Insecticide sensitivity monitoring
3.1 Objective
Monitoring the resistance of vector Anopheles for insecticides and understanding the sensitivity of the main local vectors to insecticides will help to guide the work of mosquito control and rational use of insecticides.
3.2 Tested mosquitoes
The adult Anopheles blood-sucked are captured from the site (monitoring site). After spawning, breed the eggs to the first generation. In addition, the pupae are determined within 24 hours after emergence. If there are difficulties in breeding, Anopheles mosquitoes which have just sucked blood can be collected in the field on the night before the experiment and brought back to the laboratory for determination in the morning, when the stomach blood is digested in stage III-IV of Sheila’s.
The WHO scheme pointed out that the age, physiological status and sex of the tested mosquitoes all affected the results of resistance testing. In the preliminary sensitivity test of differentiated concentration, it is recommended to use adult female mosquitoes without blood feeding at the age of 3-5 days (sugar water is fed and starve them for 6 hours). In addition, the determination of 5 and 10 times concentration can be carried out with non-blood-fed female mosquitoes. In the on-the-spot environment, adult female mosquitoes caught in the field can be tested directly (only non-blood-sucking female mosquitoes; sugar water can be fed and starve them for several hours before testing), which is typical of the representativeness epidemiology. In order to obtain the standard aging results, it is recommended to use the same batch of female mosquitoes or offspring raised by the mosquitoes collected in the field to carry out the experiment.
3.3 Preparation and test concentration of insecticide paper
White oil and ether are mixed at 1:2 as solvent. The insecticide shall be dissolved into it according to the required amount, mixed into a certain concentration gradient. In addition, 2.14 mL shall be evenly dripped on the filter paper of 16 cm×12.5 cm. After 2 hours, the ether could be volatilized completely. The dose standard of WHO on the paper is calculated on the basis of the concentration of insecticides in white oil, for example, 1% of the mixture is actually 0.33%.
3.4 Experimental method
The adult mosquito filter paper contact method shall be used to determine the differentiated dose of WHO 5 or 10 times. Each experiment shall be repeated at least 4 times (the total number of single mosquito species in the experimental group shall be not less than 100, to ensure the accuracy of mosquito species identification). At the same time, there are two groups of blank controls in the same experiment, and the number of mosquitoes in each group shall be the same as that in the experimental group. The specific operations are as follows.
3.4.1 Install the recovery tube on one side of the square partition. In addition, use a mosquito attractor to take 20-25 female mosquitoes. Gently blow the mosquitoes into the tube facing light and close the partition.
3.4.2 In the control group, the filter paper shall be rolled into a tube and inserted into the contact, and the filter paper should be closely attached to the tube wall; put on disposable gloves. The filter paper of the experimental group shall be rolled into a barrel and inserted into the contact, and the filter paper should be closely attached to the tube wall. (Note: a. Put the medicine face inside. b. Operators try to avoid contact with drugs on filter paper.)
3.4.3 Install the contact tube lined with medicine paper on the other end of the square partition, remove the partition, gently blow the mosquitoes against light into the contact tube, and close the partition quickly. Then remove the recovery tube.
3.4.4. The contact tube shall be upright. The respective numbers of mosquitoes exposed to 10 min, 15 min, 20 min, 30 min, 40 min, 50 min and 60 min shall be recorded. The knockdown rate shall be calculated. (Formula 4-16)
3.4.5. After contact (60 min), pull off the partition, blow all mosquitoes into the recovery tube, and close the partition. Then remove the contact tube.
3.4.6 Keep the recovery tube upright, feed with 10% glucose water, keep the room temperature between 25 ℃±2 ℃ (WHO recommended 27 ℃±2 ℃), relative humidity 70%-80% (WHO recommended 75% ±10%) in the laboratory (record room mild relative humidity).
3.4.7 The respective numbers of dead mosquitoes and surviving ones are checked after 24 hours. The mortality rate shall be calculated.
