Larvicidal Activity in vivo of Ethanolic and Aqueous Extracts From Moringa (Moringa oleifera Lam.) on Aedes aegypti L. (Diptera: Culicidae)

The insecticides properties of Moringa oleifera (moringa) were evaluated in Aedes aegypti larvae, throughout an entirely randomized trial, represented by ethanolic and aqueous extracts obtained from leaves, flowers, barks, seeds and moringa roots, besides the control group. Five batches of 10 in 3 stadium larvae were distributed in distilled water, added an extract concentration of 50 mL L, and the control treatment (distilled water). The test reading was measured after 24, 48 and 72 hours after larvae exposure, were considered dead those who did not respond to a mechanical stimulation of a clamp. The seed ethanolic extract produced the best performance after 24 hours (34% mortality), but after 48 hours, the flower extract was the more potent (38% mortality). The largest larvicidal activity was observed with the extract concentration of 90 mL L. The results indicate that moringa has larvacides properties against Aedes, but its chemical constituents need to be isolated and tested separately to enhance your larvicidal activity.


Introduction
Aedes aegypti L. is one of the most important global public health vectors, it can transmit to man, the diseases yellow fever, dengue, chikungunya and zika. Among the arboviruses mentioned, dengue incidence has increased 30-fold in the past 50 years, with increasing geographical expansion to new countries and zika which has been associated with several cases of Guillain-Barré syndrome and microcephaly in Brazil (WHO, 2012;WHO, 2017).
With the exception of yellow fever, which has a virus vaccine, the main strategy to combat other arboviruses is focused on vector elimination. Or reduction of infestation rates to very low levels, through the use of chemical insecticides as a priority measure. Currently, an integrated vector control has been adopted, bringing together various strategies, including educational, but ineffective in some countries.
In 2015, was first registered in Mexico a vaccine against dengue, but still there is no prediction to public distribution, leaving as main tool the vector control (Wichmann et al., 2017).
Studies indicate the larvicidal activity of several vegetal extracts (Costa et al., 2013;Krinski et al., 2014;Silva et al., 2015). Among the researched vegetal species, M. oleifera Lam. is an Asian tree, widely cultivated in Brazil due to its low production cost and high adaptability to climate and soil conditions, it is not toxic to humans, (Coelho, 2007) and its larvicidal properties against A. aegypti have been proven in previous research (Ferreira et al., 2009;Santos et al., 2009;Coelho et al., 2009;Pontual et al., 2014).
Therefore, considering the well-known properties of moringa, our purpose was to evaluate the larvicidal activity of ethanolic and aqueous extracts from different parts of the plant on A. aegypti populations from Cariri, Ceará, Brazil.

Egg Collection
One hundred ovitraps has been installed in districts with high rates of vector infestation in Crato-CE, selected after consulting the A. aegypti Infestation Index Rapid Survey (LIRAa). The ovitraps were consisted of a black plastic cylinder container (400 mL capacity) with a wooden paddle (Eucatex, 3 × 11 cm), fixed vertically inside for deposition of eggs, immersed in water. The installation of traps occurred at strategic locations of homes and collected five days later. The straws were brought to the laboratory of Agricultural Entomology of the Federal University of Cariri (UFCA) for identification and preparation of eggs for larvae hatching.

Extracts Production
Ethanolic and aqueous extracts were obtained from leaves, flowers, barks, seeds and roots of M. oleifera. Leaves and roots came from seedlings with 90 days, grown on the premises of the Institute Novo Sol (INSOL) in Juazeiro do Norte, Ceará. The remaining parts of the moringa were collected from adult plants also from the INSOL. The vegetal material was stored in a forced air circulation oven at 60 ºC, until constant weight and it was subsequently milled. The powder was mixed with water and ethanolic at a concentration of 10% (w/v), left to rest for 24 hours in the dark at ambient condition. Subsequently, the supernatant was separated by filtering, obtaining an aqueous and ethanolic extract at 10% (w/v).

