1. Introduction
Aedes aegypti (Linnaeus 1762) (Diptera: Culicidae) is considered an important vector regarding public health, as it transmits several arboviruses that affect humans. These arboviruses include the Dengue virus, and
Ae. aegypti is an important vector for urban Yellow fever [
1,
2]. This mosquito is also a vector for the Chikungunya virus (CHIKV)—the most common symptoms are fever and characteristically severe joint pain—and the Zika virus (ZIKV)—which has been associated with cases of microcephaly among newborns from women infected with this virus, along with other neurological disorders such as the Guillain–Barré syndrome [
3,
4]. The prevalence of these diseases has been increasing across the world where
Ae. aegypti is endemic, covering tropical and subtropical areas [
5]. About half of the world’s population is now at risk of Dengue with an estimated 100–400 million infections occurring each year in more than 100 countries [
2].
Vector control programs play an important role in the prevention of mosquito-borne diseases [
6]. Therefore, efforts to control the population of the mosquito
Ae. aegypti are essential for preventing outbreaks of the Dengue, Zika and Chikungunya viruses [
6,
7]. Current interventions include the elimination of mosquito larvae by applying larvicides to larval breeding sites. In general, the main larvicidal agents are based on organophosphate chemicals and bacterial agents [
8]. These agents have undesirable effects, such as toxicity to the environment and to non-target organisms, including humans. [
9]. Faced with these challenges, there has been a quest for safer and more sustainable alternatives for arboviral vector control.
One effective intervention is the use of natural plant products that can act as insecticides but are environmentally safe [
10,
11]. Plant products have been evaluated for their toxic properties against insects, especially in the form of essential oils [
12]. Essential oils, also known as volatile oils or ethereal oils, are complex mixtures of volatile compounds, lipophilic, generally odoriferous and liquid, originating from the secondary metabolism of plants [
13,
14]. They present acute contact and fumigant toxicity to insects, repellent and antifeedant activities, as well as present development and growth inhibitory activity [
15]. Thus, essential oils have gained attention as potential bioactive agents against
Ae. aegypti, either in their crude form or by means of purified substances [
16]. Essential oils are mainly composed of terpenoids, particularly monoterpenes and sesquiterpenes [
13], and they are known to exhibit many activities; for example, they act as nematicides [
17], leishmanicides [
18] and insecticides [
19]. In addition, they have been widely used for medicinal and cosmetic applications in the pharmaceutical, sanitary, cosmetic, agricultural and food industries [
20]. Some oils, such as thymol and carvacrol, are food flavorings that are generally recognized safe (GRAS) and are an indication of low mammalian toxicity for starting materials [
21]. In view of these facts, it was of our interest to test essential oils’ constituents on
Ae. aegypti. Initially, 27 plant oil constituents were screened, and the most successful ones were focused on this study: dihydrojasmone, p-cymene, carvacrol, thymol, farnesol and nerolidol (unpublished study).
The elucidation of the mode of action of natural products in
Ae. aegypti larvae has a fundamental importance to intensify its effects and for the development of a larvicide. As a tool for this, morphological studies help to understand the toxic effects and possible mechanisms of action [
22,
23,
24]. Thus, the present study aimed to characterize the larvicidal effects of dihydrojasmone, p-cymene, carvacrol, thymol, farnesol and nerolidol on third-stage
Ae. aegypti larvae and their effects on the morphology of the mosquito using electron transmission microscopy.
4. Discussion
The emergence of many arboviral diseases transmitted by
Ae. aegypti and their capacity to resist synthetic chemical insecticides has increased the interest in exploring new products against this mosquito. Furthermore, extensive usage of synthetic insecticides has caused risks to human health, animals and the environment [
27].
Plants are an important and rich source of bioactive chemical compounds that can act effectively towards controlling
Ae. aegypti, with lower impacts on human health and the environment [
10,
11].
According to the results from the present study, dihydrojasmone presented high efficacy regarding larvicide activity (LC50 = 66 µg/mL) against Ae. aegypti, with a duration of 1 to 6 days after the initial application. It is important to show that among the essential oils’ constituents of this study, dihydrojasmone showed the greatest delay in larval development. It should be noted that until now, there had not been any reports in the literature regarding the activity of dihydrojasmone against Ae. aegypti larvae.
In relation to the monoterpene p-cymene, with an LC
50 of 23 μg/mL. These data demonstrate its larvicidal potential in comparison with p-cymene from
Clausena excavata, which presented LC
50 = 43.3 μg/mL against fourth-stage larvae (L4) of
Ae. aegypti and LC
50 = 34.9 μg/mL against fourth-stage larvae (L4) of
Aedes albopictus [
28].
According to Govindarajan et al. (2016) [
29], carvacrol was seen to have larvicidal activity against different species of mosquitos, including
Anopheles stephensi (LC
50 = 21.15 μg/mL),
Anopheles subpictus (LC
50 = 24.06 μg/mL),
Culex tritaeniorhynchus (LC
50 = 27.95 μg/mL) and
Culex quinquefasciatus (LC
50 = 26.08 μg/mL). Tang et al. (2011) [
30] found similar results for carvacrol against
Aphis craccivora and
Leucania separata at median lethal concentrations (LC
50) of 16.8 and 12.7 μg/mL, respectively. In the present study, carvacrol presented a higher LC
50 than those mentioned previously (LC
50 = 40 μg/mL). However, in leaf immersion assays, the action found against
Pochazia shantungensis nymphs reached LC
50 = 56.74 μg/mL [
31].
