Integrated Control of Aedes Aegypti With Parasitic Nematodes, Biological Control Agents, Chemical Insecticides and Plant Essential Oil

Aedes aegypti can transmit dengue fever, yellow fever, Chikungunya fever, Zika virus disease and vector density control is the most effective way to prevent these infectious diseases. However, the extensive use of chemical pesticides has caused a series of problems, such as environmental pollution, killing non-target organisms and so on. In this study, a parasitic nematode, Romanomermis wuchangensis was used in the larviciding evaluation of Ae. aegypti, while the activity of four chemical insecticides and biological control agents were tested. Besides, Mentha haplocalyx essential oil was isolated and its olfactory physiological function with OBP1 protein of Ae. aegypti antenna was measured by the prokaryotic expression and uorescence competitive binding assay. Compared with the control group, R. wuchangensis indicated high eciency and environmental friendliness in the control of Ae. aegypti. After the second instar larvae were parasitized, the mortality of two treatment groups exceeded 75%. Compared to control group, the quantitative real-time PCR analysis results demonstrated that SOD, POD and CAT genes had obvious high expression levels in the nematodes parasitic groups. The antioxidant enzyme test results also exhibited obvious difference of SOD, CAT and POD during the nematode parasitic period. Besides, Bacillus thuringiensis (Bti) and chemical insecticide experimental results also showed great insecticidal ecacy against mosquito larvae. Five chemical components including Menthol, Pinene, Limonene, Isopulegol and Pulegone were identied from M. haplocalyx and exhibited great binding ability with AaegOBP1 protein. Present results illustrated that the integrated application of these various mosquito vector control methods in the future has broad prospects. nematodes which parasitized and killed mosquito larvae 21 . In our laboratory, the mermithid nematode R. wuchangensis has also been successfully used as an ecosystem-friendly biocontrol agent for mosquito control. Our previous study indicated that R. wuchangensis could infect C. quinquefasciatus and Ae. albopictus, and the infection rate and fatality rate of C. quinquefasciatus reached 49.18% and 100% in the eld experiment 29 . The results demonstrated that the control of mosquito by nematodes had the characteristics of environmental friendliness and long-term sustainability, suggesting they maybe widely used in eld biological control of mosquito pests. different concentrations of plant essential oil can obviously repel adult mosquitoes, which is helpful for the prevention of mosquito pests. In present study, we also further explore the olfactory protein (OBP1) of Ae. aegypti and performed uorescence competitive binding experiments with ligand compounds with higher content in plant essential oil. Fluorescence competitive binding analysis suggested that AaegOBP1 protein have strong binding ability to Menthol, Pinene, Limonene, Isopulegol and Pulegone from Mentha haplocalyx. These results indicated that AaegOBP1 protein could specically distinguish the volatile odor molecules of plant essential oil through the recognition of different ligand compounds, involving in the olfactory repellent behavior of adult Ae. site-directed mutation


Introduction
The mosquito Aedes aegypti were considered to be one of the most dangerous medical insects in the world 1 , as they were vectors of several globally important vector-borne diseases, including dengue virus (DENV) 2 , yellow fever virus 3 and chikungunya virus (CHIKV) 4 . In the past 50 years, the dengue epidemic caused more than one billion infections and one million deaths, and the number of cases have increased 30 times without any signs of slowing down 5,6 . Moreover, in Asia and the Americas, the burden of dengue was approximately 1300 disability-adjusted life years (DALYs) per million population, which was similar to the other childhood and tropical diseases in this regions 7 . Since 1978, dengue has been detected for nearly forty years in China and it also occasionally erupts in Guangdong Province every year. Geographically, the dengue outbreaks have gradually expanded from Guangdong, Hainan, and Guangxi in Southern coastal regions of China to other regions including Fujian, Zhejiang and Yunnan Provinces 8 .
Due to the outbreak of dengue fever in Guangdong Province in 2014, more than ten thousands of people were infected by the dengue epidemic and the local government spent >$30 million US dollars to control mosquito populations 9 .
