Routine in vivo bioassay technology for pesticides is a technology developed during the long-term pesticide screening process. Some classic methods and supporting equipment still occupy a very important position in current applications. Various methods are now introduced as follows:
(1) Contact action. Since many pesticides have contact activity, there are many methods and instruments for measuring toxicity in this area. Commonly used methods include spray method, powder spray method, liquid immersion method, drip method, and medicine film method.
① Spray method: The spray method and powder spraying method are both based on simulating the actual pesticide application conditions in the field, and try to make the dosage of pesticides obtained on the insect's body surface the same and uniform. The main spray equipment currently used for testing includes Hoskings horizontal cylinder sprayer, Campbeil rotary disk sprayer, precipitation fog reduction device, and Potter spray tower.
②Powder spray method: The purpose of powder spray method and spray method is roughly the same. The advantage of this method is that the spray is not only uniform, but also not limited to the upper surface, but can be sprayed on all surfaces of the object being sprayed.
③Dipping method: The objects used in the dipping method are generally aquatic insects (such as mosquito larvae, water fleas), stored grain pests, scale insects and aphids, and eggs of test insects. For mosquito larvae, long-term immersion contact treatment can be performed, that is, directly immersed in a certain concentration of medicinal solution, and the results will be observed after a certain period of time. Other terrestrial insects have shorter periods of time, generally ranging from seconds to minutes.
④Drip method: The principle is to drop a certain dose of the agent on the insect body wall to observe the contact toxicity of the agent on penetrating the insect body wall. This method is the most accurate method for insecticidal virulence testing and is also a commonly used testing technology. It can be used for most target insects and mites.
Insects should generally be anesthetized before dripping the medicinal solution to prevent the insect's own movement from affecting the accuracy of the experimental operation. The general methods of anesthesia include freezing, ether treatment, and carbon dioxide treatment. In addition, it is also necessary to pay attention to the holding method of insects, and try not to cause mechanical damage to the test insects. For larger insects, you can hold them with your hands. For smaller insects, you can suck the insects onto the mouth of the straw and add medicine. Adding fine gauze will work better.
⑤Injection method: This method is also widely used, and the instrument used is similar to the drip method, and it also has the characteristics of accurate dosage. However, the injection method is to inject the drug into the body of the test insect instead of dripping it on the body surface.
⑥Drug film method: This method is a contact treatment method. The principle is to apply a certain amount of medicine on filter paper, glass plate or wax paper through spraying, powder spraying, dripping, smearing, immersion and other methods to form a uniform medicine film, and then place the test insects on it and crawl A certain period of time, and then transferred to a normal environment. After a certain period of time, the death of the insects is observed and recorded.
This method is suitable for all crawling insects, and can also be used for some flying insects (such as mosquitoes, flies and other adults). You can make a square glass box with the inside of the 6 sides of the box passed The drug film treatment ensures that no matter where the insects rest, they are still exposed to the drug. In addition, after the drug film has been left for different periods of time, or after being weathered or sun-dried, the test insects can then be put in to determine the long-lasting effectiveness of the drug.
(2) Stomach action. The principle is to take advantage of the voracious nature of insects, ingest pharmaceuticals while the insects are eating normal food, and detect whether the pharmaceuticals can pass through the insect's digestive tract to produce toxicity to the insects. General methods include feeding method, feeding and drinking method, and oral injection method.
① Feeding method: Feeding method is divided into unrestricted feeding method and quantitative feeding method. According to the feeding method, it is divided into leaf clamping method and single leaf feeding method. Among them, the sandwich method is the most commonly used. The leaf clamping method is to add a certain amount of pesticide evenly between two leaves and feed the insect. The pesticide will be eaten by the insect along with the leaves. The amount of drug swallowed is calculated based on the area of ??the swallowed leaf. In order to make the amount of pesticide applied to the leaves even, the pesticide is usually applied to one side of the leaf by powdering, spraying, coating or dripping the pesticide.
After the liquid is dry, apply paste or gelatin to one side of the other leaves and stick them together, then feed the insects. The advantage is that it avoids direct contact between the agent and the insect body, and eliminates the contact killing effect of the agent on the insects.
