A method for assessing feeding behavior of mosquito larvae and applications thereof

Abstract

The application provides a method for evaluating the feeding behavior of mosquito larvae and application thereof, and belongs to the technical field of insect feeding. The application provides a method for evaluating the feeding behavior of mosquito larvae, wherein after yeast dyed by aniline blue is used as feed to feed mosquito larvae, the feeding level is reflected according to the content of aniline blue in the mosquito larvae; the application also provides application of the method in screening and / or developing drugs for regulating the feeding behavior of mosquito larvae. The examples show that the method can accurately reflect the change of the feeding behavior of mosquito larvae, and has important significance for screening and / or developing drugs for regulating the feeding behavior of mosquito larvae and then inhibiting the growth and development of the mosquito larvae.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of insect feeding, and particularly relates to a method for evaluating the feeding behavior of mosquito larvae and application thereof. BACKGROUND

[0002] Among mosquito insects, female aedes, anopheles and culex insects can transmit diseases such as dengue fever, malaria, yellow fever, filariasis and Japanese encephalitis in the process of biting humans, which seriously endangers human health. Mosquito insects usually live in water areas in the larval stage, and have a small range of activities, and complete growth and development by feeding on organic debris and plankton. Adult mosquitoes are good at flying and have a large range of activities. Therefore, the comprehensive prevention and control of mosquito larvae is an effective way to prevent mosquitoes and resist diseases.

[0003] Inhibiting the feeding behavior of mosquito larvae can effectively prevent the larvae from completing the normal growth and development process, and screening active compounds and determining their influence on the feeding amount of mosquito larvae is of great significance for preventing mosquitoes and resisting diseases. However, there are few studies on the determination method of the feeding amount of mosquito larvae at present, although there are various methods for detecting the feeding amount of fruit flies and other insects in the prior art, such as capillary method and dye method, but the living environment of mosquito larvae is completely different from that of fruit flies, and the life activities of mosquito larvae are limited in water, so the conventional detection method cannot be applied to the accurate evaluation of the feeding level of mosquito larvae. Therefore, it is urgent to provide a method for accurately evaluating the feeding behavior of mosquito larvae. SUMMARY

[0004] The purpose of the present application is to provide a method for evaluating the feeding behavior of mosquito larvae, which can accurately evaluate the feeding level of mosquito larvae.

[0005] In order to achieve the above purpose, the present application provides the following technical scheme:

[0006] The present application provides an application of aniline blue dyed yeast as feed in evaluating the feeding behavior of mosquito larvae.

[0007] Preferably, the preparation method of the aniline blue dyed yeast is to dye the yeast with aniline blue solution for 8-14h.

[0008] Preferably, the concentration of the aniline blue solution is 1-10mg / mL.

[0009] The dyeing concentration of the yeast is 0.8-1mg / mL.

[0010] The present application provides a method for evaluating the feeding behavior of mosquito larvae, comprising the following steps:

[0011] The mosquito larvae are divided into an experimental group, a calibration group and a blank group:

[0012] The experimental group uses the aniline blue dyed yeast in the application as the feed to feed the mosquito larvae, and the spectrophotometry is used to determine the absorbance value M1 of the aniline blue in the mosquito larvae after feeding;

[0013] The calibration group uses the aniline blue dyed yeast in the application as the feed to feed the mosquito larvae after starvation treatment, and the spectrophotometry in the experimental group is used to determine the absorbance value M of the aniline blue in the mosquito larvae after feeding;

[0014] The blank group uses the undyed yeast as the feed to feed the mosquito larvae after starvation treatment, and the spectrophotometry in the experimental group is used to determine the absorbance value M0 of the aniline blue in the mosquito larvae after feeding;

[0015] The M1, M and M0 are substituted into the formula I to calculate the feeding index of the mosquito larvae:

[0016] Feeding index=(M1-M0) / (M-M0) Formula I

[0017] The feeding index is proportional to the feeding level of the mosquito larvae, and the higher the feeding index, the higher the feeding level of the mosquito larvae.

[0018] Preferably, the feeding time is 10-20 min.

[0019] Preferably, the added amount of the feed is greater than or equal to 0.04 mg per mosquito.

[0020] Preferably, the starvation treatment time is 4-8 h.

[0021] Preferably, the age of the mosquito larvae includes any one of the following: the second instar, the third instar and the fourth instar.

[0022] The application provides an application of the method in screening or evaluating a drug for regulating the feeding behavior of mosquito larvae.

