Properties for Sourcing Nigerian Larvicidal Plants

Aedes aegypti is the primary vector of chikungunya, yellow and dengue fevers. Dengue fever is the major cause of child morbidity and hospitalisation in some Asian and African countries, while yellow fever is prevalent in Nigeria. The development of resistance to the available insecticides has necessitated the continued search for safer ones from plants. Eighteen plant extracts with ethnomedical claims of or demonstrated febrifuge, antimalarial, insecticidal and insect repellent biological activities were tested for activity against the fourth instar larvae of Aedes aegypti. About 61% of the eighteen extracts demonstrated high to moderate larvicidal activity. Extracts of Piper nigrum and Abrus precatorius seeds were the most active and the larvicidal constituent(s) of the latter should be determined.


Introduction
Aedes aegypti Linn. (Culicidae) is the primary vector of chikungunya, yellow and dengue fevers [1]. Dengue fever is the major cause of child morbidity and hospitalisation in some Asian and African countries, while yellow fever is prevalent in Nigeria as well as other tropical countries. The absence of established diagnostic facilities has hindered the detection of dengue virus in Nigeria, as its symptoms have been often mistaken with malaria, thyphoid, etc. [2]. The conventional insecticides used in mosquito control are limited, with reported adverse effects on the ecosystem. Also, these vectors have developed resistance to the available insecticides [3]. These problems have therefore necessitated the OPEN ACCESS continued search for safer insecticides in eradication or reduction of the vectors' populations. Since they are highly biodegradable, well tolerated by the ecosystem and have low mammalian toxicity, medicinal plant extracts have been reported as safer alternatives [4]. More successes have been reported with plants tested for activities suggested by their ethnomedicinal/folkloric uses [5][6][7], a statement agreed to by the WHO [8]. Also, the larvicidal, pupicidal and adulticidal activities of many plant constituents were found comparable to those of the standard drugs [4,[9][10][11][12][13] Since larvicidal activity is not traditionally known, it was suggested that ethnomedical claims or febrifuge, antimalarial, insecticidal and insect repellent biological activities could be considered as part of the factors for sourcing plant larvicides [14]. Hence this study is a further attempt in testing this hypothesis by assessing the larvicidal activity of an additional fourteen plants, listed in Table 1, against fourth instar larvae of A. aegypti.

Results and Discussion
Dead larvae were those that could not be induced to move when probed with a needle in the siphon or the cervical region while moribund larvae were those incapable of rising to the surface or not showing the characteristic diving reaction when the water was disturbed [15]. According to WHO [15], the percentage mortality should be calculated by adding the moribund larvae to those that are dead. However, similar to an earlier work [14], the larvae that were moribund after 48 h were added to the living, hence a more stringent assay of larvicidal activity with higher LC 50 and LC 90 values for the extracts. The LC 50 values of extracts of the leaves of L. owariensis, C. indica, C. patens, H. opposita, M. indica and A. boonei, woods of A. altilis and E. heterophylla, E. chlorantha stem bark, and leaf and stem of C. afer at 24 h were significantly higher than those at 48 h, similar to their LC 90 values ( Table 2). This showed that longer exposure to these extracts benefitted their larvicidal activities, probably indicating cumulative effects in their activities. On the other hand, the LC 50 values of extracts of the seeds of P. nigrum and A. precatorious, C. longa rhizome, A. altilis stem bark, whole plant of S. biafrae, leaves of D. cumminsii and M. koenigii as well as that of Endosulphan at 24 h were comparable with those at 48 h, similar to their LC 90 values at 24 and 48 h ( Table 2). This may indicate short larvicidal activities of these extracts [14,16]. Extracts with LC 50 < 2 mg/mL were regarded as very active [14]. Therefore, LC 50 of 0.01 and 0.85 mg/mL at both 24 and 48 h for P. nigrum and A. precatorius seed extracts, respectively qualified them as the most active of the eighteen extracts tested. Endosulphan, a commercial insecticide, with LC 50 of 0.93 and 0.90 mg/mL at 24 and 48 h, respectively had larvicidal activity that is comparable (p > 0.05) to those of P. nigrum and A. precatorius (Table 2). Insecticidal activity has been reported for these two plants [17,18], which are also either used ethnomedicinally to treat malaria or fever (Table 1), making them possible good plant larvicides and sources of larvicidal compounds. This result therefore lends credence to the hypothesis proposed for sourcing plants larvicides [14].