Until now, because of no vaccine or speci c treatment for dengue fever, many indirect prevention and control measures have been taken, such as population surveillance, disease prevention and rapid outbreak response are used to improve mosquito vector control 1 . The use of insecticides has often been the only feasible method of disease control. For instance, wide-scale house spraying of DDT during the 1950s to 1960s dramatically reduced malaria incidence in Asia, and the pyrethroids were introduced to Cambodia in the late 1980s for malaria and dengue control 5 . In China, more than 27, 000 kg of pyrethroids were used for ultra-low volume spraying in the dengue control of Guangzhou city in 2014, and a large amounts of temephos and fenthion were also used as larvicides to prevent the mosquito spread 10 . However, the use of chemical pesticides not only polluted the environment but also caused great harm to non-target insects including butter y, honeybee, bumblebee and other pollinators. The widespread use of chemical pesticides could easily interfere normal physiological behavior of butter y and bee, such as visiting owers, foraging, pollination and other ecological function 11,12 .
Insect growth regulators (IGRs) have been widely used in pest control in many areas, which were highly effective larviciding agents of mosquito larvae and showed low mammalian toxicity and more safety to most of non-target organisms 13,14 . Pyriproxyfen and S-methoprene were two kinds of effective juvenile hormone mimic with low toxicity to mammals, which could affect the hormonal balance in insects, thus strongly inhibit embryogenesis, metamorphosis and adult formation 15,16 . Di ubenzuron was also a readily available insect growth regulator, which could inhibit chitin synthesis and exhibited the ovicidal and larvicidal properties 17 . Besides, there were also some bioinsecticides used for larvae control, such as the microbial agent Bacillus thuringiensis (Bti) has become the most commonly used larvicide worldwide, which can form spores which contain crystals, predominantly comprising one or more Cry and/or Cyt proteins that have potent and speci c insecticidal activity 18,19 . Plant-derived essential oils were also an environmentally friendly control agent, especially for repelling adult mosquitoes, indicating that they can be an important supplement for mosquito pest control 20 .
In addition, nematodes also played a key role in the eld prevention of mosquito pests, such as Romanomermis iyengari (Mermithidae) was one of entomopathogenic nematodes which parasitized and killed mosquito larvae 21 . In our laboratory, the mermithid nematode R. wuchangensis has also been successfully used as an ecosystem-friendly biocontrol agent for mosquito control. Our previous study indicated that R. wuchangensis could infect C. quinquefasciatus and Ae. albopictus, and the infection rate and fatality rate of C. quinquefasciatus reached 49.18% and 100% in the eld experiment 29 . The results demonstrated that the control of mosquito by nematodes had the characteristics of environmental friendliness and long-term sustainability, suggesting they maybe widely used in eld biological control of mosquito pests.
Reactive oxygen species (ROS) produced by insect cells was the rst line of defense against the invasion of insecticides and parasites, including hydrogen peroxide (H 2 O 2 ), free hydroxyl (OH − ) and superoxide anion (O 2− ) 22 . Insects mainly protected themselves by antioxidant enzymes, such as Superoxide dismutase (SOD) could effectively remove O 2− and convert it into H 2 O 2 , Catalase (CAT) and Peroxidase (POD) work together to remove H 2 O 2 , which these antioxidant enzymes coordinated to regulate ROS in insects to keep them in dynamic balance 23,24 . Early studies have shown that in the process of parasitizing the host, the death of the host was usually caused by oxidative damage, including Sarcophagha crassipalpis, Nasonia vitripennis, Plutella xylostella,, Cotesia plutellae, Tenebrio Molitor and Scleroderma guani, etc [25][26][27] . However, there was no report about the oxidative damage effect of nematode on the mosquito, though it may play a vital role of biological control of mosquitoes.