[0023] The application provides an application of the method in screening or evaluating a drug for inhibiting the growth and development of mosquito larvae.

[0024] Preferably, the drug includes diantanan.

[0025] Beneficial effects:

[0026] The application provides an application of aniline blue dyed yeast as feed in evaluation of feeding behavior of mosquito larvae. The yeast cells are dyed with aniline blue, and after the mosquito larvae are fed with the aniline blue dyed yeast, the content of the aniline blue is continuously increased with the increase of the feeding amount, and the feeding level of the mosquito larvae can be accurately reflected according to the content of the aniline blue in the mosquito larvae. In the embodiment of the application, the mosquito larvae are fed with the yeast dyed with aniline blue and the yeast dyed with fluorescence respectively, and the absorbance value in the mosquito larvae is detected, and the results show that the content of the aniline blue in the mosquito larvae can be detected in the aniline blue dyed group, and the content is increased with the increase of the feeding amount of the mosquito larvae, while the background fluorescence of the mosquito larvae in the fluorescence dyed group interferes with the fluorescence of the labeled yeast, so that the detection of the fluorescence intensity cannot accurately reflect the feeding level of the mosquito larvae. Therefore, the yeast dyed with aniline blue as the feed is beneficial to accurately reflecting the feeding behavior and the feeding amount of the mosquito larvae.

[0027] The application provides a method for evaluating the feeding behavior of mosquito larvae, and the feeding index calculated by the method can accurately evaluate the feeding behavior of the mosquito larvae. The blank group and the calibration group data are introduced into the feeding index calculation formula, so that the interference of the mosquito larvae itself and different batches of experimental operations on the detection results can be avoided, and the feeding level of the mosquito larvae can be more accurately reflected. The feeding behavior of the mosquito larvae subjected to starvation treatment and the mosquito larvae not subjected to starvation treatment is evaluated by the method, and the results show that the feeding index of the mosquito larvae not subjected to starvation treatment is significantly lower than that of the mosquito larvae subjected to starvation treatment. The feeding behavior of the mosquito larvae at the second instar, the third instar and the fourth instar is evaluated by the method, and the results show that the feeding amount of the mosquito larvae is significantly increased from the second instar to the fourth instar, and it can be seen that the evaluation method can accurately reflect the feeding behavior of the mosquito larvae.

[0028] The application provides an application of the method in screening or evaluating drugs for regulating the feeding behavior of mosquitoes.

[0029] The application also provides an application of the method in screening or evaluating drugs for inhibiting the growth and development of mosquito larvae. The insufficient intake of food by adult mosquitoes during the larval stage will greatly affect the normal emergence and mating of adult mosquitoes, and it is difficult to complete the normal growth and development. Therefore, the method can be used for screening or evaluating drugs for regulating the feeding behavior of mosquitoes and thereby inhibiting the growth and development of mosquito larvae. The influence of DEET on the feeding behavior of mosquito larvae is evaluated by the method, and the results show that after DEET is added to the food and / or the environment, the feeding level of the mosquito larvae is significantly reduced. It can be seen that DEET is a drug that can effectively reduce the feeding level of mosquito larvae, and the method has important significance for studying drugs for regulating the feeding behavior and growth and development of mosquito larvae. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 The amount of aniline blue dyed yeast and A600 a relationship diagram between the yeast and the Aedes albopictus larvae;

[0031] Figure 2 a time dynamic diagram of the amount of aniline blue dyed yeast consumed by the Aedes albopictus larvae, wherein ** represents P<0.05;

[0032] Figure 3 a relationship diagram of the added amount of the yeast and the amount of the Aedes albopictus larvae consumed;

[0033] Figure 4 a result diagram of the influence of the starvation treatment and the non-starvation treatment on the feeding index of the Aedes albopictus larvae;

[0034] Figure 5 a difference diagram of the feeding index of the Aedes albopictus larvae in different age stages;

[0035] Figure 6 a diagram of the influence of the mosquito repellent DEET on the feeding index of the Aedes albopictus larvae;

[0036] Figure 7 a result diagram of the fluorescence in the intestinal tract of the Aedes albopictus larvae after the larvae consume the unmarked yeast and the diacetate fluorescein marked yeast. DETAILED DESCRIPTION

[0037] The present application provides an application of aniline blue dyed yeast as a feed in evaluating the feeding behavior of mosquito larvae.

