Momordica charantia L. extracts against Aedes aegypti larvae Author Mituiassu, L. M. P. Universidade de Vassouras, Laboratório de Insetos Vetores, Vassouras, RJ, Brasil & Universidade de Vassouras, Mestrado Profissional em Ciências Ambientais, Vassouras, RJ, Brasil Author Serdeiro, M. T. Universidade de Vassouras, Laboratório de Insetos Vetores, Vassouras, RJ, Brasil & Fundação Oswaldo Cruz - FIOCRUZ, Instituto Oswaldo Cruz - IOC, Laboratório Interdisciplinar de Vigilância Entomológica em Diptera e Hemiptera, Rio de Janeiro, RJ, Brasil Author Vieira, R. R. B. T. Fundação Educacional Dom André Arcoverde, Faculdade de Medicina de Valença, Centro de Ensino Superior de Valença, Valença, RJ, Brasil Author Oliveira, L. S. Universidade de Vassouras, Pró-Reitoria de Ciências da Saúde, Vassouras, RJ, Brasil Author Maleck, M. Universidade de Vassouras, Laboratório de Insetos Vetores, Vassouras, RJ, Brasil & Universidade de Vassouras, Mestrado Profissional em Ciências Ambientais, Vassouras, RJ, Brasil & Fundação Oswaldo Cruz - FIOCRUZ, Instituto Oswaldo Cruz - IOC, Laboratório de Entomologia Médica e Forense, Rio de Janeiro, RJ, Brasil text Brazilian Journal of Biology 2022 e 236498 2022-12-31 82 1 6 http://dx.doi.org/10.1590/1519-6984.236498 journal article 10.1590/1519-6984.236498 1678-4375 11552757 Extraction from the flowers and fruits (EFF) of M. charantia yielded 330 mg of AcOEt extract, 290 mg of MeOH extract and 42 mg of HEX extract. The AcOEt extract used at a concentration of 100 µg/mL demonstrated behavioral alterations among larvae,with low mobility and lethargy within 24 hours of larval treatment (L3).Concerning the mosquito development cycle, the larval period was reduced by four days (7±1.2 days; P<0.0001) at a concentration of 100 µg/mL when compared with the testimony control group (11±2 days). In turn, the L3-adult period was reduced by three days (10±1.8 days; P<0.001) (as shown in Table 1 (1A)) compared with the testimony control group (13±1.9 days). The same extract presented 96.7% (P<0.001) and 86.7% (P<0.001) larval mortality (L3) at concentrations of 200 µg/mL and 100 µg/mL, respectively, for up to 48 hours of treatment (as shown in Table 1 (1B)). This extract revealed an LC 50 value of 37.2 µg/mL. In addition, bioassays with the AcOEt extract demonstrated larval viability (L3-L4) of only 3.3% (200 µg/mL) and 13.3% (100 µg/mL). At lower concentrations, the AcOEt extract at 50 µg/mL reduced the larval period by four days (8.1±1.7 days; P<0.0001) as compared with the control testimony (12.5±3.1 days) (as shown in Table 2 (2A)). The time required for L3-to-adult development showed a reduction at concentrations of 10µg/mL (12.9±2.8 days; P<0.1) and 50 µg/mL (10.8±1.9 days; P<0.0001), in relation to the control testimony (15.3±3.3 days) (as shown