Control of Aedes albopictus Mosquito Larvae with Carpesium abrotanoides L.

Using the Carpesium abrotanoides L. plant for mosquito larvae control can effectively reduce the Aedes albopictus mosquito population and provide a foundation for designing plant-derived insecticides.

As a vital vector of dengue fever, yellow fever, and other mosquito-borne diseases, Aedes albopictus (Diptera: Culicidae) can be broadly distributed worldwide and cause a severe threat to public health. To date, considering the fast-emerging insecticide resistance in the mosquito, the development of new botanical insecticides to control and reduce Ae. albopictus is urgent and crucial. Therefore, to investigate the decoction effect of the plant C. abrotanoides L. on mosquito larvae killing, we detected the mortality of larvae after treatment with different concentrations (60 mg/mL, 120 mg/mL, and 180 mg/mL) of decoction within a series of time points (12 h, 24 h, 36 h, and 48 h). We found that 24 h with 180 mg/mL C. abrotanoides L. decoction treatment killed 92.35% of mosquitoes relative to the control treatment. Meanwhile, 36 h with 120 mg/mL could also kill more than 90% of mosquitoes. Furthermore, Carassius auratus populations were exposed to 120 mg/mL C. abrotanoides L. decoction for 36 h and were not dead. The mortality evaluation indicated that this concentration is not a harmful level of ecological environmental pollution. This study provides a possible plant candidate that could be used for designing plant-derived insecticides. Additionally, these methods can be altered and applied to other mosquito species.

Aedes albopictus, also known as "Asian tiger mosquito", can spread a variety of diseases, such as dengue fever, chikungunya fever, and Zika virus disease, by sucking human and animal blood¹. Due to the wide distribution of Ae. albopictus, the epidemic situation of mosquito-borne diseases such as dengue fever has become increasingly serious in recent years, posing a severe threat to the life and health of people worldwide². At present, for most mosquito-borne diseases, there is no effective vaccine or specific therapeutic drug. Killing mosquitoes with chemical insecticides is still the main means of controlling mosquito-borne diseases³. The chemical control method using chemical insecticides can kill mosquitoes quickly and efficiently and has become the main means of mosquito vector control⁴. However, the long-term and large-scale use of insecticides has led to a decline in the sensitivity of Ae. albopictus to insecticides and insecticide resistance, which has become the greatest obstacle to controlling mosquito-borne diseases. Therefore, it is of great practical significance to develop a new type of mosquito insecticide with high efficiency, safety, and environmental protection.

In nature, plants are the primary producers. Insects and many animals eat plants. When plants suffer from various "natural and man-made disasters", they produce secondary metabolites to survive. These substances often have the ability to resist other organisms' feeding, disease, and insect resistance. They not only have effects on a variety of pests but also have a low risk of environmental toxicity⁵. Carpesium abrotanoides L. is a perennial herb of the Carpesium abrotanoides genus of Compositae, also known as "toad blue", "deer living grass", "wild tobacco", etc. It is widely distributed in China and East Asia. Its stems and leaves can be used as insecticides in these areas to treat abrasions and fever⁶. Its fruit is locally known as "Bei-He-Shi" in China and is used to treat tapeworm and Ascaris lumbricoides in folk medicine^7,8. It has been reported that the plant is rich in monoterpenes, sesquiterpenes, phenols, and other characteristic components and has effective pharmacological effects, such as anti-inflammatory, anti-fungal, anti-parasitic, antitumor, and antiviral effects⁷. Recent studies have found that it has an antifeedant effect on Spodoptera exigua⁹, contact toxicity to Sitophilus zeamais¹⁰, killing ability for cysticercus cellulosae of Taenia asiatica⁸, antifeedant activity, and contact toxicity to armyworm and Plutella xylostella¹¹. Preliminary progress has been made in studying the toxic effects of C. abrotanoides L. on some parasites, agricultural pests, and sanitary pests, which can be used to control the larvae of Aedes albopictus.

This study discusses the protocol for controlling Ae. albopictus larvae with C. abrotanoides L. In this protocol, C. abrotanoides L. decoction was used to act on the fourth instar larvae of Ae. albopictus, and larval death was detected after treatment with C. abrotanoides L. decoction at different concentrations (60 mg/mL, 120 mg/mL, and 180 mg/mL) at a series of time points (12 h, 24 h, 36 h, and 48 h). Determining the killing effect of C. abrotanoides L. decoction on Ae. albopictus larvae provide a new idea for further mosquito control with high efficiency, low toxicity, and the use of environmentally friendly botanical insecticides.

