Background

Mosquitoes are responsible for lymphatic filariasis (Culex quinquefasciatus Say), yellow fever, dengue (Aedes aegypti Linn.) and malaria (Anopheles gambiae Giles) (Ndione et al., 2013). These infections are debilitating and deprive the sufferers the beauty of enjoying life and being useful to themselves, their families and their countries. Synthetic chemical insecticides are the tools of choice to control the vectors and prevent the mosquito transmitted infections (Brattsten and Hamilton, 2012). However, because of their indiscriminate use, mosquitoes have developed resistance, rendering the management strategy less effective. Moreover majority of the synthetic chemicals have undesirable effects on non-target organisms and even foster environmental and human health concerns (Yang et al., 2002). Because of these unfortunate effects, recent search for potent malaria vector control methods have shifted to botanicals that have been found to be quantitatively and qualitatively rich in volatile constituents (Choochote et al., 2005; Praveen et al., 2012). The extracts are also biodegradable and nontoxic to the environment (Choochote et al., 2005; Krishnappa et al., 2013; Elumalai et al., 2013).

Additionally, plant extracts have demonstrated toxicity against mosquito eggs, larvae, pupae and adults (Elumalai et al., 2012a; Elumalai et al., 2012b; Gokulakrishnan et al., 2012; Ramar et al., 2012; Ramar et al., 2014; Balu et al., 2015; Pravin et al., 2015). They also have demonstrable repellency (Pitasawat et al., 2003; Omolo et al., 2004; Traboulsi et al., 2005), and oviposition deterrents (Cavalcanti et al., 2004; Ansari et al., 2005) effects. Indeed plant products had been envisioned as possible replacement of synthetic chemicals for the management of mosquito immature (Anupam et al., 2012).

Phytolacca dodecandra plant extracts has proven effective not only against immature of filarial vector Cx. guinquefasciatus Say (Diptera: Culicidae) (Misganaw et al., 2012) but also to An. gambiae larvae (Yugi et al., 2015) and adults (Yugi et al., 2016). Reports however, on its effectiveness as larvicide has largely been on the different larval stages (Larval instars 1 to 4) without a mention of the non-feeding stage, the pupae. This study was therefore designed to test and report on the effects of crude ethanol and water extracts of P. dodecandra parts on pupae of An. gambiae in the laboratory. This was to provide information on toxicity of P. dodecandra extracts on this crucial link to the An. gambiae life history.

1 Results

This experiment was conducted for a period of three weeks using 3,690 An. gambiae pupae. It was observed that water extracts of mature green fruits of P. dodecandra were more toxic compared to ethanol extracts of the same part. Extracts of P. dodecandra from the lowlands (Nyando) was more potent than that from the highlands (Eldoret). It was also found that the activity of P. dodecandra extracts was dose dependent with mean percent mortality increasing with increased concentrations irrespective of part of P. dodecandra or solvent used. Only concentration of 40 mgs/100 mls from mature green fruits sourced from the highlands (Eldoret) killed > 80% of exposed pupae irrespective of solvent used (Table 1). Concentrations of 20 mgs/100 mls and higher from mature green fruits sourced from the lowlands (Nyando) killed > 80% of exposed pupae irrespective of solvent used (Table 2). Mortalities from exposure to Neem leaf extract was less than 80%. Exposure of pupae to the different treatments differed significantly (p <0.001) irrespective of part, geographical source or solvent used in the extraction of P. dodecandra except for the negative control (p >0.05) (Table 3).

Water extracts of P. dodecandra was needed in low doses for observed toxicity against pupae compared to ethanol extracts. Toxicity strength of P. dodecandra plant parts extracts was of the order; fruits > leaves of the shoot > leaves of the mid-section irrespective of solvent of extraction or source of P. dodecandra. Dose of treatments did not significantly influence pupae mortality (p > 0.05) except for water extracts of mature green fruits from Nyando (Table 4). All calculated goodness of fit determined by χ² were less than the χ² critical value of 22.4 (df = 13; p < 0.05) and therefore the hypothesis of no relationship (H₀) between dose and mortality of An. gambiae pupae was retained.

2 Discussions

In the current study it was demonstrated that water extracts of mature green fruits of P. dodecandra was more potent as pupicide compared to ethanol extracts as was extracts of P. dodecandra from the lowlands (Nyando) compared to that from the highlands (Eldoret) irrespective of dose. Plenty of studies exist that demonstrate bioefficacy of plant extracts against vector mosquitoes. For example Carvalho et al. (2003) reported larvicidal activity of the essential oil from Lippiasidoides, against Ae. aegypti Linn, Mann and Kaufman (2012) of essential oil and Jenson et al. (2006) of crude extracts of various plants extracts against different mosquito species in the field of vector control. In all these cases the levels of potency of plant extracts were correlated with type and part of plant and solvent used in the extraction (Jeyabalan et al., 2003). In this study we also demonstrate that crude extracts of P. dodecandra is toxic against An. gambiae pupae irrespective of solvent used in extraction or geographical origin of P. dodecandra plant.