Name of Plant and Part
The respective LC 50 = 0.56 and 0.65 ppm (5.6 and 6.5 × 10 −4 mg/mL) reported for Aedes albopictus and Culex quinquefasciatus with P. nigrum [31] may indicate that these larvae were more susceptible than A. aegyptii (Table 2). Furthermore, the LC 50 of 0.06 and 0.03 mg/mL given for aqueous and ethanolic extracts of its dried ripe fruits against C. quinquefasciatus, respectively [32] confirmed that the plant has a high larvicidal activity against many larvae. Piperine has been isolated as the active adulticidal constituent of the plant [33]. After 24 h, the methanolic extracts of A. precatorius shoot and seeds had LC 50 of 0.03 and 0.02 mg/mL, respectively against C. quinquefasciatus and Anopheles vagus, while the ethylacetate extract of its seed had LC 50 of 0.14 mg/mL against Culex vishnui [34,35], indicating that these larvae were more susceptible (Table 2). Additionally, the fruit and seed extracts of A. precatorius were found toxic to adult mosquitoes [36], suggesting it as a good plant larvicide and adulticide.
The 2.0 < LC 50 < 4.2 mg/mL given by the extracts of C. longa rhizome, A. altilis stem bark and leaves of L. owariensis and C. indica indicated moderate activity while the remaining extracts (LC 50 > 4.2 mg/mL) were inactive ( Table 2). This is the first reported activity of the leaf extracts of L. owariensis and D. cumminsii, leaf and stem of C. afer, A. altilis stem bark and S. biafrae whole plant against any larvae. At 48 h, C. afer leaf and stem, H. opposita leaf, C. patens leaf and E. chlorantha stem bark were also moderately active (Table 2), indicating benefit of longer exposure to these extracts and the slow acting nature of their active principles. At 48 h, the larvicidal activities of M. indica and E. heterophylla were also improved. The C. longa rhizome oil (LC 50 = 0.017 mg/mL) was reported more toxic to A. gambiae larvae than the leaf oil (LC 50 = 0.029 mg/mL) [37]. The lower activity obtained for the extract of this rhizome (Table 2), may indicate that the active constituents were probably more in the volatile oil, although difference in the larvae used may also play a role. Furthermore, its oil hydrolates had LC 50 of 24.7% and 35.5% v/v against A. albopictus and C. quinquefasciatus, respectively [38]. The extract of its rhizome exhibited high but varying activities against the larvae of Nilaparvata lugens, Plutella xylostella, Myzus persicae and Spodoptera litura, different species of stored grain pests while the insecticidal principle was identified as ar-tumerone [39].
At 24 h, the petroleum ether extract of C. indica leaf displayed higher activity (LC 50 = 0.056, LC 90 = 0.248 mg/mL) against C. quinquefasciatus larvae [40] than the LC 50 = 3.84, LC 90 = 6.41 mg/mL obtained for its methanolic extract used in this study (Table 2), indicating differences in the susceptibility of the larvae. The A. boonei leaf extract did not kill the larvae after 48 h ( Table 2). The aqueous extracts of A. boonei stem bark and leaf significantly (p < 0.01) reduced the survival and weights of the Sesamia calamistis larvae, the pink stalk borer, in a dose dependent manner. Equally high concentrations of the stem bark (2.8% and 2.1%) and leaf (5.6% and 3.5%) have been reported to kill 50% of the larvae at 10 and 20 days after introduction, respectively [41], which would give LC 50 of 28, 21 and 56, 35 mg/mL for the stem bark and leaf, respectively in 10 and 20 days. The LC 50 values of 2.70, 11.33 and 12.54 mg/mL given by A. boonei leaf extracts, respectively at 24 h against Anopheles arabiensis indicated ethanol > aqueous > methanol extracts as the order of larvicidal activity [42]. Similarly, the LC 50 value of E. heterophylla whole plant extract at 24 h could not be determined while its LC 50 = 5.75 mg/mL at 48 h indicated non-activity (Table 2). Its ethanolic and petroleum ether extracts displayed high activity (LC 50 = 0.024 and 0.025 mg/mL, respectively) against Cx. quinquefasciatus [43], indicating higher susceptibility of this larvae. The inactive methanolic extract of M. indica leaf ( Table 2) has also been reported to be inactive against C. quinquefasciatus [34,44]. The stem bark of A. altilis had better activity than its wood while the activity of leaf and stem of C. afer was comparable (Table 2). Lastly, the extracts of D. cumminsii leaf and the whole plant of S. biafrae, which were used as negative internal controls for the hypothesis of properties considered to be useful in choosing plant larvicides (Table 1), were inactive (Table 2). This situation was similar to that of Euphorbia macrophylla, used for the same purpose in an earlier report [14], and may further confirm the reliability of these four properties as factors for consideration in choosing plant larvicides.

Plant Collection
The whole plant or different parts of the plants listed in Table 1

Plant Extraction
The leaves and seeds were air-dried while the whole plants, stems and rhizomes were oven dried at 40 °C. They were subsequently powdered and 500 g of each plant material was extracted in methanol (2 L) at room temperature for 3 days, with agitation. The extract was filtered and concentrated in vacuo. This process was repeated two times and the combined dried extract for each plant part [45] was kept in the refrigerator (4 °C) until needed for the larvicidal assay [16].

Larvicidal Test
The eggs of A. aegypti collected from the National Medical Centre, Yaba, Lagos, Nigeria were suspended in water for 24-48 h to hatch. The larvae were fed with rabbit pellets (Bendel feeds, Edo State), until they reached the 4th instars stage. Larvicidal tests were done using slight modification [16] of the standard method [15]. Stock solutions (25 mg/mL) of the extracts were prepared by solubilising in dimethylsulphoxide (DMSO) and diluted with distilled water to give a 0.02% final concentration of DMSO. They were thereafter serially diluted to obtain 25 mL of different concentrations (0-5 mg/mL) of the test agents and twenty five larvae were introduced into each cup. The toxicity of endosulphan, a commercial insecticide, was evaluated as the positive control at 0.312, 0.625, 0.937, 1.25, 1.56 and 1.88 mg/mL. Mortality was recorded after 24 and 48 h of exposure during which no nutritional supplement was added [15]. The mean and standard error of the mean for six replicates were calculated while the percentage mortalities, LC 50 and LC 90 values, representing the concentrations for 50 and 90% larval mortalities, were predicted using Microsoft Excel program 2007 [16]. No mortality was observed with the negative control.

Statistical Analysis
The larvicidal activities of the extracts were compared with that of Endosulphan using one way analysis of variance (ANOVA) followed by Student-Newmann-Keul post-hoc test [14]. p < 0.05 was considered as significant.

Conclusions
A 61% of the eighteen extracts of the plants either used ethnomedically or reported to have antimalarial, febrifugal; insecticidal and insect repellent activities demonstrated high to moderate larvicidal activity, in agreement with Adebajo et al.'s [14] hypothesis of using these factors to source plant larvicides. Extracts of P. nigrum and A. precatorius seeds were the most active and would be interesting to determine the larvicidal constituent(s) of the latter.