The goat blood used to feed female mosquitoes was collected from a local abattoir in Duyun City, Guizhou, China, and used following the ethical guidelines and regulations of the Key Laboratory of Human Parasitic Diseases in Qiannan Prefecture, Duyun, Guizhou, China.

1. Preparation of reagents

NOTE: Refer to the Table of Materials for a list of equipment, reagents, and other consumables used in this protocol.

1. Extraction and identification of C. abrotanoides L.
   1. Collect the whole grass of C. abrotanoides L. Whole grass of C. abrotanoides L. in this study was collected from Sandu Shui Autonomous County, Guizhou Province (N: 25.95, E: 107.87) in April 2021.The photos from the collection site for C. abrotanoides L. in Figure 1.
      NOTE: According to the resource distribution, medicinal properties, tissue structure, and physical and chemical identification characteristics of C. abrotanoides L., the identification of C. abrotanoides L. was as follows: The whole plant is 30-100 cm high, with upright stems, many branches in the upper part, alternate leaves in the lower part of the stem, slightly petiolate, oval leaves, 10-15 cm long and 5-8 cm wide, full or irregularly serrated, leaves in the upper part of the stem are nearly sessile, long oval, gradually smaller upwards, and the head is axillary, nearly sessile, with drooping flowers, special gas, light taste and pungent. The plant was identified as C. abrotanoides L. by teachers specializing in traditional Chinese medicine at our affiliations.
2. C. abrotanoides L. cleaning, drying, and crushing
   1. Remove the impurities such as dead leaves and soil from the collected C. abrotanoides L. and clean it. Air-dry it for 10-15 days under ventilated conditions or dry it in a dry oven at 95 °C for 6 h.
      NOTE: During the drying process of C. abrotanoides L., it is necessary to turn it over so that it can dry evenly. If C. abrotanoides L. tissue is not easily crushed during the crushing process, it can be dried again and then crushed.
3. Preparation of C. abrotanoides L. essence decoction
   1. Weigh 360 g of C. abrotanoides L. refined powder, add 1000 mL of distilled water, and decoct with slow fire (power 800 W) for 2 h.
   2. Filter the decoction with ordinary filter paper and then concentrate in a rotary evaporator to make an extractum. Dilute the extractum with distilled water to 1000 mL, and prepare a C. abrotanoides L.refined water decoction containing 360 mg/mL of the original medicinal material.
   3. Take 50 mL, 100 mL, and 150 mL of C. abrotanoides L. refined water decoction and dilute them to prepare water decoctions containing 120 mg/mL, 240 mg/mL, and 360 mg/mL of the original medicinal materials, 150 mL each for standby.
      ​NOTE: During the boiling process, the heat should be well controlled. After boiling, the heat was reduced, and the liquid was kept at 100 °C and slightly boiled.

2. Mosquito sample preparation

1. Source of mosquitoes
   1. Collection of Ae. albopictus larvae. In this study, the Ae. albopictus larvae were collected in the residential area of Duyun City (N: 26.25, E: 107.52), Guizhou Province, in August 2020.
   2. According to the distribution, larval and adult morphological characteristics of Ae. albopictus, the identification of Aedes albopictus was as follows: The base of Ctenophora of Aedes albopictus larvae has a finedraw; incomplete tail saddle; the breathing tube is black, short, and thick; the adults of Aedes albopictus are small- to medium-sized mosquitoes. The female mosquito is medium-sized and black. The midthoracic scutellum has a white longitudinal stripe, usually tapering gradually at the rear and forking in the anterior area of the small scutellum. The foot joint has a base white ring, and all or most of joint 5 is white. The mosquitoes were identified as Ae. albopictus by teachers of Parasitology and mosquito specialty at our affiliations.
   3. Maintain conventional feeding at a temperature of 25 ± 1 °C and relative humidity of 70% ± 10%, and use 80 W fluorescent lamp as the light source (light: dark = 14 h:10 h).
   4. Feed the larvae with chicken liver powder, and adult mosquitoes with 10% sugar water.
2. Mosquito eggs collection
   1. When the larvae of Ae. albopictus had grown into pupae, suck the pupae out using a disposable plastic Pasteur pipette and transfer them to the adult mosquito cage.
   2. After 3-5 days of adult mosquito emergence, the female and male mosquitoes mate. At this time, feed the female mosquitoes with blood for 4-8 h.
      NOTE: The blood added with anticoagulant was put into a culture dish, heated with 37 °C warm water, and covered with a layer of gauze cloth for female mosquitoes to suck blood.
   3. Collect the eggs of Aedes albopictus using an egg collecting cup (homemade: 300 mL straight-sided opaque plastic cup containing 250 mL of distilled water, and then the filter paper was immersed halfway around the inner edge of the cup).
      NOTE: The eggs of Ae. albopictus can be stored for 3-6 months.
3. Mosquito hatching and feeding
   1. During the experiment, Ae. albopictus usually hatched 1 week in advance. Place the eggs of Aedes albopictus on an enamel plate, add 1000 mL of tap water, and allow it to stand for more than 24 h. The eggs hatch into larvae in 1-2 days.
   2. The larvae molted once every 1-2 days, that is, increased one instar. Select 1000 larvae at the end of the third instar or the beginning of the fourth instar after breeding for 2-3 generations without contacting any chemical insecticides in the laboratory for the test.
      ​NOTE: If the density of larvae is too high, divide them into plates to allow the larvae to grow normally.