[0038] In the present application, the insect species of the mosquito family preferably comprises at least one of the following: Aedes, Anopheles and Culex, and more preferably Aedes. In the embodiment of the present application, the aniline blue dyed yeast is used to evaluate the feeding behavior of the Aedes albopictus larvae.

[0039] The present application does not limit the type of yeast, and as an optional embodiment, the yeast is Saccharomyces cerevisiae. The active dry yeast of the Saccharomyces cerevisiae is configured into a bacterial solution to feed the mosquito larvae as a feed. In the embodiment of the present application, the yeast is purchased from Angel Yeast Co., Ltd.

[0040] In the present application, the preparation method of the aniline blue dyed yeast is preferably to dye the yeast with an aniline blue solution for 8-14 hours. The dyeing time is more preferably 12 hours. The dyeing temperature is preferably 16-30℃, and more preferably 25-27℃. The concentration of the aniline blue solution is preferably 1-10 mg / mL, and more preferably 2-6 mg / mL, and most preferably 3 mg / mL. The dyeing concentration of the yeast is 0.8-1 mg / mL, and more preferably 0.9 mg / mL. The yeast cells are fully dyed by dyeing for a certain time at a specific concentration of aniline blue, and the aniline blue dyed yeast as a feed is conducive to accurately reflecting the feeding level of the mosquito larvae.

[0041] In the present application, the feeding time is preferably 10-20 min, more preferably 15-20 min, and most preferably 20 min. Due to the storage capacity of the digestive tract of mosquito larvae and excretion, too long feeding time will cause part of the dye to be excreted out of the body, resulting in inaccurate reflection of the feeding level of mosquito larvae. In an embodiment of the present application, mosquito larvae are fed with yeast dyed with aniline blue for different times (10 min, 20 min, 30 min, 40 min, 50 min and 60 min), respectively, and the change in the aniline blue content in mosquito larvae is determined, and the results show that the aniline blue content in mosquito larvae linearly accumulates within 20 min before feeding, which indicates that the feeding amount reflected by aniline blue within 20 min is not affected by excretion and the storage capacity of the digestive tract of mosquito larvae, and the feeding level of mosquito larvae measured within this period is more accurate.

[0042] In the present application, the added amount of the feed is preferably ≥0.04 mg per mosquito, and more preferably 0.1-0.2 mg per mosquito. Too little added amount of the feed is not conducive to distinguishing the difference in the feeding level of mosquito larvae in different treatment groups, and providing sufficient amount of the feed can accurately reflect the feeding level of mosquito larvae. In an embodiment of the present application, different added amounts of the feed (0.01 mg per mosquito, 0.02 mg per mosquito, 0.04 mg per mosquito, 0.08 mg per mosquito, 0.12 mg per mosquito, 0.16 mg per mosquito and 0.2 mg per mosquito) are compared, and the aniline blue content in mosquito larvae is determined after the mosquito larvae are fed for 20 min, and the results show that an added amount of the feed lower than 0.04 mg is insufficient for mosquito larvae to feed for 20 min, and too low added amount of the feed cannot accurately reflect the difference in the feeding behavior of mosquito larvae in different treatment groups when the feeding behavior of mosquito larvae is evaluated; when the added amount of the feed is ≥0.04 mg, the aniline blue content in mosquito larvae no longer increases with the increase in the added amount of the feed. Therefore, an added amount of the feed ≥0.04 mg per mosquito is appropriate for mosquito larvae to feed for 20 min, and the preferred added amount of the feed for mosquito larvae is ≥0.04 mg per mosquito.

[0043] In the present application, after yeast dyed with aniline blue is fed to mosquito larvae, the feeding level of mosquito larvae can be accurately reflected according to the aniline blue content in mosquito larvae. In an embodiment of the present application, the yeast dyed with aniline blue is extracted with methanol, and the absorbance value is detected, and the results show that the amount of yeast dyed with aniline blue is proportional to the absorbance at 600 nm (absorption peak of the coloring group of aniline blue). Therefore, the amount of yeast dyed with aniline blue ingested by mosquito larvae can be determined by A 600Numerical characterization. The present application further adopts aniline blue dyed yeast and fluorescent dyed yeast to feed mosquito larvae, detects the absorbance value in mosquito larvae, and the results show that the aniline blue dyed group can detect that the aniline blue content in mosquito larvae increases with the increase of the feeding amount, while the background fluorescence of the fluorescent dyed group mosquito larvae itself interferes with the fluorescence of the fluorescent marker, and the detection of the fluorescence intensity cannot reflect the feeding level of the mosquito larvae, therefore, the aniline blue as a dye for yeast dyeing is beneficial to accurately reflecting the feeding behavior of the mosquito larvae.