in Table 2 (2A)). The same extract showed low mortality among L 3 larvae at concentrations of 1 µg/mL and 10 µg/mL (6.7% and 10%), respectively (as shown inTable 2 (2B)). Similarly, mortality was low in the case of L 4 larvae (10 and 5%) (as shown in Table 2 (2B)). At the concentration of 50 µg/mL, the extract presented moderate mortality for L3 (40%) (P<0.01), but low mortality for L4 and pupae (13.3% and 3.5%), respectively (as shown in Table 2 (2B)). Regarding the larval development period, the MeOH extract at concentration of 200 µg/mL (8.5 ± 0.9 days; P<0.0001) reduced the larval period, in comparison with the testimony control (11.8 ± 1.9 days). Besides, the pupal period at concentrations of 100 µg/mL (2.1 ± 0.4 days; P<0.01) and 200 µg/mL (4.5 ± 2.2 days; P<0.0001) were extended when compared with the testimony control group (1.7 ± 0.6 days) (as shown in Table 3 (3A)). Twenty-four hours after treatment, the larvicidal activity of the MeOH extract of M. charantia resulted in low mobility and lethargy among the larvae. Mortality of L 3 larvae reached 70% (P<0.001) at the concentration of 200 µg/mL (as shown in Table 3 (3B)), with 20% viability (P<0.01) for L3-adult development, resulting from larval disintegration and thus reducing the chances of larval emergence. This extract exhibited low toxicity at the concentration of 100µg/mL, with only 1.7% L3 larval mortality, 10% L4 larval mortality, and 3.3% pupal mortality (as shown in Table 3 (3B)). Furthermore, the MeOH extract presented LC 50 = 129.6 µg/mL. Table 1. Duration of development (A) and mortality (B) of Aedes aegypti in L3 larvae treated in a rearing environment with crude ethyl acetate extract (AcOEt) from the flowers and fruits of Momordica charantia .

Application	Larval (days)		Pupal (days)	L3-Adult (days)
1A	X ± SD	R	X ± SD	R	X ± SD	R
Control	12.2±2a	4-15	1.7±0.5a	1-3	13.5±1.8a	5-16
Testimony	11±2b	6-14	2.3±0.7b	1-4	13±1.9ab	9-15
100 µg/mL	7±1.2c****	5-8	2.8±0.9b	2-5	11±1.8c***	8-13
200 µg/mL	10±1.4bc	9-11	3±1.4b	2-4	13±2.8ab	11-15
L3	L4	Pupa
1B
X ± SD	R	%	X ± SD	R	%	X ± SD	R	%
Control	0 ± 0a	0	0	0	0	0	0.3 ± 0.5	13-13	1.7
Testimony	0 ± 0ab	0	0	0	0	0	0	0	0
100 µg/mL	17 ± 1.0c***	2-7	86.7	0	0	0	0	0	0
200 µg/mL	19 ± 1.0d***	2-5	96.7	0	0	0	0	0	0

Experiments with 20 A. aegypti larvae (L3) for each test and control group were performed in triplicate and with three repetitions. Mean and standard deviation (X ±SD).Range (R). Values followed by the same letter do not present significant differences. Significance levels through the Tukey test, represented as ****P<0.0001; ***P<0.0001. Table 2. Duration of development (A) and mortality (B) of Aedes aegypti in L3 larvae treated in a rearing environment with crude ethyl acetate extract (AcOEt) from the flowers and fruits of Momordica charantia .