The larvicide activity of thymol (LC
50 = 45 μg/mL) found in the present study corroborates what was seen in the study by Waliwitiya et al. (2009) [
32] regarding
Ae. aegypti larvae. It has also been reported that thymol has toxic activity against
Spodoptera litura [
33],
Musca domestica [
34],
Drosophila melanogaster,
P. shantungensis [
29] and the mosquito
C. quinquefasciatus [
35]. Testing of this monoterpene against the larvae of
An. subpictus,
Aedes albopictus and
C. tritaeniorhynchus mosquitoes gave rise to LC
50 of 22.06, 24.83 and 28.19 μg/mL, respectively [
36].
The bioassays with farnesol reported LC
50 = 21 μg/mL for larvicide activity, while Simas et al. (2004) [
37] reported the same activity over
Ae. aegypti with LC
50 = 13 μg/mL. In the study by Park et al. (2020) [
38], farnesol showed mosquito larvicidal activities and caused retardation of ovarian growth of female
Ae. albopictus by modulating the formation of the JH receptor complex.
The sesquiterpene nerolidol showed high efficiency, with LC
50 = 11 μg/mL. This result agrees with what was found in the study by Simas (2004) [
37], who reported LC
50 = 17 μg/mL for
Ae. aegypti. Larvicidal activity caused by nerolidol was also reported by Chantraine et al. (1998) [
39] and Ali et al. (2013) [
40] but with LC
50 = 9 μg/mL and 13.4 μg/mL, respectively. These values were lower than those observed in the present study and in the study by Simas et al. (2004) [
37]. Nerolidol isolated from the seeds of
Magnolia denudata also showed larvicidal activity against the larvae of
Culex pipiens pallens,
Ae. aegypti,
Ae. albopictus and
Anopheles sinensis [
41].
According to the classification by Cheng et al. (2003) [
42], the essential oils’ constituents evaluated in the present study, namely farnesol (LC
50 = 21 µg/mL), p-cymene (LC
50 = 23 µg/mL), nerolidol (LC
50 = 11 µg/mL), thymol (LC
50 = 45 µg/mL) and carvacrol (LC
50 = 40 µg/mL) are highly active substances for controlling
Ae. aegypti, since they presented an LC
50 < 50 μg/mL, while dihydrojasmone (LC
50 = 66 µg/mL) is merely active since it presented an LC
50 < 100 μg/mL.
Essential oils frequently show a broad spectrum of bioactivity in relation to the development of insects of importance for public health and agriculture. Their lipophilic nature facilitates their interference in the basic metabolic, biochemical, physiological and behavioral functions of insects [
43]. Essential oils can interfere with the feeding behavior of arthropods, act as a growth regulator or act as a neurotoxin, among the toxicity mechanisms; they can also act as protein denaturants and enzyme inhibitors, in addition to promoting the disintegration of the plasma membrane [
44,
45]. These diverse mechanisms of action contribute to preventing the emergence of resistant vectors. Among the various biological parameters of the effects of essential oils and their constituents on insects, the present study showed the interference of the p-cymene, carvacrol, thymol, farnesol and nerolidol, using electron microscopy, over the morphology of the third-stage larvae (L3).
The larvae treated with the different essential oils’ constituents presented alterations to the external wall of the tegument, thus demonstrating that these substances act directly on the cuticle.
In addition to this aspect, the ultrastructure of the treated larvae also showed alterations to the midgut, with signs of cell destruction and vacuolization of epithelial cells. These signs were indicative of cell disorganization, with spaces between cells and an accumulation of granules in some places in the cytoplasm. There were also pale nuclei, which are characteristic of nuclear degeneration. The microvilli presented an altered appearance, which could hinder food absorption and thus directly affect the nutrition of the larvae. Swollen or destroyed mitochondria hinder ion transportation, which is an important function of epithelial cells. Narciso et al. (2014) [
23] evaluated the morphological effect of the neolignan burchellin, isolated from leaves of
Ocotea cymbarum (Lauraceae), and presented cell disorganization and destruction in the midgut region, spaces between cells and vacuolization of epithelial cells. Maleck et al. (2014) [
24] also reported the cytotoxic effect of the amide piperlonguminine, isolated from
Piper tuberculatum and
Piper acutifolium, on the epithelial cells of the digestive system of
Ae. aegypti. That study showed that vacuolization of the cytoplasm and mitochondrial edema occurred.
The severe vacuolization of epithelial cells and the presence of myelin figures indicated cell distress, suggesting the toxicity of essential oils on mosquito larvae.
The microscopic study conducted by Seye et al. (2021) [
46] revealed that the oil of
Cymbopogon citratus (Lemongrass) acts by causing cell destruction in the larval tissue of
Ae. aegypti mosquitoes. The study observed significant damage in various cellular structures, including the intestinal microvilli, mitochondria, genetic material, and the body fat mass. According to Cantrell et al. (2010) [
47] larvicidal substances can be absorbed through the cuticle of insects, through the respiratory tract, or even through ingestion. Inside the larvae, these substances can reach their site of action or cause systemic effects by means of diffusion in different tissues [
48].