The amount of drug swallowed is calculated by multiplying the leaf area swallowed by the test insect by the dose per unit area of ??the leaf, thereby obtaining the dose consumed by each test insect per unit volume (μg/g). There are several methods for calculating leaf area: 1) Graph paper method: Place the remaining leaves eaten by test insects on graph paper, count the number of graph papers eaten, and calculate the area of ??the grid, 2) Leaf area measurement Instrument method: Use a leaf area meter to directly measure the area of ??devoured leaves; 3) Computer scanning method: Use a digital camera to take photos of the remaining leaves after the test insects in each treatment have devoured them, and calculate the leaf area through a computer.
The method of calculating the lethal dose is to rank the worms in the order of the dose swallowed by each head, and indicate the life and death of the test worms at this dose, thereby dividing the worms into survival groups, life and death groups and death groups.
No. 1 to 8 are the survival group, No. 9 to 40 are the life and death group, and No. 41 to 47 are the death group. There are both living and dead people in the life and death groups. Calculate the average amount of medicine swallowed by the surviving individuals and dead individuals in the life and death groups, respectively, and record them as A and B. That is, A=(sum of the dose of live insects in the living and dead groups)/total number of live insects in the living and dead groups, B=(sum of the dose of dead insects in the living and dead groups)/total number of dead insects in the living and dead groups, LD50=(A+B)/2.
Demonstration of the response of pesticides to certain insects
A= (0.21＋0.23＋0.28＋……＋0.64)/16=6.00/16=0.375
B=（0.20＋0.22＋0.25＋……）＋0.65/17=5.89/17=0.368
LD50=（A＋B）/2=（0.375＋0.368 )/2=0.372 (μg/g)
The disadvantage of this method is that the unrestricted feeding method cannot control the food intake of a single test insect, and the rationed feeding method cannot eliminate the vomiting behavior of insects after swallowing. There are other unknown factors, and the method of calculating the area of ??leaves eaten by insects is not very accurate, thus affecting the dosage of pesticides eaten. In addition, it is also more troublesome and time-consuming. This method is only suitable for insects with chewing mouthparts.
In the feeding method, in addition to feeding leaves, there are also methods such as feeding poisonous bait.
②Feeding and drinking method: This method is suitable for flies, stink bugs and other insects with sucking habits. It is also divided into free feeding and drinking methods and forced feeding and drinking methods. The free feeding and drinking method is an unrestricted feeding and drinking method that allows the insects to eat the prepared sugar solution mixed with pharmaceuticals. The amount of pharmaceuticals sucked by the insects is calculated by measuring the weight of the insects or the volume of the pharmaceutical liquid before and after sucking. The forced feeding method is to fix the insect and place the mouth of the pipette containing the medicinal solution next to the insect's mouth. After the insect has sucked a certain volume, remove the pipette. This can ensure that each insect is The amount of potion absorbed is the same. In addition, there is also an oral injection method, which is to inject sugary liquid directly into the insect's mouth with a syringe to force it to swallow it. Generally, the needle tip is required to be smooth to avoid puncturing the tissue in the oral cavity, and the operation skills are required to be skilled.
(3) Fumigation action. Fumigants can be gases, liquids or solids, but they must ultimately pass through the respiratory system of insects in gaseous form to kill them. The easy diffusion of gas determines that fumigants must be used in a closed environment. Therefore, the detection of the fumigation effect of the agent must also be carried out under closed conditions, and it is required not to be in direct contact with insects in a solid or liquid state. All fumigation effect measurement methods are designed based on this point.
At present, the instruments and devices for fumigants mainly include static gas measurement method devices and fumigators with air inflow and outflow control. Generally, some simple fumigators belong to the former type. They fill a closed container with saturated gas through a certain method, and control the absorption of the filled gas or adjust the gas in the container by controlling the time when insects are put in for testing. concentration to achieve the purpose of dose control.
In addition, the adjustment of the physical and chemical properties of the soil should also be considered when designing experimental methods for soil fumigants.
(4) Systemic action. The basic principle of systemic action is that after the agent is passed through a specific part of the plant (roots, stems, leaves or seeds), the agent can be absorbed by the plant and reach other parts through conduction, allowing insects to feed on the parts not directly treated with the agent. Observe whether the insect has died after being poisoned to determine whether the agent has this property.