The findings of the current study is also consistent with those of extracts of berries of Endod against Immature filarial vector, Cx. Quinquefasciatus Say (Diptera: Culicidae) (Misganaw et al., 2012), extracts of essential oils from leaves of Plectranthus glandulosus and Callistemon rigidus against pupae of Ae. aegypti and Cx. quinquefasciatus (Pierre et al., 2014) and essential oils from seven plants against pupae of Cx. quinquefasciatus and An. stephensi (Ramar et al., 2014).

The observation that water extracts of mature green fruits of P. dodecandra was more potent than ethanol extracts of the same parts was however inconsistent with an earlier finding of Anupam et al. (2012) and Mgbemena (2010) that concluded that ethanol extracts of bioactive from plants are generally more potent compared to water extracts from the same plants. However, results of this study resonates with an observation made by Anupam et al. (2012), Mgbemena (2010) and Were (2008) on the influence of geographical disposition of a plant on the concentration and distribution of phytochemical therein. The observation that extracts of P. dodecandra sourced from the lowlands (Nyando) was more potent compared to that sourced from the highlands (Eldoret) was however inconsistent with that of Were (2008) in that whereas she demonstrated that extracts of parts of P. dodecandra sourced from the highlands were more potent to that sourced from the lowlands the results herein demonstrates otherwise. The findings herein leads to the conclusion that crude ethanol and water extracts of P. dodecandra is toxic against An. gambiae pupae and is employable as a tool for effective disruption of An. gambiae life cycle.

3 Materials and Methods

3.1 Study area and experimental mosquitoes

The experiments were conducted in the Entomology laboratory at Centre for Global Health Research/Kenya Medical Research Institute (CGHR/KEMRI). An. gambiae mosquitoes maintained at the laboratories were used. Mosquito culture was done following standard techniques (Das et al., 2007) at temperatures of 28-30°C, relative humidity of 70-80% and photoperiod of 12:12 (L:D).

3.2 Plant extracts preparation

Phytolacca dodecandra plant parts were sourced from Eldoret [+0.518829^ON, 35.284927^OE] and Nyando [-0.250393^ON, 34.870190^OE] and Azadirachta indica (Neem) from Nyando [-0.250393^ON, 34.870190^OE]. The plant parts were identified, voucher specimen number JOY2012/001 for P. dodecandra and JOY2012/002 for A. indica issued and thereafter deposited in the herbarium at the School of Biological Sciences, University of Nairobi, Kenya. The plant parts were prepared and extracted using standard procedure describe by Das et al. (2007) and Parekh et al. (2005). The extracts were then kept in airtight glass bottles to serve as stock quantity.

3.3 Pupicidal bioassays

Twenty early stage pupae (pupae that metamorphosed from L4 larvae within a two hour window) of An. gambiae were placed individually using a dropper in transparent plastic containers measuring 6 cm top × 5.7 cm bottom × 3.5 cm height. The containers with pupa were arranged in sets of threes and each set of containers received approximately 33 mls of a particular concentration of treated rain water.

The concentrations were prepared by taking 80 mgs of freeze-dried stock, dissolving it in 200mls of rain water and serially diluting to obtain concentrations of 40, 20, 10, 5 and 2.5 mg in 100 mls of rain water. Positive (ethanol and water extracts of Neem) and negative controls (untreated rain water) were also set simultaneously. The set up was replicated ten times.

The mouth of each container was covered with mosquito netting to prevent emerged adult mosquito from escape. The exposed pupae were left to stay in the treatments on experimental tables overnight and mortality rate registered after 24 hours post exposure. Pupae mortality was calculated for each concentration using the formula;

Mortality was corrected using Abbotts formula (Abbott, 1925).

In addition, standard WHO procedures were used to assess effectiveness of the extracts as pupicide at a threshold mortality rate of > 80% (WHO, 2005).

3.4 Data analysis

Data obtained from the bioassays was entered in excel spreadsheets and the relationship between pupicidal effect of the extracts with part of P. dodecandra plant used and concentration determined using descriptive statistics. One way analysis of variance (ANOVA) was used to determine the level of significance of the effects of treatments on pupae mortality. Regression (probit) analysis was used to calculate the lethal concentration (LC₅₀) and χ² statistics of the extracts used. Results on concentrations of extracts were expressed as mean ± SD and those on effect of dose expressed as mean ± SE of three replicates in each treatment. P-values were considered significant at p < 0.05. All statistical analysis was performed using Statistical Package for Social Scientist (SPSS) version 16.

Authors’ contributions

Y.J.O., conceived, designed the experiments, wrote manuscript and analyzed the data, K.J.J., and M.C., conducted the experiments. . All authors read and corrected the manuscript.

Competing interest

The authors declare that they have no competing interest.

Acknowledgments

I thank Mr. Richard Amito, Mr. Dalton Ochieng’, Ms Charlotte Awuor, Ms. Patience Akoth and Mr. Trevor Omondi for culturing mosquitoes used in this study. I also thank Centre for global Health Research /Kenya Medical Research Institute (CGHR/KEMRI) for laboratory space, mosquitoes and equipments for conducting the experiments and National Commission for Science Technology and Innovation (NACOSTI) for funding the project (Grant contract # NCST/5/003/3^rd Call PhD/056 to YJO).

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