Fig. 4-1 The operation process is shown in the flow chart above(The picture comes from WHO guidelines)
3.5 Result collation
The mortality rate of the control group is more than 20%, so the experiment is invalid. The test results should be recorded and the experiment be repeated. If the mortality rate of the control group is between 5% and 20% (including 5%), that of the treatment group will be corrected by Abbott formula (formula 4-17), and the corrected mortality rate (%) be calculated. If the control mortality rate is less than 5%, there is no need to correct the test results. When reporting the mortality count, the sample size must be given, preferably an estimate of the 95% confidence interval.(https://www.daowen.com)
Mortality of experimental group (%) = total number of dead mosquitoes/total number of mosquitoes in experimental group×100%
3.6 Valuation Indexes of WHO Pesticide Resistance
3.6.1 Sensitivity detection of differentiated dose (diagnostic dose)
In order to determine the tolerance of a species to a given pesticide, baseline sensitivity data (dose differentiation) for the pesticide must first be established in the normal or sensitive population of the given species. Methods: the pesticide-nonresistant vectors shall be exposed to filter paper impregnated with continuous concentrations of specific insecticide compounds. In addition, the mortality percentage curve shall be drawn on logarithmic probability paper, which can be used to estimate the concentrations needed to produce different degrees of killing. It can also be done using a logarithmic probability statistical model. With this method, a concentration corresponding to 99.9% mortality rate (LC99.9) can be obtained; at this concentration, all individuals in the susceptible population are most likely to be killed. Twice this concentration is often referred to as differential (or diagnostic) concentration.
3.6.1.1 Mortality rate ≥ 98%: mosquitoes are sensitive to the pesticide.
3.6.1.2 Mortality rate 90%-97%: there may be resistance, which needs further verification; after further verification, if the mortality rate is still lower than 98%, it is confirmed that the resistance exists; further resistance strength testing and resistance mechanism research are carried out.
3.6.1.3 Mortality rate ＜ 90%: resistance exists, and it is not necessary to use other bioassay methods to confirm the existence of pesticide resistance genes in the test population. Additional samples need to be further tested at concentrations of 5 and 10 times.
Moreover, the mechanism and distribution of pesticide resistance need to be further studied.
After pesticide resistance has been identified, remedial action should be taken as far as possible to manage insecticide resistance in accordance with GPIRM guidelines to ensure the effectiveness of malaria-transmitting mosquito insecticides.
3.6.2 Determine the resistance index of dose (5 times dose)
Any resistance phenotype detected at differentiated concentrations of the insecticide can expose sufficient or additional mosquitoes to 5 times and 10 times higher concentrations of the insecticide to assess its deep significance. Higher concentration exposure will produce the information of resistance intensity, which can be defined as the intensity of resistance phenotype. When the dose is determined to be 5 times, especially 10 times, there is still resistance, indicating that the control failed. As a consequence, there is an urgent need to develop an appropriate resistance management plan.
3.6.2.1 Mortality ≥ 98%: low resistance intensity;
3.6.2.2 Mortality ＜ 98%: medium to high resistance intensity.
3.7 Note
3.7.1 The pesticide paper should be used within 6 hours after preparation. If necessary, 10 to 20 pieces of pesticide paper can be clamped with a glass plate of the same size. In addition, the gap can be sealed with transparent glue and put into a black paper bag. At a lower temperature, them can be stored for about a month. However, once opened, them cannot be stored and must be used within 2-3 days. If the experimenter has loaded them into the contact tube, them can only be used once.
3.7.2 The contact time should be paid special attention to. If it is too short, such as within 15 minutes, the result is often affected by the difference of individual residence time on the pesticide paper and the speed of operation; if it is too long, such as more than 4 hours, it is also inconvenient to operate and may increase death. Generally speaking, the contact time should be between 15 minutes and 2 hours.
Table 4-3 Record Sheet for Determination of Pesticide Resistance of Vector Anopheles Mosquitoes
Table 4-4 Monitoring Statistics of Resistance of Vector Anopheles to Common Insecticides