3. Detection of the toxic effect of C. abrotanoides L. decoction on Ae. albopictus larvae by the dipping method

1. Set grouping
   1. Set the experimental group: Prepare C. abrotanoides L. decoctions (50 mL) with three drug concentrations (120 mg/mL, 240 mg/mL and 360 mg/mL).
   2. Blank control group: Use dechlorinated water (homemade: tap water standing for more than 24 h) with the same volume as the drug.
   3. Positive control group: Prepare the drug deltamethrin, a common chemical insecticide, at a final concentration of 0.05 mg/mL.
2. Adding liquid medicine
   1. Use several disposable plastic cups with a volume greater than 200 mL, and add 35 Ae. albopictus larvae at the end of the third or the beginning of the fourth instar to 50 mL of dechlorinated water.
   2. In the experimental group, add 50 mL of C. abrotanoides L. refined water decoction of each concentration, shake gently and mix evenly for a total volume of 100 mL. The final concentrations of the C. abrotanoides L. refined water decoction are 60 mg/mL, 120 mg/mL, and 180 mg/mL.
   3. In the blank control group, add 50 mL of dechlorinated water to a total volume of 100 mL and mark it as blank control.
   4. In the positive control group, add 50 mL of deltamethrin solution with a concentration of 0.1 mg/mL, shake gently and mix evenly for a total volume of 100 mL to a final concentration of 0.05 mg/mL and mark it as positive control.
3. Observations
   1. Gently transfer the marked plastic cups to the observation room with the same feeding environment, and record the deaths of Ae. albopictus larvae in each experimental and control group at 12 h, 24 h, 36 h, and 48 h, respectively. Observe the dead individuals under the microscope and compare them with the normal Ae. albopictus larvae.
   2. At the above observation time point, if the larvae were stirred with a sharp fine needle and could not escape or had no response, consider them as dead. Repeat the test three times.
      ​NOTE: If the mortality of larvae in the control group exceeded 20%, the test results were invalid and needed to be repeated. The overall approach is summarized in Figure 2.

4. Outdoor simulation experiment

1. Apply the three concentrations of C. abrotanoides L. decoction to Ae. albopictus larvae under outdoor conditions to observe larval death. Compare whether there is any difference between the toxic effect and that in the laboratory.
2. Apply the three concentrations of C. abrotanoides L. refined water decoction to wild crucian carp under outdoor conditions to observe whether it impacted the growth of crucian carp. This is to preliminarily judge whether the C. abrotanoides L.refined water decoction causes damage to the ecological environment.
   ​NOTE: The concentrations were similar to the concentrations mentioned in step 3.2.2. The time points used are 12 h, 24 h, 36 h, and 48 h, respectively. The effect of the field simulation experiment was judged by comparing the mortality of Ae. albopictus treated with C. abrotanoides L. decoction under laboratory feeding conditions and outdoor natural conditions.