[0044] The present application provides a method for evaluating the feeding behavior of mosquito larvae, comprising the following steps:

[0045] The mosquito larvae are divided into an experimental group, a calibration group and a blank group:

[0046] The experimental group uses the aniline blue dyed yeast in the application as feed to feed the mosquito larvae, and the absorbance value M1 of the aniline blue in the mosquito larvae after feeding is determined by spectrophotometry;

[0047] The calibration group uses the aniline blue dyed yeast in the application as feed to feed the mosquito larvae after starvation treatment, and the absorbance value M of the aniline blue in the mosquito larvae after feeding is determined by the spectrophotometry in the experimental group;

[0048] The blank group uses undyed yeast as feed to feed the mosquito larvae after starvation treatment, and the absorbance value M0 of the aniline blue in the mosquito larvae after feeding is determined by the spectrophotometry in the experimental group;

[0049] The M1, M and M0 are substituted into formula I to calculate the feeding index of the mosquito larvae:

[0050] Feeding index = (M1-M0) / (M-M0) formula I

[0051] The feeding index is proportional to the feeding level of the mosquito larvae, and the higher the feeding index, the higher the feeding level of the mosquito larvae.

[0052] In the present application, the age of the mosquito larvae includes any one of the following: the second instar, the third instar and the fourth instar. The species of the yeast, the added amount of the feed and the feeding time are the same as those in the application of the aniline blue dyed yeast as feed in evaluating the feeding behavior of the mosquito larvae. The number of mosquito larvae in the three groups is preferably ≥8, more preferably 9-20, and most preferably 10. The method for determining the absorbance of the aniline blue dyed yeast after being fed by the mosquito larvae is preferably that the mosquito larvae after feeding are extracted by an alcohol solution, the supernatant is centrifuged to measure the absorbance at 600 nm; the alcohol solution preferably includes methanol.

[0053] In the present application, in the experimental group, the state of the larvae before feeding the aniline blue dyed yeast includes a normal normal feeding state or a starvation state.

[0054] In the present application, the time of the starvation treatment in the blank group and the standard group is 4-8h, more preferably 4h. The appropriate starvation time can make the mosquito larvae in the starvation state, and improve the feeding level of the mosquito larvae. Too long starvation time will lead to the decrease of the activity of the mosquito larvae, and affect the feeding level of the mosquito larvae. In the blank group, the absorbance of the mosquito larvae after feeding on the non-stained yeast can avoid the interference of the mosquito larvae itself on the absorbance value. In order to avoid the interference of the residual aniline blue in the environment into the mosquito larvae on the determination of the feeding results, when the blank group feeds the mosquito larvae with the unstained yeast as the feed, the yeast suspension stained with aniline blue and the unstained yeast suspension are centrifuged respectively, the supernatant of the unstained yeast is removed, the supernatant of the yeast stained with aniline blue is sucked and added into the unstained yeast tube, and the yeast suspension of the unstained yeast is obtained to feed the mosquito larvae. In the standard group, the absorbance of the mosquito larvae after feeding on the yeast stained with aniline blue is determined, and the absorbance value of the standard group can reflect the feeding level of the mosquito larvae in the starvation state. The setting of the blank group and the calibration group and the calculation of the feeding index according to the formula I can avoid the interference of the mosquito larvae itself and different batches of experimental operations on the absorbance value, and more accurately reflect the feeding level of the mosquito larvae.

[0055] The method of the present application can accurately reflect the difference in the feeding level of the mosquito larvae with different starvation degrees and different ages.

[0056] In an embodiment of the present application, the feeding behavior of the mosquito larvae with starvation treatment and the mosquito larvae without starvation treatment is evaluated by the method of the present application, and the results show that the feeding index of the mosquito larvae without starvation treatment is significantly lower than that of the mosquito larvae with starvation treatment. It can be seen that the method of the present application can accurately reflect the feeding level of the mosquito larvae.

