Introduction
Synthetic insecticides (Poopathi and Archana, 2010) have for a long time been used to manage extensive morbidity and mortality within disease-endemic countries (Boutayeb, 2006) due to Mosquito-borne diseases. Though effective this strategy is accompanied with serious harm to human and other animals in the environment (Cartilla and De la Cruz, 2012). In addition continued use of synthetic insecticides has been demonstrated to lead to reduced control due to development of resistant in insect populations (Charles and Nielsen-LeRoux, 2000).

The increasing negative effect of synthetic insecticides has made scientists seek for alternative eco-friendly pest control methods (Poopathi and Archana, 2010; Montasser et al., 2011). Botanicals such as Phytolacca dodecandra here after Endod have been found promising as alternative larvicidals against Culex quinquefasciatus (Misganaw et al., 2012) and An. gambiae (Yugi et al., 2015) and pupicidals against Culex quinquefasciatus (Misganaw et al., 2012). There is also evidence of demonstrable microbial activity (Joshi et al., 2011). Though efficacy of Endod extracts on An. gambiae larvae has been demonstrated, no information exists on the dose or amount of the extracts

that are effective on each larval stage. It is in line with this that this study was designed to study and report on the effective dose of crude ethanol and water extracts of mature green fruits and leaves of Endod on larvae of An. gambiae in a laboratory set up.

1 Results
The experiment was conducted for a period of eight months using a total of 84,240 larvae (L1s, L2s, L3s and L4s). WHO threshold of >80% mortality was used as standard to decide on the effectiveness of the various doses against the exposed larvae. For L1s, concentration of 20 mgs and 40 mgs of ethanol extracts of mature green fruits of Endod from Eldoret and Nyando respectively killed over 80% of the exposed larvae.  For aqueous extracts of Endod mature green fruits and leaves of the shoot, mortality of over 80% was observed for concentrations of 40 mgs only irrespective of source (Figure 1).

Figure 1 Mortality of An. gambiae first instars (L1) exposed to different concentrations of crude ethanol and water extract of Endod. Error bars represents standard error of means

For L2s, concentrations of 20 mg and 40 mg of ethanol extracts of mature green fruits from Eldoret and Nyando killed over 80% of exposed L1s respectively. Mortality of over 80% was observed for concentrations of 40 mgs of extracts of leaves of the shoot and midsection of Endod from Nyando and Eldoret respectively. Concentrations of 20 mg and 40 mgs of aqueous extracts of mature green fruits from Eldoret and Nyando killed over 80% of exposed L2s respectively (Figure 2).

Figure 2 Mortality of An. gambiae second instars (L2) exposed to different concentrations of crude ethanol and water Endod extract. Error bars represents standard error of means

For L3s, concentrations of 10 mg and 20 mg and above of ethanol extracts of mature green fruits and leaves of the shoot of Endod from Eldoret and Nyando respectively killed over 80% of exposed L3s. The same was observed for concentration of 40 mgs of ethanol extracts of leaves of the midsection of Endod (Figure 3). For the L4s, more than 80% mortality of exposed larvae was observed for concentration of 10 mgs and above for ethanol extracts of mature green fruits irrespective of source (Figure 4).

Mortality of over 80% was observed for L1s and L3s for ethanol and aqueous extracts respectively for Neem while Deltamethrin killed all exposed larvae irrespective of stage or concentration. The effects of the extracts were considered significantly different at p < 0.05 (Table 1) for all exposed larval stages.

Figure 3 Mortality of An. gambiae third instars (L3) exposed to different concentrations of crude ethanol and water Endod extracts. Error bars represents standard error of means

Figure 4 Mortality of An. gambiae fourth instars (L4s) exposed to different concentrations of crude ethanol and water Endod extract. Error bars represents standard error of means

Table 1 Duncan’s statistics on mortalities due to exposure of An. gambiae larvae to crude extracts of parts of Endod Note: 1 P is the probability for the level of significance. P was considered significant at p < 0.05 2 df is the degree of freedom of the considered units

2 Discussions
Ethanol extracts of mature green fruits of Endod at higher concentrations killed all exposed aquatic stages of An. gambiae. Mortality percentages however, reduced with continued dilution. This trend was similar to Ethanol extracts of Endod leaves although observed mortalities were lower than that of fruit extracts. Earlier the same trend had been reported for powdered extracts of Endod on An. gambiae larvae (Were, 2008). This demonstrates that extracts of ethanol and water extracts of Endod have similar efficacies as Chenopodium ambrosioides Linn (Jude et al., 2013) and Jatropha curcas L (Zewdneh et al., 2011) against An. gambiae and Anopheles arabiensis larvae respectively.