5. Statistical analysis

1. Express the quantitative data in Mean ± SD and the count data as percentages (%).
   NOTE: The statistical inference method used here was p < 0.05, and the difference was statistically significant.
2. Use appropriate software for statistical analysis of the data and the toxicities of three different concentrations of C. abrotanoides L. decoction to Ae. albopictus larvae were used to preliminarily determine which concentration of decoction had the best toxicity to Ae. albopictus larvae.

Here, whole grass of C. abrotanoides L. in this study was collected from the wild (Figure 1). After the identification of C. abrotanoides L., the decoction of C. abrotanoides L. was obtained by the decoction method (Figure 2) and prepared in different concentrations (60 mg/mL, 120 mg/mL, and 180 mg/mL). It was applied to Ae. albopictus larvae by the larval dipping method. It was found that 24 h with 120 mg/mL C. abrotanoides L. decoction treatment could make dead larvae black, and stiffness was observed through morphological assessment (Figure 3). Then, the mortality of larvae was detected after treatment with different concentrations (60 mg/mL, 120 mg/mL, and 180 mg/mL) of decoction within a series of time points (12 h, 24 h, 36 h, and 48 h). It was found that 36 h of exposure to 120 mg/mL could kill more than 90% of mosquitoes. Carassius auratus populations were exposed to 120 mg/mL C. abrotanoides L. decoction for 36 h, and they were not dead. The mortality evaluation indicated that this concentration is not a harmful level of ecological environmental pollution (Figure 4). The median lethal concentrations (LC₅₀) of Ae. albopictus larvae treated with C. abrotanoides L. decoction for 24 h, 36 h, and 48 h were 117.49 mg/mL, 83.09 mg/mL, and 73.34 mg/mL, respectively. Furthermore, the LC₅₀ decreased with prolonged treatment time (Table 1).

Figure 1: The photos from the collection site for C. abrotanoides L.. The whole grass of C. abrotanoides L. in this study was collected from the Sandu Shui Autonomous County, Guizhou Province (N: 25.95, E: 107.87) in April 2021. Please click here to view a larger version of this figure.

Figure 2: Flow chart of C. abrotanoides L. decoction acting on Ae. albopictus larvae. (A) Picking and identification of C. abrotanoides L. and the decoction of C. abrotanoides L. were obtained by the decoction method. (B) Concentrations of C. abrotanoides L. decoction (120 mg/mL, 240 mg/mL, and 360 mg/mL). (C) Ae. albopictus larvae were treated using the larval dipping method and set as a blank control group. Please click here to view a larger version of this figure.

Figure 3: Normal and dead larval morphologies of Ae. albopictus. C. abrotanoides L. decoction (120 mg/mL) was used to act on Ae. albopictus larvae for 24 h. Observed through morphological assessment, the dead larvae were black and appeared stiff. A blank control was set, which did not have any added C. abrotanoides L. decoction. Bar = 10x. Please click here to view a larger version of this figure.

Figure 4: Poisonous effect of C. abrotanoides L. decoction on Ae. albopictus larvae. The poisonous effect on Ae. albopictus larva was examined in the presence of C. abrotanoides L. decoction (60 mg/mL, 120 mg/mL, and 180 mg/mL) at 12 h, 24 h, 36 h, and 48 h. Each experiment was performed in triplicate, and the results represent the mean of three individual experiments. Data are representative of one experiment with at least three independent biological replicates. Data are represented as mean ± SEM, n = 6. The x-axis shows the time (h) when the Ae. albopictus larvae were examined in the presence of C. abrotanoides L. decoction, and the y-axis shows the mortality (%) of the Ae. albopictus. Please click here to view a larger version of this figure.

Table 1: The median lethal concentrations (LC₅₀) of Ae. albopictus larvae treated with C. abrotanoides L. decoction. The median lethal concentrations (LC₅₀) of Aedes albopictus larvae treated with C. abrotanoides L. decoction for 24 h, 36 h, and 48 h were 117.49 mg/mL, 83.09 mg/mL, and 73.34 mg/mL, respectively. Furthermore, the LC₅₀ decreased with prolonged treatment time.

Currently, Ae. albopictus has become one of the 100 most invasive species in the world. According to World Health Organization (WHO) statistics, in 2020, the areas most affected by Ae. albopictus in Asia will account for approximately 70% of the global disease burden¹². As a vital vector of dengue fever, yellow fever, and other mosquito-borne diseases, Ae. albopictus can be broadly distributed worldwide and cause a severe threat to public health. Here, the development of new botanical insecticides to control and reduce Ae. albopictus can provide safer and environmentally friendly options. Therefore, a new method was developed to generate botanical insecticides to investigate the poisoning effect of C. abrotanoides L. on Ae. albopictus larvae to provide a possible plant candidate that could be used for designing plant-derived insecticides.