[0057] In the whole growth process of the mosquito insects, the mosquito larvae start feeding as soon as they hatch from the eggs, the second instar mosquito larvae have strong appetite, and the third instar and the fourth instar enter the binge period, the body of the mosquito larvae grows rapidly, and the feeding level of the fourth instar mosquito larvae is the highest. In an embodiment of the present application, the feeding behavior of the second instar, the third instar and the fourth instar mosquito larvae is evaluated by the method of the present application, and the results show that the feeding amount of the mosquito larvae increases significantly from the second instar to the fourth instar, which further proves that the evaluation method of the present application can accurately reflect the feeding level of the mosquito larvae.

[0058] The present application provides the application of the method in screening or evaluating the drugs for regulating the feeding behavior of the mosquitoes.

[0059] In the present application, the drugs include drugs that inhibit the feeding behavior of mosquito larvae, which can inhibit the growth and development of mosquito larvae and thus achieve the prevention and control of mosquitoes. The drugs also include drugs that promote the feeding behavior of mosquito larvae, such as attractants and phagostimulants, which can attract mosquito larvae and thus achieve the prevention and control of mosquitoes.

[0060] The present application also provides the use of the method in screening or evaluating drugs that inhibit the growth and development of mosquito larvae.

[0061] The feeding behavior of mosquito larvae is an output behavior guided by external food chemical stimuli and the intrinsic state of the organism. Insufficient intake of food by adult mosquitoes during the larval stage will greatly affect the development of adult mosquitoes, such as normal emergence and mating. Therefore, the method for evaluating the feeding behavior of mosquito larvae can accurately determine the effect of the addition of drugs on the feeding behavior of mosquito larvae. Since the reduction of feeding behavior can easily affect the growth and development of mosquito larvae, the method for evaluating the feeding behavior of mosquito larvae can also be used to screen or evaluate drugs that inhibit the growth and development of mosquito larvae by regulating the feeding behavior of mosquitoes.

[0062] In the present application, the drugs include DEET. The chemical formula of DEET is C 12 H 17 NO, the English name is diethyltoluamide (DEET), and the alias is N, N-diethyl-3-methylbenzamide. The use of DEET is preferably sprayed on the skin or clothes to avoid bites by insects and mosquitoes. The concentration of DEET is preferably ≥10 -3 (v / v), more preferably 10 -3 (v / v). The results of evaluating the effect of DEET on the feeding behavior of mosquito larvae using the method of the present application show that the feeding level of mosquito larvae is significantly reduced after adding DEET in the food and environment, which indicates that DEET can be used to prepare drugs that reduce the feeding level of mosquito larvae, and can also be used to prepare drugs that inhibit the growth and development of mosquito larvae.

[0063] In the present application, all raw materials / components are commercially available products well known to those skilled in the art unless otherwise specified.

[0064] In order to further illustrate the present application, the technical solutions provided by the present application are described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present application.

[0065] 1. Experimental materials and environmental explanation

[0066] Yeast is active dry yeast, which is purchased from Angel Yeast Co., Ltd.;

[0067] Aniline blue, fluorescein diacetate (FDA) and DEET were purchased from Shanghai McLean Biochemical Technology Co., Ltd.

[0068] Centrifuge (Eppendorf, Germany)

[0069] Cell 6-well culture plate (Corning, USA)

[0070] Micro UV / visible spectrophotometer (Yuanzhi instrument, Shanghai)

[0071] The normal food for mosquito larvae before being fed with yeast alone was fish food and yeast mixed at a ratio of 2:1.

[0072] Experimental site: 318 insect room of Biology Building, Nankai University;

[0073] Experimental time: 14:00 to 18:00;

[0074] Indoor conditions: temperature 27±1℃, relative humidity 70±5%.

[0075] Example 1

[0076] 1. Preparation of aniline blue dyed yeast

[0077] Prepare a yeast suspension with a concentration of 1 mg / mL and dH2O as the solvent; prepare an aniline blue solution with a concentration of 5 mg / mL and PBS as the solvent;

[0078] Add 6.4 mL of the above yeast suspension and 1.6 mL of the above aniline blue solution to a 15 mL centrifuge tube, place it in an incubation bed at 15 rpm, and incubate overnight at room temperature (27℃) in the dark;

[0079] After incubation, centrifuge at 4000 rpm for 5 min, wash the yeast cells with distilled water to remove excess dye until the supernatant is clear, and obtain aniline blue dyed yeast.