Application	Larval (days)		Pupal (days)		L3-Adult (days)
2A	X ± SD	R	X ± SD	R	X ± SD	R
Control	11.7±4.8ª	2-23	2.8±0.7a	1-5	14.4±4.9a	5-25
Testimony	12.5±3.1ab	7-19	2.9±0.6ab	2-4	15.3±3.3ab	9-22
1 µg/mL	13.1±4.4ab	5-22	2.4±0.8ac*	1-4	15.7±4.5ab	8-24
10 µg/mL	10±1.9ac**	7-15	3±1.5ab	1-9	12.9±2.8ac*	8-21
50 µg/mL	8.1±1.7c****	5-11	2.6±0.8ab	1-4	10.8±1.9d****	8-14
L3	L4	Pupa
2B
X ± SD	R	%	X ± SD	R	%	X ± SD	R	%
Control	0.3 ± 0.5ª	1-1	3.3	0.3 ± 0.5a	1-1	1.7	0.3 ± 0.5a	1-1	1.7
Testimony	0.3 ± 0.5ab	1-1	1.7	0a	0-0	0	1 ± 0a	1-1	5
1 µg/mL	1 ± 1ab	1-3	6.7	2 ± 2a	1-4	10	0a	0	0
10 µg/mL	2 ± 3ab	1-5	10	1.2 ± 1a	1-2	5	0.3 ± 0.5a	1-1	2
50 µg/mL	8 ± 4c**	3-12	40	3 ± 0.5a	2-3	13.3	0.3 ± 0.5a	1-1	3.5

Experiments with 20 A. aegypti larvae (L3) for each test and control group were performed in triplicate and with three repetitions. Mean and standard deviation (X ±SD).Range (R). Values followed by the same letter do not present significant differences. Significance levels through the Tukey test, represented as ****P<0.0001; **P<0.01; *P<0.1 vs testimony control AcOEt:DMSO (1:3). Table 3. Duration of the development (A) and mortality (B) of Aedes aegypti in L3 larvae treated in a rearing environment with crude methanol (MeOH) extract from the flowers and fruits of Momordica charantia .

Application	Larval (days)		Pupal (days)		L3-Adult (days)
3A	X ± SD	R	X ± SD	R	X ± SD	R
Control	13.2±1.9a	5-16	1.7±0.6a	1-3	14.5±1.8a	6-17
Testimony	11.8±1.9b	6-14	1.6±0.6b	1-4	13.5±1.8b	7-16
100 µg/mL	11.2±1.9b	7-13	2.1±0.4c**	2-4	13.4±1.7b	9-15
200 µg/mL	8.5±0.9c****	7-11	4.5±2.2d****	1-7	13±2.5b	9-15
L3	L4	Pupa
3B
X ± SD	R	%	X ± SD	R	%	X ± SD	R	%
Control	0a	0	0	0a	0	0	0.3 ± 0.5a	13-13	1.7
Testimony	0ab	0	0	0ab	0	0	0ab	0	0
100µg/mL	0.3 ± 0.5ab	1-1	1.7	2 ± 3ab	6-6	10	0.3 ± 0.5ab	1-1	3.3
200µg/mL	13 ± 4c***	1-6	70	1 ± 0ab	1-2	8.3	1 ± 1ab	2-2	1.7

Experiments with 20 Ae. aegypti larvae (L3) for each test and control group were performed in triplicate and with three repetitions. Mean and standard deviation (X ± SD).Range (R). Values followed by the same letter do not present significant differences.Significance levels through the Tukey test, represented as **** P<0.0001; ***P<0.001; **P <0.01 vs testimony control MeOH:DMSO (1:3). The results indicated that the HEX crude extract showed low larval toxicity at a concentration of 100 µg/mL and resulted in 78-90% larval emergence. Considering the development period, these results were statistically similar to those obtained from the testimony control group. 4. Discussion Diseases transmitted by mosquitoes are a threat to human health. There are many strategies to control mosquitoes like A. aegypti and its immatures forms ( Brasil , 2009 ). However, the synthetic chemical insecticides currently in use have some disadvantages. Some factors such as vector resistance, toxicity to humans and non-target organisms drive the interest in exploring new control alternatives (Pavela, 2016; Benelli, 2018 ). Plants are rich sources of resource for biologically active substances that show a potential to control A. aegypti , being considered attractive alternatives to the conventional chemical insecticides ( Muangmoon et al., 2018 ). In some regions of Africa, plant-based methods such as burning raw materials, crude extracts, and oil preparations have demonstrated repellency against mosquitoes and provided protection for humans. In rural communities, these traditional methods are accessible and easily available ( Pavela and Benelli, 2016 ). Many studies have documented the effectiveness of plant extracts and their isolated substances in controlling A. aegypti . In this context, Azevedo et al. (2019) evaluated the larvicidal activity of extracts from 16 native plants from the Araripe National Forest, Ceará , Brazil .Among the plants that were evaluated, the ethanolic extract of Ocotea sp . was the most efficient against A. aegypti , presenting 100% larval