As a traditional Chinese herbal medicine, C. abrotanoides L. can be used to treat bruises and fever. Its extract material is also friendly to the environment and can be used as a plant-derived insecticide with good safety. It has been demonstrated that the toxicity of C. abrotanoides L. extract to humans was low^13,14. The reason for choosing C. abrotanoides L. decoction is that the first three stages of Ae. albopictus must be in the water for growth and development, which is closely related to its life cycle. The experimental results show that C. abrotanoides L. decoction has a good poisoning effect. This method of application can poison Ae. albopictus larvae and effectively control the Ae. albopictus mosquito population. Therefore, Ae. albopictus larvae were selected for the research objective, which is a representative species. C. abrotanoides L. decoction was chosen for mosquito larvae control to better simulate the ecological environment. Second, the efficacy of the substance itself can be preserved naturally and with no additional modifications. Furthermore, the control group showed that C. abrotanoides L. decoction for Carassius auratus populations was not poisonous and was environmentally friendly.

For the choice of method, several concentrations from low concentration to high concentration (60 mg/mL, 120 mg/mL, and 180 mg/mL) were set by multiplying the initial chosen concentration. A more suitable poison concentration may be found in experiments in the field. In addition, according to relevant literature, a series of time points (12 h, 24 h, 36 h, and 48 h) were chosen. However, for the poisoning of Ae. albopictus larvae in the wild, the time may need to be extended to better observe the poisoning effect. The poisoning effect of C. abrotanoides L. on mortality can be judged by mosquito larvae and the number of adult eclosions.

In addition to the C. abrotanoides L. decoction, methanol, ethyl acetate, and petroleum ether can be used to extract the active compounds of C. abrotanoides L. Methanol is a hydrophilic organic solvent, and ethyl acetate and petroleum ether are lipophilic organic solvents. The types of effective compounds extracted by each of these solvents are different. Methanol mainly extracts alkaloids, ethyl acetate extracts flavonoids, and petroleum ether extracts volatile oils and sesquiterpenes. For example, relevant literature reports that plant-derived sesquiterpenoids have effective anti-inflammatory, anti-parasitic, antitumor and other drug activities^15,16,17,18. The sesquiterpenoids isolated from C. abrotanoides L. have strong antimalarial activity against the Plasmodium falciparum D10 strain, and their activity against parasites is more than ten times that of mammalian cell lines, so they have the potential for large anti-parasitic applications and broad application prospects¹⁹. Terpenoids have rich structural types and diverse biological activities and have important application value for the research and development of new medicines^20,21. The latest research has found that Carpesium abrotanoides essential oil and its main components can protect against a dengue vector, Ae. aegypti (Diptera: Culicidae). The essential oil (EO) and its main components have mosquito repellent potential²². Another study also found that EOs had a concentration-dependent larvicidal effect on Ae. aegypti larvae. Aromatic compounds found in EOs triggered a significant response to mortality. EOs can be used as a green pesticide to effectively control vector-borne diseases²³. These findings provide new ideas for follow-up research.

Here, the results provide a possible plant candidate that could be used for designing plant-derived insecticides. Regarding the limitations of this method, first, only the toxic killing effect of water extract on Ae. albopictus has been verified, which can be used to control the number of Ae. albopictus larvae. More work will be needed to develop commercial mosquito control products. Second, in the process of preparation of C. abrotanoides L. essence decoction, some active components in C. abrotanoides L. may be lost with the volatilization of water vapor, which may be the reason why the mortality observed in this experiment was lower than in the actual situation. Finally, the popularization and application of this method are still in the stage of small-scale experiments. If this method of controlling Ae. albopictus can be demonstrated to be highly reliable; it will be applied to a wider range and other mosquito species.

The authors have no conflicts of interest to declare.

We thank Dr. Xin-Ru Wang from University of Minnesota, for insightful suggestions and guidance. This work was supported by the scientific research fund of Qiannan Medical College for Nationalities (Qnyz202112, QNYZ202205), and Science and Technology Fund of Guizhou Provincial Health Commission (qwjh [2022] No. 101, project gzwkj2023-251).