[0080] 2. Relationship between the amount of aniline blue dyed yeast and A 600

[0081] Dilute the aniline blue dyed yeast to a final concentration of 0, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, and 1 mg / mL, respectively;

[0082] Take 1 mL of aniline blue dyed yeast with different concentrations into a 1.5 mL centrifuge tube, centrifuge at room temperature at 12000 rpm for 1 min, remove the supernatant, and add 50 μL of methanol to vortex and extract thoroughly;

[0083] ​After extraction, add 50 μL of methanol and vortex to mix. Centrifuge at 12,000 rpm for 5 min at room temperature. Take 20 μL of the supernatant and measure the absorbance (A) at 600 nm (the absorption peak of the aniline blue chromophore). 600 ), 3 repetitions per group;

[0084] Using unstained yeast at different concentrations as a control group, in Graphpad software, the concentration of yeast stained with aniline blue was plotted on the x-axis, and A... 600 Plot the graph on the ordinate and perform regression analysis to obtain the relationship and R². 2 value.

[0085] The amount of yeast stained with aniline blue and A 600 See the relationship between them Figure 1 ,according to Figure 1 The results showed that the number of yeast cells stained with aniline blue was related to A... 600 It is directly proportional, meaning that the amount of aniline blue-stained yeast ingested by mosquito larvae can be considered to be directly proportional to A. 600 The magnitude of a numerical value.

[0086] Example 2

[0087] 1. Temporal dynamics of mosquito larvae feeding on aniline blue-stained yeast

[0088] Four mL of yeast stained with 0.5 mg / mL aniline blue was transferred to a 5 mL tube and filled into a single well of a 6-well plate. After the yeast settled, ten third-instar mosquito larvae that had been starved for 4 hours were introduced. The experiment was terminated after the mosquito larvae fed for 10, 20, 30, 40, 50, and 60 minutes, respectively. Each group was repeated in triplicate.

[0089] Methanol extraction of whole mosquito larvae tissue fluid and absorbance determination: Mosquito larvae were transferred to a new six-well plate filled with dH2O and washed. Finally, they were transferred to clean filter paper. Using a brush, the larvae were carefully dipped into a 1.5 mL centrifuge tube containing 50 μL of methanol for extraction. The larvae were thoroughly ground and extracted, then another 50 μL of methanol was added and vortexed. Finally, the mixture was centrifuged at room temperature at maximum speed for 5 min, and 20 μL of the supernatant was used to measure the absorbance at 600 nm (A). 600 );

[0090] The time-dynamic changes in the amount of aniline blue-stained yeast consumed by mosquito larvae are shown in the figure. Figure 2 ,according to Figure 2 The results showed that aniline blue accumulated linearly in mosquito larvae within 20 minutes before feeding. The amount of food consumed, as reflected by aniline blue, was not affected by the larvae's excretion or digestive tract's ability to store food. Therefore, measurements of larval feeding during this period were relatively accurate. Consequently, subsequent feeding experiments were conducted within this timeframe.

[0091] 2. The relationship between the amount of yeast added and the amount of food consumed by mosquito larvae.

[0092] Add 0.1 mg, 0.2 mg, 0.4 mg, 0.8 mg, 1.2 mg, 1.6 mg and 2 mg of aniline blue-stained yeast to each well of a 6-well plate. After the yeast has settled, 10 third-instar mosquito larvae that have been starved for 4 hours are introduced and fed for 20 minutes. Each group has 3 replicates.

[0093] The relationship between the amount of aniline blue-stained yeast added and the amount of food consumed by mosquito larvae is shown in [reference needed]. Figure 3 .according to Figure 3 The results showed that when the feed addition was 0.04 mg / larva or more, the aniline blue content in the mosquito larvae no longer increased with the increase of feed addition. It can be seen that the feed addition of 0.04 mg / larva or more is excessive for mosquito larvae to feed for 20 minutes. The amount of yeast introduced for aniline blue staining should be ≥0.04 mg / larva.

[0094] Example 3

[0095] Establish a quantitative method for mosquito larval feeding behavior and verify the effectiveness of the method.

[0096] 1. Establish a method for assessing the feeding behavior of mosquito larvae.

[0097] Mosquito larvae were divided into an experimental group, a calibration group, and a control group:

[0098] In the experimental group, aniline blue-stained yeast was used as feed to mosquito larvae, and the absorbance value M1 of aniline blue in the mosquito larvae after feeding was measured by spectrophotometry.

[0099] After starvation treatment for 4 hours, mosquito larvae in the calibration group were fed with aniline blue-stained yeast. The absorbance value M of the mosquito larvae after feeding was determined by the spectrophotometric method of the experimental group.