Determination of Larvicidal Activity
The experiments were conducted at the Entomology Agricultural Laboratory of UFCA, under controlled conditions of temperature (25±1 °C), relative humidity (70±10%) and 12-hour photoperiod in a chamber type B.O.D. during the period from February to May of 2016.
For each treatment (ethanolic and aqueous extract), five batches of third stadium larvae (L3) were introduced in disposable cups with capacity of 50 mL of distilled water. The extract was added to the vessel in a concentration of 50 mL L -1 , with only distilled water as the remaining control. The test reading was measured after 24, 48 and 72 hours after larvae exposure, were considered dead those who did not respond to a mechanical stimulation of a pipette, being observed the percentage of mortality. The treatments were conducted with five repetitions per each extract.
The experiments were, the best performance larvicide was reached with the ethanolic extract of moringa seed (24 hour exposure), various concentrations were tested, with a variation of 10 to 100 mL L -1 , to identify the one that caused the higher mortality rate, and lethal concentrations. The trials were conducted according to the same criteria described above, this time with 11 treatments and 4 replications, totaling 44 experimental plots.

Data Analysis
The data were subjected to analysis of variance using the F test (P < 0.05). The comparison of averages between extracts from different parts of the plant was done by Tukey test, at 5% probability. The data significant of the mortality at 24, 48 and 72 hours after exposure of the larvae were assessed by models of regression.
Subsequently, the data were subjected to analysis of variance using the F test (P < 0.05). The comparison of averages between extracts from different parts of the plant was done by Tukey test, at 5% probability. If significant, effects of mortality at 24, 48 and 72 hours after exposure of the larvae were assessed by models of regression (P < 0.05) represented by the equation: y = y 0 + ax + bx 2 , ethanolic extract of leaf and flower, where: y = response variable; x = hours after larvae exposure. While: y = y 0 + ax, for root and seed.
The mortality efficiency in the treatments of larvae control was determined in percentage through the Abbott formula (1925): where, E% = Mortality efficiency; NC = Number of alive individuals in the control treatment; NT = Number of alive individuals treated.
Lethal concentrations (LC 10 , LC 50 and LC 90 ) in the seed ethanolic extract were determined by linear log-Probit regression, through StatPlus v5 program (Analyst Soft Inc.), significance level of 0.05.

Larvicidal Activity of Ethanolic and Aqueous Extracts From Moringa oleifera
The results show that the most potent extract came from the seed (Tables 1 and 2), resulting in larval mortality of 34%, after 24 hours of exposure. The flower and root extracts resulted in 14% and 10% mortality rate. However, after 48 hours of exposure, the flower extract reached 38% mortality, a greater efficiency than that of the seed, resulting in 38% of mortality in the larvae, yet seed extract did not differ (Table 1).  After 72 hours of exposure, the treatments did not differ, but again the flower and seed extracts has obtained the higher mortality rate, 10% and 6%, respectively. The vegetal insecticides generally exhibit rapid degradation in the environment, many applications may be required to obtain a satisfactory control (Morais & Marinho-Prado, 2016). Probably, during the exposure period, the extracts lost their insecticidal activity due to the increased time of exposure to light on the molecules (not synthetic), causing to be unstable in the environment with low residual power. Considering overall efficiency of three exposure periods, both seed and flower extracts showed the same mortality efficiency (62%) ( Table 1). Figure 1 illustrates the relationship between A. aegypti larvae mortality percentage subjected to ethanolic extracts of flower, seed, leaf and root from M. oleífera and the hours after larvae exposure. It is noticed a quadratic relationship between the evaluation time and ethanolic extracts of flower and leaf, and linear correlations between the seed and root extracts. The moringa seeds contains, besides lectins, glycoproteins of nonimmune origin, which bind carbohydrates by two binding sites, agglutination of animal cells and/or vegetables,

Larvic
The ethan analysis, d minimum mL L -1 , i.e     Vol. 11, No. 8;2019 Studying the mortality efficiency of essential oil concentrations, on larvae of A. aegypti, Silva et al. (2017), obtained data behavior similar to those obtained in this study, where the increase in the concentration of the product resulted in a greater control efficiency of larvae of this vector.

Conclusions
Moringa has larvicidal activity against Aedes aegypti until 48 hours of exposure. The greatest bioactives concentration seems to be present in seeds and flowers, because get better results with extracts produced with those parts. Aqueous extracts are less efficient in the control, even seed made. It is believed that the isolation of plant constituents can guide further research on the persistence and influence increase of environmental conditions.