In this study, we present statistics on the incidence of dengue fever in China from 2015 to 2018 prior to the novel Coronavirus outbreak. Besides, we compared the control effects of chemical insecticides, IGRs, B. thuringiensis and plant essential oil on the larvae and adult of Ae. aegypti. The mortality rate of Ae. aegypti was observed during parasitized by R. wuchangensis, and the activities of SOD, POD and CAT during the lethal period of the host were tested to determine whether oxidative damage was involved, then explore the potential cause of oxidative damage to the host through the content of Malondialdehyde (MDA). Simultaneously, Real-time quantitative PCR were used to investigate the expression patterns of SOD, POD and CAT genes of Ae. aegypti larvae under different parasitic period by R. wuchangensis. In addition, the effective components of plant essential oil was identi ed from Mentha haplocalyx and chemoecological functional analysis of olfactory protein (OBP1) of Ae. aegypti were also investigated.

Ethics statement
The Ae. aegypti strain was provided by China CDC and reared in the laboratory of Hubei provincial center for disease control and prevention (Wuhan City, China). Romanomermis wuchangensis was a parasitic nematode and maintained in our laboratory incubator of Central China Normal University (Wuhan City, China). All animal experiments were carried out in accordance with the experimental guidance and regulations of Hubei CDC. Mentha haplocalyx was a common medicinal plant that grown in the experimental eld and the extracted plant essential oils were carried out in accordance with relevant guidelines and regulations. All volunteer study in the repellent experiments of mosquito were conducted with the informed/written consent of all subjects and all experimental methods were carried out in accordance with relevant guidelines and regulations. All experimental protocols and animal procedures were also approved by the institutional committee of school of life science at Central China Normal University in China (CCNUIRB).
Mosquito populations were maintained at insectary conditions (26°C±2°C; relative humidity 70%±10% L:D 14:10) and females were fed on mouse blood to complete their gonotrophic cycle. The mouse blood was collected from experimental animal center of Hubei provincial center for disease control and prevention (Wuhan City, China) and were also approved by the Institutional Review Board at Central China Normal University in China (CCNUIRB). Second instar larvae of Ae. aegypti was infected by R. wuchangensis by the ratio of 1:5 (mosquito: nematode). Then the infected mosquitos were maintained in the Plastic basin. When the nematodes emerged from Ae. aegypti, the nematodes of each developmental stage were independently collected and stored at 4°C. Insecticides were used in this study: deltamethrin, permethrin, temephos, propoxur, pyriproxyfen, S-methoprene, di ubenzuron and 7000 ITU/mg Bacillus thuringiensis (Bti). All tested insecticides were provided by China CDC.
IGRs tests 50 second instar larvae of Ae. aegypti were infected by 250 and 500 R. wuchangensis in glass breakers, by the ratio of 1:5 (mosquito: nematode) and 1:10, respectively. No nematodes were added to the control group and each beaker was lled with 50 mL chlorine-free water. Both the control group and treatment group were performed as three replicates and reared in the insectary. After 48 hours, add chlorine-free water to 100 mL. The mosquito larvae were observed every 24 hours and count the death of larvae.
A series of glass breakers (250 mL) were lled with 99.9 mL chlorine-free water, either 0.1 mL stock solution insecticide or 0.1 mL acetone was added. The acetone solution served as a control. For each concentration of the insecticide stock solution, three replicates were performed. Twenty-ve third instars of uniform size were exposed to the insecticide solution in each replicate. The glass breakers were covered with medical gauze (to contain emerged adults) and a perforated plastic cap (to minimize evaporation). Emergence was determined after insecticide exposure as complete emergence had occurred in all controls at this time.

Chemical insecticides and Bti bioassays
In order to compare the insecticidal effects of biological insecticides, four representative chemical insecticides (temephos, permethrin, propoxur and deltamethrin) were used for comparative analysis.
Chemical insecticides bioassays were set up as described above for the IGRs bioassays, except that mortality was determined after 72 h of exposure, and larvae were considered dead if they were unresponsive to touching with a probe 28 . Bacillus thuringiensis was set up as described above except that the solvent is changed from acetone to chlorine-free water.

