvae are divided into at least 5 treatment groups.
12. The method according to claim 1, wherein in the step (3), the test feed is prepared by: dissolving the tested pesticide by using distilled water or/and an organic solvent which is nontoxic to bee larvae, preparing one or more tested pesticide solutions with different concentrations, and respectively adding the tested pesticide solutions into basic daily ration to prepare tested feed, wherein 0.5-5 mu L of the tested pesticide solution is contained in 41-61 mu L of the tested feed.
13. The method according to claim 12, wherein in step (3), the feed amount of each larva of bee in the continuous rearing process in step (3) is 41-61 μ L, and the feed is replaced every 24 hours.
14. The method according to claim 1, wherein in step (4), the pupation plate holes are padded with dry sterilized paper.
15. The method according to claim 14, wherein in step (4), the larvae entering pupation stage are first blotted to dry their body surface feed before transferring to pupation plates.
16. The method according to claim 15, wherein in step (4), the larvae transferred to the pupation plate do not move until the bee larvae have fully developed.
17. A method for artificially feeding bee larvae in a laboratory is characterized by comprising the following steps:
(1) preparation of bee larvae: inserting an empty honeycomb into a beehive containing a bee colony, fixing a queen bee in a spawning period on the empty honeycomb for spawning, discharging the queen bee out of the honeycomb containing bee eggs after 20-28 hours, and continuously keeping the honeycomb containing the bee eggs in the beehive until the bee eggs in the honeycomb hatch into bee larvae;
(2) laboratory rearing of bee larvae: taking out the inserted bee spleen from the beehive, picking the bee larva, carrying out adaptive breeding for 3 days by using basic ration, carrying out health examination on the 4 th day, and selecting the healthy larva to continue breeding for 3 days;
(3) pupating and observing: transferring the larvae which are about to enter the pupation stage after continuously breeding for 3 days in the step (2) to plate holes of a pupation plate for preparing pupation, observing the processes of defecation, silking, pupation and eclosion of the larvae in the plate holes, and counting the survival, pupation or/and eclosion conditions of the larvae.
18. The method according to claim 17, wherein in step (2), the bee larvae are selected from 1 day old later stage larvae, which are white in body color; in the step (3), the pupation plate holes are padded with dry sterilized paper.
19. The method of claim 18, wherein the basal ration comprises, by mass: 50 plus or minus 5 percent of fresh royal jelly, 37 plus or minus 2 percent of sterile water, 6 plus or minus 1 percent of glucose, 6 plus or minus 1 percent of fructose and 1 plus or minus 0.5 percent of yeast extract.
20. The method of claim 19, wherein the basal ration comprises, by mass: 50% of fresh royal jelly, 37% of sterile water, 6% of glucose, 6% of fructose and 1% of yeast extract.
21. Use of the laboratory method of artificially feeding larvae of honeybees according to claim 17 for evaluating the toxicity of pesticides to the larvae of honeybees.
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| CN108739680B (en) * | 2018-05-29 | 2020-06-09 | 中国农业科学院蜜蜂研究所 | Test method for determining influence of pesticide on bee larva development |
| CN110122430A (en) * | 2019-06-19 | 2019-08-16 | 北京林业大学 | A method of utilizing the genotoxicity of Drosophila melanogaster evaluation organic solvent |
| CN112098638B (en) * | 2020-07-30 | 2024-06-18 | 山东省农业科学院植物保护研究所 | Method for determining toxicity of pesticide to wallbee larvae |
| CN114208785A (en) * | 2022-02-17 | 2022-03-22 | 中国农业科学院蜜蜂研究所 | Culture method capable of improving birth weight and kiss length of Chinese bees |
| CN114451361B (en) * | 2022-02-25 | 2023-03-21 | 中国农业科学院蜜蜂研究所 | Method for detecting nutritive value of pollen to bees |
| CN115530126A (en) * | 2022-09-14 | 2022-12-30 | 广东省科学院动物研究所 | Application of Bombella Apis in promoting growth and development of bee larvae |
| CN118091105A (en) * | 2024-04-27 | 2024-05-28 | 山东亿嘉农化有限公司 | Analysis method and analysis device for toxicity of pesticide to bees |
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