mortality in all tested concentrations. In a study conducted by Cruz et al. (2019) , the steroidal alkaloid solasodine, isolated from the fruit of Solanum paludosum , caused 63% mortality of the 4 th instar larvae at a concentration of 150 µg/mL. Regarding the insecticidal activity of M. charantia, Pari et al. (2020) studied the activity of the ethanolic extract of M. charantia seed against the immature forms of An. stephensi , C. quinquefasciatus and A. aegypti and the results in the third instar showed LD 50 = 246.757 ppm, LD 50 = 239.018 ppm and LD 50 = 228.001 ppm, respectively. Singh et al. (2006) reported that this plant revealed larvicidal activity against three mosquito species: An. stephensi , C. quinquefasciatus and A. aegypti . Maurya et al. (2009) evaluated the larvicide activity of M. charantia fruit against An. stephensi and C. quinquefasciatus in petroleum ether, carbon tetrachloride and methanol extracts. The methanol extract presented activity against An. stephensi (LD 50 =142.82 µg/mL, LD 90 =524.54 µg/mL) and against C . quinquefasciatus (LD 90 =579.93 µg/mL).This means that higher concentrations of the methanol extract of M. charantia fruit would produce a more potent larvicidal activity, as demonstrated by the study conducted by Subramaniam et al. (2012) , where the larvicide activity of M. charantia leaves was tested against An. stephensi in the four larval and pupal stages. They found different activities for the methanol extract between the various stages: 1 st instar, LD 50 =93.45 µg/mL; 2 nd instar, LD 50 =123.74 µg/mL; 3 rd instar, LD 50 =167.17 µg/mL; 4 th instar, LD 50 =216.15 µg/mL; and pupae, LD 50 =256.66 µg/mL.Methanol extract at a concentration of 100 µg/mL presented low toxicity, while at 200 µg/mL a moderate toxicity was observed. Similar data were obtained from Rahuman and Venkatesan (2008) working with the Cucurbitaceae family, who have demonstrated the existence of larvicide activity against fourth-stage A. aegypti larvae with a methanol extract (LD 50 =199.14µg/ mL, LD 90 =780.10 µg/mL), although in the current study an LD 50 was obtained at a lower concentration. Kamaraj and Rahuman (2010) found that ethyl acetate extract from the leaves of five plant species of the family Cucurbitaceae which were tested, including M. charantia , produced high mortality(L4)at a concentration of 500µg/mL against Culex gelidus and C. quinquefasciatus . This result was similar to that of the present study regarding the ethyl acetate extract from M. charantia flowers and fruits at concentrations of 100 µg/mL and 200 µg/mL (87% and 97%, respectively). It is important to note that the ethyl acetate extract of flowers and fruits showed the same activity at lower concentrations. On the other hand, the hexane extract of M. charantia did not show toxicity for L 3 larvae and only a low toxicity for L 4 larvae and pupae was observed. According to the work reported byKamaraj and Rahuman (2010), the hexane extract presented low toxicity against C. gelidus and C. quinquefasciatus . Singh et al. (2006) demonstrated that the hexane extract of M. charantia fruits had better larvicidal activity than the crude aqueous extract, and that An. stephensi larvae were more susceptible than C. quinquefasciatus and A. aegypti larvae. The difference in results can be explained based on the chemical composition of plants which may vary according to environmental factors such as soil type , humidity, solar irradiation, wind, temperature and atmospheric pollution, among others ( Barreto, 2005 ). The ethyl acetate extract of M. charantia demonstrated toxicity against A. aegypti larvae, thus confirming the efficacy of this species of the Cucurbitaceae family as a source of active natural plant products and its importance as a potential new larvicide for controlling the mosquito vector of dengue virus, zika, chikungunya, and urban yellow fever.