Repellent activity of plant essential oil against mosquitos
The plant essential oil was isolated from the fresh leaves of Mentha haplocalyx by a Clevenger-type apparatus 20,29 and stored at -80°C until use. The chemical composition of this essential oil was analyzed by gas chromatography-mass spectrometry (GC-MS). All Ae. aegypti adults were kept in the cage (30 x 30 x 30 cm) and randomly separated into four groups of twenty-ve each and were fasting for 1 day before the test. The M. haplocalyx essential oil (1.25µl, 2.5µl, 5µl, 10µl, 20µl) were applied on the ventral part of volunteer forearm and inserted into the cage. The repellent activity of essential oil against Ae. aegypti was measured using the method described by Nasrul et al. 30 . The alcohol was conducted as control and each test concentration was repeated three times.

Assays of antioxidant enzyme activities and oxidative damage parameter
A series of glass breakers (250 mL) were lled with 99.9 mL chlorine-free water, either 0.1 mL stock solution temephos (0.003 mg/L, Preliminary experiments indicated this was the maximum sublethal concentration of temephos) or 0.1 mL acetone was added. The acetone solution served as a control. For each of the stock solution, three replicates were performed. 50 third instar larvae of uniform size were exposed to the insecticide solution in each replicate. Then, 50 second instar larvae of Ae. aegypti were infected by R. wuchangensis in glass breakers, by the ratio of 1:10 (mosquito: nematode). No nematodes were used for the blank control comparison and each beaker was lled with 50 mL chlorine-free water. Both the control group and treatment group were performed as three replicates, reared in the insectary. All larvae in one glass breaker were assembled and homogenized in ice-cold buffer (0.8% NaCl, pH 7.4) in a proportion of 0.1 g body weight to 1 mL of buffer. After the homogenates were centrifuged at 10,000×g for 15 min at 4°C, the supernatant was collected for test. Antioxidant enzyme activities and oxidative damage parameter were measured using a commercially available assay kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China).

Real-time quantitative PCR analysis of antioxidant enzyme genes
The Ae. aegypti larvae (second instar) was infected by R. wuchangensis by the ratio of 1:10 (mosquito: nematode). Expression patterns of three antioxidant enzyme genes (SOD, AY745980.1, POD, XM_021849339.1, CAT, XM_001663550.3) were determined by real-time quantitative PCR analysis. Total RNA was extracted from different treatments with Trizol test kit and six primers (SOD-YF, SOD-YR, POD-YF, POD-YR, CAT-YF and CAT-YR, Rps3 gene used as the reference, Table 1) were used to test their relative abundance of three antioxidant enzyme genes in mRNA level. Actin gene was used as the reference and real-time quantitative PCR was measured on a Bio-Rad CFX 96 PCR system. Three technical replicates and three biological replicates were conducted to check reproducibility. The experimental process refers to the method in our previous literature and the real-time PCR data was analyzed by the 2 −ΔCt method 31 .
Olfactory recognition of Ae. aegypti OBP1 protein and potential repellent ligands from plant essential oil The Ae aegypti olfactory gene OBP1 (genebank number: AY189223.1) was used for prokaryotic expression and a nity chromatography puri cation. The target gene was induced by IPTG and expressed in E. coli prokaryotic expression system using by their technical methods described by Zhou et al 32 . After puri cation, the target protein was tested for its binding capacity with chemical ligands with potential repellent activity in M. haplocalyx essential oil. The uorescence competitive binding assay was tested on the uorescence spectrophotometer Hitachi F-4500 according to our laboratory previous method of Mao et al 31 . The uorescent probe N-phenyl-1-naphthylamine (1-NPN) and potential repellent ligands from chemical composition of plant essential oil were used to measure their competitive a nities to Ae. aegypti OBP1 protein.