[0100] After starvation treatment for 4 hours, mosquito larvae in the control group were fed with unstained yeast. The absorbance value M0 of the mosquito larvae after feeding was determined by spectrophotometry using the experimental group.

[0101] Substitute M1, M, and M0 into Formula I to calculate the feeding index of mosquito larvae:

[0102] Feeding index = (M1-M0) / (M-M0) Formula I

[0103] Each mosquito larva was fed 0.2 mg of food over a period of 20 minutes.

[0104] The feeding index is proportional to the feeding level of mosquito larvae, and the higher the feeding index, the higher the feeding level of mosquito larvae.

[0105] 2. Validation of the effectiveness of the quantitative method for mosquito larvae feeding behavior

[0106] The experiment verifies the effects of starvation state, age (second, third and fourth instar) and repellent DEET on the feeding behavior of mosquito larvae.

[0107] Calibration group: pick 40 three instar mosquito larvae in a glass cup, wash off the food residue, starve for 4 hours, then put into a single well of a 6-well plate with 2 mg of aniline blue dyed yeast (0.5 mg / mL), 10 mosquito larvae per well, and feed for 20 minutes;

[0108] Blank group: pick 40 three instar mosquito larvae in a glass beaker, wash off the food residue, starve for 4 hours; during the experiment, centrifuge 4 mL of 0.5 mg / mL aniline blue dyed yeast and undyed yeast, remove the supernatant of the undyed yeast, and add the supernatant of the aniline blue dyed yeast tube to the undyed yeast tube to obtain the undyed yeast suspension, then put the undyed yeast suspension into a single well of a 6-well plate, 10 mosquito larvae per well, and feed for 20 minutes.

[0109] Experimental group 1 is divided into starvation group and non-starvation group

[0110] Starvation group: the starvation group is treated the same as the calibration group;

[0111] Non-starvation group: pick 40 three instar mosquito larvae, transfer them to a single well of a 6-well plate, add appropriate food, wash the mosquito larvae after feeding for 4 hours, and put them into a single well of a 6-well plate with 2 mg of aniline blue dyed yeast (0.5 mg / mL), 10 mosquito larvae per well, and feed for 20 minutes.

[0112] Experimental group 2 is divided into second instar group, third instar group and fourth instar group

[0113] Second instar group: the second instar group is treated the same as the calibration group, with the only difference being that the instar of the mosquito larvae is second instar;

[0114] Third instar group: the third instar group is treated the same as the calibration group;

[0115] Fourth instar group: the fourth instar group is treated the same as the calibration group, with the only difference being that the instar of the mosquito larvae is fourth instar.

[0116] Experimental group 3 is divided into no DEET addition group and DEET addition group

[0117] No DEET addition group: the no DEET addition group is treated the same as the calibration group;

[0118] The DEET adding group: 40 normal fed 3rd instar mosquito larvae were selected in a glass beaker, and the food residues were washed off, and the larvae were starved for 4 hours; 4 mL of the yeast dyed with 0.5 mg / mL aniline blue was centrifuged, and the supernatant was taken out and dissolved with DEET to a final concentration of 10 -3 (v / v), and then the supernatant with dissolved DEET was added into the yeast dyed with aniline blue to obtain a DEET-treated yeast suspension dyed with aniline blue, and the suspension was added into a single hole of a 6-hole plate, and 10 mosquito larvae were put into each hole for feeding for 20 minutes.

[0119] After the experiment was completed, the absorbance values of each group were determined by the method of extracting the whole worm tissue liquid of the mosquito larvae with methanol in Example 2, the absorbance value of the blank group was M0, the absorbance value of the calibration group was M, and the absorbance value of the experimental group was M1, which were substituted into the above formula I to calculate the feeding index. SPSS was used for significance analysis, and single sample T test and one-way analysis of variance were used for comparison, and each group had 3 repeats.

[0120] The results of the feeding index of the starved group and the non-starved group are shown in Figure 4 , and the results of the feeding index of the 2nd instar, 3rd instar and 4th instar mosquito larvae are shown in Figure 5 According to the results of Figure 4 and Figure 5 , the feeding amount of the non-starved group of mosquito larvae was significantly reduced, and the feeding amount of the mosquito larvae increased significantly from the 2nd instar to the 4th instar. It can be seen that the method of the present application can accurately reflect the changes of the feeding behavior of the mosquito larvae.