The processing method based on this principle is usually called the direct determination method. Different application areas require different treatment methods. If the treatment method is to absorb from the roots, the top of the main root of the plant can be cut off, and the remaining part can be inserted into the little finger-shaped tube containing the medicinal solution, and the lateral roots can be saved and immersed in the nutrient solution, or the medicinal solution can be added to the nutrient solution to prepare the required concentration. The lateral roots are fully immersed in it; if the pesticide is applied from the leaves, the pesticide can be applied to the front or back of the leaves by drip irrigation or a brush dipped in the pesticide or even partial spray; if the pesticide is applied from the stem, woody plants can Wrap the stem with a circle of absorbent cotton containing a certain concentration of chemicals, and then wrap the outside with plastic cloth. If the plant is young and tender, you can apply the chemical solution on the stem by smearing it. For older trees, you need to scrape off a layer of old bark. After applying the pesticide; if the systemic effect of the seeds is tested, the seeds can generally be treated with a soaking liquid. The medicinal liquid is generally equivalent to twice the weight of the seeds, or the seeds can be completely submerged during soaking.
The other method is called the indirect determination method. Parts of the plant are treated with medicinal liquid. After a certain period of time, the untreated parts are taken and ground, diluted into juices of different concentrations, and then a certain number of larvae are added. Or observe the death of water fleas, and use this immersion method to indirectly determine whether the agent has a systemic effect.
(5) Antifeedant effect. Antifeedant effects are usually designed in the same way as gastrotoxic effects. However, the results of observation are usually not the mortality of the test insects, but often the amount of food taken by the insects or the analysis of the parts of food eaten by the insects and changes in insect weight, etc., to determine whether the agent has antifeedant activity against the test insects. basis.
The general method for measuring the antifeedant activity of leaf-eating insects with chewing mouthparts is the leaf disc method. That is, apply the pesticide evenly on the leaves, make leaf discs of the same size, place them in a moisturizing petri dish, and then add the test insects. The method of alternately placing treated leaf discs and control leaf discs in the same petri dish is called selective antifeedant activity assay. The method of placing only control or treated leaf discs is called non-selective antifeedant activity assay. For example, black circles represent leaves treated with pesticides, and white circles represent leaves without pesticides. A represents the measurement of selective antifeedant activity, and B and C represent the measurement of non-selective antifeedant activity. After allowing the test insects to feed for a certain period of time, remove the remaining leaves and calculate the area of ??the leaves fed by the insects. Calculate the feed rejection rate through the formula:
Leaf Disk Method
(6) Attraction effect. Attractants can basically be divided into sex attractants, food attractants and spawning attractants.
A rough measurement can be done by setting up temptation traps. For example, a shelf with glue is placed in the sarong, and cotton dripping with chemicals is stuck in the center of the glue to attract the target insects. For larger insects, the glue can be replaced by a gas bottle, and the effectiveness of the pesticide can be judged by counting the number of trapped insects.
Most of these methods are operated in the field and are easily affected by climatic factors and the number of insects in the field. Olfactometers are used in many laboratory measurement methods. The basic principle is to place insects at the bifurcation of two alternative roads, one of which contains the agent to be tested and the other without. Observe which branch the insect enters. If the agent has no effect, the probability of insects entering the two branches is equal. Once the attractant has an effect, the stronger the effect, the more insects will be attracted.
(7) Repellent effect. Its principle is similar to that of attractants, and an olfactometer can also be used in experimental design, with roughly similar methods.
(8) Interfering effect on growth, development and reproduction. Compounds with this property usually do not directly kill insects, but interfere with the synthesis of insect brain hormones, juvenile hormone, ecdysone and chitin, leading to changes in insect growth, metamorphosis, diapause and other physiological phenomena, indirectly leading to reduced damage. purpose.
Test methods can be used to observe the effects of pesticides through the gastric poisoning, contact, fumigation, systemic and other treatment methods introduced above, and by observing the changes in insect molting, pupation, emergence, changes in morphology, egg laying volume, egg hatching rate and other indicators. effect.