Statistical analysis
The mortality rate of tested larvae was analyzed using SPSS 20.0 software. Then the estimation of the lethal and emergence inhibition concentration of 50% con dence limits was obtained. All the data were analyzed by one-way ANOVA with the SPSS 20.0 and the gures were drawn by GraphPad Prism software. The result in the graphs represents the mean values ± SE. Statistical testing of signi cance was analyzed using Tukey's multiple range test.

Results
Overview of dengue fever cases A total of 16936 cases of dengue fever were reported in mainland China from 2015 to 2018, of which 12984 cases were local cases and 4042 were imported cases. The epidemiological characteristics of dengue fever cases were statistically analyzed throughout the year, and the epidemic began to rise in August and reached its peak in September every year. The main imported countries were Myanmar and Cambodia, and the proportion of imported cases showed an increasing trend (  Dengue cases outbreak typically occur from August to November (89% of the total reported cases), with September being the peak. In China, the ratio of male and female affected by Dengue was 1.14:1, and the age was mainly from 20 to 65 years old (81%). The occupational distribution of reported cases were housework and unemployment (19%), business services (16%), farmers (14%), workers (10%), retirees (9%) and students (7%), accounting for 75% of the total reported cases.

Biological insecticides assays
In biological insecticides assays, the larvicidal activities of R. wuchangensis to second instar larvae of Ae. Aegypti were shown in gure 3, from the fth day after infection, the Ae. Aegypti larvae gradually appeared dead individuals. In the different affected ratio groups (mosquito: nematode, 1:5 and 1:10), their mortality rate were 75.79% and 96.L00%, respectively. As shown in Table 3, three novel IGRs could obviously control the number of mosquito pests. Among them, the pyriproxyfen was the most toxic larvicide (EC 50 =0.000023 mg/L), followed by di ubenzuron (0.001659 mg/L) and S-methoprene (0.0856 mg/L). The e cacy of pyriproxyfen on Ae. aegypti larvae was more e cient than the other two IGRs. Chemical insecticides bioassay Four representative chemical insecticides including temephos, permethrin, propoxur and deltamethrin were tested for their insecticidal e cacy against Aedes aegypti. Toxicities of four novel insecticides (temephos, permethrin, propoxur, deltamethrin) to third instar larvae of Ae. aegypti were shown in Table 4, and LC 50 values were statistically analyzed. The deltamethrin was the most effective larvicide (LC 50 =0.001658 mg/L), followed by permethrin (0.009222 mg/L), temephos (0.009371 mg/L) and propoxur (0.585596 mg/L). The dosage of propoxur was signi cantly higher than that of the other three insecticides. Chemical composition identi cation of M. haplocalyx essential oil and repellent activity A total of 16 compounds in the M. haplocalyx leaf essential oil were identi ed and their abundances were shown in Table 5. Analysis of the essential oil by GC-MS revealed that Menthol, Pinene and Cyclohexanone were the most abundant three major constituents in the essential oil, consisting 32.87%, 25.04% and 10.95% of the total oil, respectively. Other main compounds in this essential oil contained Limonene (3.83%), Menthyl acetate (2.87%), Longifolene (1.79%), Isopulegol (1.64%) and Pulegone (1.43%). Repellent testing of M. haplocalyx essential oil against Ae. aegypti indicated its excellent protection at greater doses of 20µl, 10µl and moderate protection at 5µl and 2.5µl. In contrast, the protection time was signi cantly reduced at the lowest dose 1.25µl. For all the tested groups, the repellent activity gradually increased with increasing concentrations (Fig. 4). Therefore, the essential oil of M. haplocalyx may prove useful in the development of mosquito repellents as an effective personal protection measure against mosquito bites. Antioxidant enzyme activities and oxidative damage parameter