[0121] The results of the feeding index of the group without adding DEET (control group) and the group with adding DEET are shown in Figure 6 According to the results of Figure 6 , under the condition of 10 -3 DEET, the feeding index of the mosquito larvae was significantly reduced, which indicated that DEET could effectively inhibit the feeding of the mosquito larvae, and could be used for preparing a drug for reducing the feeding level of the mosquito larvae, and could be used for preparing a drug for regulating the feeding behavior of the mosquito larvae and then inhibiting the growth and development of the mosquito larvae.

[0122] Comparative Example 1

[0123] Preparation of yeast labeled with fluorescent dye

[0124] 0.5 mg / mL yeast suspension was prepared, and the solvent was dH2O; 10 mM FDA stock solution was prepared, and the solvent was DMSO;

[0125] 1 mL of yeast and 20 μM final concentration of fluorescein diacetate (FDA) were added into a 2 mL centrifuge tube, and the mixture was dyed at 37°C in the dark for 1 hour; after dyeing, the mixture was centrifuged at 10,000 rpm for 1 minute, and the yeast cells were washed with distilled water to remove excess dye;

[0126] Take 1 mL of fluorescently labeled yeast (0.5 mg / mL) and unlabeled yeast (0.5 mg / mL) in a single well of a 4-well plate, place a single mosquito larva in each well, and after 20 min of feeding, detect the fluorescence intensity of the mosquito larva intestine under an inverted fluorescence microscope, with an excitation wavelength of 450-490 nm (GFP filter). The results of the intestinal fluorescence of mosquito larvae after feeding on unlabeled yeast and FDA-labeled yeast are shown in Figure 7 .

[0127] From Figure 7 The results show that mosquito larvae that feed on unlabeled yeast have strong background fluorescence, which cannot be significantly separated from the fluorescence of fluorescently labeled mosquito larvae. Therefore, the method of using fluorescently labeled yeast has strict requirements for the fluorescent group. Commonly used green and red fluorescent groups cannot be used in this experiment, and FDA-labeled yeast cannot be used to evaluate the feeding behavior of mosquito larvae.

[0128] Although the above embodiments have made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained under the premise of not being creative according to the present embodiments, and these embodiments all belong to the protection scope of the present application.

Claims

1. The application of an aniline blue-stained yeast as feed in evaluating the feeding behavior of mosquito larvae.

2. The application according to claim 1, characterized in that, The method for preparing the aniline blue-stained yeast is to stain the yeast with an aniline blue solution for 8–14 hours.

3. The application according to claim 2, characterized in that, The concentration of the aniline blue solution is 1–10 mg / mL; The staining concentration of the yeast was 0.8–1 mg / ml.

4. A method for assessing the feeding behavior of mosquito larvae, comprising the following steps: Mosquito larvae were divided into an experimental group, a calibration group, and a control group: The experimental group fed mosquito larvae with yeast stained with aniline blue as described in any one of claims 1 to 3, and used spectrophotometry to determine the absorbance value M1 of aniline blue in the mosquito larvae after feeding. After starvation treatment of mosquito larvae, the calibration group fed mosquito larvae with yeast stained with aniline blue as described in any one of claims 1 to 3, and used the spectrophotometric method in the experimental group to determine the absorbance value M in the mosquito larvae after feeding. After starvation treatment, mosquito larvae in the control group were fed with unstained yeast. The absorbance value M0 of the mosquito larvae after feeding was determined by spectrophotometry in the experimental group. Substitute M1, M, and M0 into Formula I to calculate the feeding index of mosquito larvae: Feeding index = (M1-M0) / (M-M0) Formula I The feeding index is directly proportional to the feeding level of mosquito larvae; the higher the feeding index, the higher the feeding level of mosquito larvae.

5. The method according to claim 4, characterized in that, The feeding time is 10 to 20 minutes.

6. The method according to claim 4, characterized in that, The amount of feed added is ≥0.04mg / animal.

7. The method according to any one of claims 4 to 6, characterized in that, The starvation treatment lasts for 4 to 8 hours. The instars of the mosquito larvae include any of the following: second instar, third instar, and fourth instar.

8. The use of the method according to any one of claims 4 to 7 in screening or evaluating drugs that regulate the feeding behavior of mosquito larvae.

9. The use of the method according to any one of claims 4 to 7 in screening or evaluating drugs that inhibit the growth and development of mosquito larvae.

10. The application according to claim 8 or 9, characterized in that, The drug includes DEET.

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