Extracts from both mature green fruits and leaves of Endod were potent against An gambiae larvae though potency of leaf extracts were lower than that of fruits. Potency of the extracts also differed with the vertical position of leaves on the Endod plant such that ethanol extracts of leaves found at the shoot were more potent than those found at the midsection of the plant.  This showed that the probable distribution of the bioactive on Endod plant are such that fruits > leaves at the shoot > leaves at the midsection.

Ethanol extracts of Endod parts were more toxic than aqueous extracts of the same parts for all concentrations against An. gambiae larvae. Solvent type used in extraction had been demonstrated to determine potency of extracted bioactives (Anupam et al., 2012) as well as larviciding potency. This had been demonstrated for Solanum xanthocarpum (Mohan et al., 2006), Euphorbia tirucalli (Singh et al., 2007), Eucalyptus globules (Maurya et al., 2007), Citrullus colocynthis (Sakthivadivel and Daniel, 2008), Azadirechta indica (Mgbemena, 2010) and Solanum nigrum (Raghavendra et al., 2009).

Ethanol extracts of Endod parts sourced from Eldoret were more potent than of the same parts sourced from Nyando. This showed that geographical origin of the plants played a role in the concentration and distribution of bioactives within the plants (Anupam et al., 2012). A similar observation had been made on Endod parts (Were, 2008), Citrus sp, Ocimum sanctum and Azadirechta indica (Mgbemena, 2010) and Jatropha sp (Sakthivadivel and Daniel, 2008) just to mention but a few.
 
This study demonstrates that crude ethanol and water extracts of Endod are potent against An. gambiae larvae at high doses. It also demonstrates that solvent type used in the extraction influences activity of extracts from Endod irrespective of parts or geographical origin.

3 Materials and Methods
3.1 Study area, experimental mosquitoes and study design
The experiments were conducted in the Entomology laboratory at Centre for Global research Institute/ Kenya medical research Institute (CGHR/KEMRI). Pink eyed An. gambiae mosquitoes maintained at the laboratories and reared following standard techniques (Das et al., 2007) were used in the experiments. A completely randomized informal ‘after-only with control’ experimental design (Kothari, 2004) was used to investigate the larvicidal effect of crude ethanol and water extracts of Endod on the mosquito larvae. The condition in the insectary was as described elsewhere (Yugi et al., 2014).
 
3.2 Deltamethrin (KOTab 1-2-3®) and plant materials acquisition and preparation
Deltamethrin (KOTab 1-2-3®) was acquired and prepared as described (Yugi et al., 2015). Fresh leaves (shoot and midsection) and mature green fruits of Endod and Fresh leaves of Azadirachta indica (Neem), were acquired, identified and voucher specimen deposited as described elsewhere (Yugi et al., 2015). The plant parts were dried in a shade at room temperature, grounded and extracts obtained using ethanol and water as described in details elsewhere (Tilahun et al., 2003; Parekh et al., 2005; Das et al., 2010; Yugi et al., 2015).

The extracts were concentrated by freeze drying using a rotary vacuum evaporator at 40-42°C to obtain essential oil that was then kept in airtight glass bottles to serve as stock quantity. From the freeze-dried stock, 80 mg were weighed and serial dilutions made to obtain different concentrations of 40, 20, 10, 10, 5 and 2.5 mg in 100mls of rain harvested water.

3.3 Larvicidal bioassays
Larvicidal activities were tested in accordance with the WHO procedure (WHO, 1996) and as described elsewhere (Yugi et al., 2015). Mortality rate were registered after 24 hour exposure period and larval mortality calculated for each concentration using the formula;

Standard WHO procedures were used to assess effectiveness of the extracts as larvicide at a mortality rate of > 80% (WHO, 2005). Moribund and dead larvae were collected and disposed off in a septic tank.

3.4 Data analysis
Data obtained from the bioassays was entered in excel spreadsheets for ease of handling. The relationship between the effective doses of the extracts with respect to parts of Endod used was determined using descriptive statistics. One way analysis of variance (ANOVA) was used to assess the level of significance of the various doses on larvae mortality. All statistical analysis was performed using SAS statistical package version 20. 

Competing interest
The authors declare that they have no competing interest.

Acknowledgements
We thank Patience Akoth, Charlotte Awuor, Dalton Ochieng’ Trevor Omondi and Richard Amito, for helping culture all mosquitoes used in this study. We thank Kisumu Polytechnic for equipments for the extractions of the crude extracts from Endod and Neem plants, Centre for Global Health Research/Kenya Medical Research Institute (CGHR/KEMRI) for mosquitoes, laboratory space and equipments, VIRED International for providing transportation and logistics for sourcing for Endod and Neem parts. We also thank National Commission for Science, Technology and Innovation (NACOSTI), Kenya for funding this project.

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