Compared to control group, we have noted the signi cant rise of MDA content in the larvae after parasitized by R. wuchangensis for 1 (p<0.01), 3 (p<0.05), and 5 (p<0.05) days (Fig. 5A). The MDA content in the larvae also signi cantly increased (p<0.01) after treated by temephos for 12 hours (Fig. 5B). A marked (p<0.05) elevation of SOD activity in Ae. Aegypti larvae was recorded when they were parasitized by R. wuchangensis for 1 and 5 days. However, the SOD activity was signi cantly (p<0.01) lower than the control group when they were parasitized by R. wuchangensis after 3 days (Fig. 6A). For temephos, SOD activity in Ae. Aegypti larvae was signi cantly increased (p<0.05) after treated for 24 hours (Fig. 6B). Besides, the POD activity was signi cantly enhanced in Ae. Aegypti larvae when parasitized by R. wuchangensis after 1 (p<0.05) and 5 (p<0.01) days, but no difference after 3 days (Fig. 7A). Temephos did not affect POD activity in Ae. Aegypti larvae (Fig. 7B). And there was no signi cant difference in CAT activity of Ae. Aegypti larvae, when parasitized by R. wuchangensis after 1 and 3 days, but after 5 days, there was also a signi cant (p<0.01) decrease in the treatment group (Fig. 8A). However, there was no signi cant difference of CAT activity in Ae. aegypti larvae after treated by temephos (Fig. 8B).

Real-time quantitative PCR analysis of antioxidant enzyme genes
Fluorescent quantitative PCR was used to detect the expression of antioxidant enzyme genes in different periods after Ae. aegypti larvae infected by nematodes. The real-time PCR results indicated that the expression levels of SOD, POD and CAT genes in the experimental group were up-regulated after parasitism (Fig. 9). Moreover, the expression levels of Ae. aegypti SOD gene in the experimental groups were signi cantly up-regulated at day 1, day 3 and day 5 (Fig. 9A) in each stage of nematode parasitization. These results suggesting that SOD, CAT and POD genes may play important roles in the resistance of Ae. aegypti to nematode invasion.
Puri cation of recombinant AaegOBP1 and uorescence competitive binding activity The AaegOBP1 gene was successfully expressed in E. coli prokaryotic expression system after IPTG induction and puri ed by Ni-NTA resin a nity chromatography after ultrasonication. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that the AaegOBP1 protein was mainly expressed in supernatant (Fig. 10). The uorescence competitive binding results demonstrated that AaegOBP1 protein could bind ve ligand volatiles from M. haplocalyx essential oil, highlighting their great binding ability (Fig. 11). The AaegOBP1 protein showed the best binding a nity to Menthol and Pinene, with Ki values (the calculated inhibition constants) of 13.19 µM and 14.55 µM (  (Table 6).

Discussion
Dengue is one of the most common vector-borne diseases and has caused substantial health, society and economic burdens to all stakeholders. Ae. aegypti is a major vector for dengue, yellow fever, and chikungunya viruses, these diseases are increasing global public health concerns due to their rapid geographical spread and increasing disease burden [33][34][35] . With abundant rainfall, suitable for Aedes breeding, and frequent foreign trade, the highest dengue fever cases were reported in Guangdong and Yunnan province in southern areas of China. These two provinces keep close connections with South East Asian countries that are in the DF-endemic regions 36 . At present, chemical insecticides are still the main method to control mosquitoes, followed by insect growth regulators, plant essential oils and biological insecticides. Therefore, the development of effective and eco-friendly mosquito control methods is required in to minimize the negative effects of currently marketed insecticides, including multidrug resistance, environment pollution, killing non-target organisms, etc 37 . In this study, we compare the e cacy of several commonly used biological control agents and chemical insecticides on Ae. Aegypti larvae.
In addition, R. wuchangensis is also an important biological control agent and can speci cally parasitize Aedes and Culex mosquito larvae, which is harmless to non-target organisms and environment. At the ratio of 1:10, the mortality rate of the mosquito larvae can exceed 95%. Ayaba et al. found that 100% L1 stage of An. gambiae larvae died within 24 hours after Romanomermis iyengari infection, and 100% of both L2 and L3 stage of larvae died within 7 days 38 . Our laboratory nematode R. wuchangensis also showed similar lethal e ciency to the R. iyengari against mosquito larvae. Except for nematodes, Bacillus thuringiensis (Bti) as an important microbial insecticide were also used to control mosquito larvae in this study. Actually, all mosquito populations were fully susceptible to Bti, which was currently used both in Brazil and Switzerland 39 . This kind of biological insecticide was only partially used in China because of its high cost. Moreover, it's worth our attention that long term use of Bti was subject to the development of resistance to Bti toxins 40 . Expect for Bti, there were entomopathogenic fungi that produced infective spores (conidia) could attach and penetrate the cuticle of mosquitoes, which released toxins that result in mosquito death 41 . Natural enemies feeding on mosquito larvae and pupae also could play important roles in reducing mosquito populations, such as sh, omnivorous copepods, Several species of copepods, etc 42 .
In addition to biological control agents, chemical repellent agents and insecticides are also used to control Aedes aegypti, Aedes albopictus and other mosquito pests in the eld. Temephos is the most widely used larvicide for mosquito control, which is often applied to prevent Ae. Aegypti larvae in the household containers 43 . Based on present result, the LC 50 was 0.009371 mg/L, and all of the larvae were dead as the concentration of 0.032 mg/L, suggesting that Ae. aegypti larvae are susceptible to temephos and it can be used to quickly control an outbreak of Ae. aegypti larvae. Similar to this study, in Argentina and French, the LC 50 of temephos to different regions of Ae. Aegypti populations were between 0.00294 mg/L-0.01017 mg/L 44 and 0.008 mg/L-0.265 mg/L 45 . Moreover, the increased MDA content and decreased activities of antioxidative enzymes also suggested that the consumption of ROS depleted and inhibited the antioxidative enzymes activities, leading to increased Ae. aegypti larvae mortality. Present results demonstrated that the e cacy of temephos and pyrethroids were much higher than that of propoxur, highlighting that these highly effective and less toxic chemical insecticides can be used to control Aedes mosquitoes in different areas of the world.
In addition to control of mosquito larvae, different concentrations of plant essential oil can obviously repel adult mosquitoes, which is helpful for the prevention of mosquito pests. In present study, we also further explore the olfactory protein (OBP1) of Ae. aegypti and performed uorescence competitive binding experiments with ligand compounds with higher content in plant essential oil. Fluorescence competitive binding analysis suggested that AaegOBP1 protein have strong binding ability to Menthol, Pinene, Limonene, Isopulegol and Pulegone from Mentha haplocalyx. These results indicated that AaegOBP1 protein could speci cally distinguish the volatile odor molecules of plant essential oil through the recognition of different ligand compounds, involving in the olfactory repellent behavior of adult Ae. aegypti. Shi et al. reported that AsinOBP1 protein of Anopheles sinensis can bind Diethyltoluamide (DEET), which provides important reference value for further exploring the olfactory mechanism of mosquito pests 46 . In the subsequent experiments, protein homology modeling, molecular docking and site-directed mutation analysis of AaegOBP1 protein will also be used to further con rm repellent molecular mechanism of Ae. aegypti.
In conclusion, we assume that parasitism has the potential to generate oxidative stress in Ae. Aegypti larvae. During the infection, oxidative stress may be a metabolic adaptation and induce oxidative stress in host insects' antioxidant response, thus improving the survival ability of insects to infection. However, when the balance is destroyed, the oxidative damage caused by ROS will increase the mortality of infected insects. With the widespread use of insecticides, the problem of insecticide resistance is becoming increasingly prominent. We recommend that multiple insecticides should be mixed-use and used in rotation, to slowing down the insecticide resistance. Obviously, the nematode R. wuchangensis is an e cient and environmentally friendly biological larvicide, highlighting it may play an important role in the prevention and control of many mosquito pests in the future.