Background

Malaria is a global health problem with an estimate of 3.2 billion people at risk worldwide. Sub-Saharan Africa populace suffers the most with a record of 88% infections and 90% deaths (WHO, 2015). In Kenya, malaria is a major cause of morbidity and mortality with prevalence upward trend in terms of age, with children aged 11–14 years showing the highest prevalence (Marufa et al., 2017). Mosquitoes are vectors to a wide range of death causing human diseases such as filariasis, malaria, dengue, yellow fever, and Japanese encephalitis in addition to malaria (Pugazhvendan and Elumali, 2013).

Malaria vector control programmes mainly involve the use of pyrethroid based synthetic chemical in the form of insecticides treated nets and indoor residual sprays (Oss`e et al., 2013; Okia et al., 2013). While these strategies have been successful, overuse (Zhao et al., 2002) has led to emergence of mosquito (Ruikar et al., 2012; Chouaibou et al., 2012; Alex et al., 2012; Brown et al., 2013; Lo and Coetzee, 2013) and parasite (Checchi et al., 2006) resistance.

To ameliorate the above trend, scientist have turned to plants for new sources of products against malaria vector for the simple reason that plants produce a broad range of bioactive chemical compounds (Altemimi et al., 2017) many of which have promising mosquitocidal activities (Tennyson et al., 2012). Indeed some phytochemical have even proven to be more effective than synthetic insecticides (Olaitan and Abiodun, 2011). Besides being target-specific (Mann and Kaufman, 2012), the plant-derived insecticides also decompose quickly (Khater, 2012) do not biomagnify (Isman, 2006) and due to poor penetrance in ecosystems, do not affect large animals (Evergetis et al., 2012).

The extracts act as environmental stressor and initiate transient states involving change in physiological responses that bring an insects near to or over the edges of their “ecological niches” (Korsloot et al., 2004). The changes affect certain crucial aspects such as fertility, fecundity, development time, and longevity. These factors are referred to as life history parameters and are important determinants of an organisms’ fitness (Grech et al., 2007). Indeed the longer the insect takes to mature, the lower the probability of the larvae emerging into adults and the higher the probability of increased exposure of emerged adults to potential predation and human interference (Padmanabha et al., 2011). This in the end impacts fitness of adult emergence and subsequent biological life cycle (Kweka et al., 2012). On the other hand if the vector does not live long enough (vectors of diseases with intrinsic incubation period like malaria), the parasite might never be able to be transmitted, and the vector competence is therefore compromised (Garrett-Jones and Shidrawi, 1969; Muturi et al., 2011; Araujo et al., 2012; Takken et al., 2013; Lefèvre et al., 2013; Moller-Jacobs et al., 2014; Breaux et al., 2014).

Knowledge of the malaria vector’s interaction with factors that influence important aspects in its life cycle such as insecticides and other environmental stressors can lead to the development of new vector control tools. In this study, the impact of ethanol and water extracts of mature green fruit and leaves of P. dodecandra on the life history of malaria vector, Anopheles gambiae (Diptera: Culicidae) mosquito eggs was evaluated.

1 Results

It was found that all the treatments impacted on the life history of the exposed mosquito population irrespective of parts of P. dodecandra used or dose. However, extracts from different parts of P. dodecandra significantly influenced mortality of the different life stages ([p<0.001] (Table 1) while dose did not ([p>0.05], Table 2). Water extract of P. dodecandra was observed to be relatively more potent than ethanol extracts though t and F (Levene’s statistics on equality of population variance) statistics (p<0.05) showed that none of the solvent significantly influenced toxicity of the extracts on the life stages (Table 3).

When the residual effectiveness of the extracts in the environment was evaluated, it was found to be dose dependent but reduced with time (Table 4). Dose of treatment nonetheless impacted significantly on mortality of all life stages (p < 0.001). When the amount of extract needed to kill at least 50% of exposed immature stages (LC₅₀) was evaluated it was found that relatively lower doses (≤ 4.6) with small differences between the lower and upper 95% CI of log concentration of the extracts were needed to effect the observed mortalities (Table 4). Chi-square test statistics on relationship between considered concentrations showed an influence on mortality for the first larval instars (L1s) for ethanol extracts of P. dodecandra fruits but not for P. dodecandra leaves or Azadirachta indica leaves (Table 5). Ch-square test statistics on water extracts of P. dodecandra fruits and leaves as well as Azadirachta indica leaves showed no significant influence of dose on mortality of all life stages of An. gambiae mosquitoes (Table 6).

2 Discussion

The present study has shown that exposure of An. gambiae mosquito eggs to water and ethanol extracts of leaf (shoot and midsection) and mature green fruits of P. dodecandra, affected the life history traits of this mosquito under laboratory conditions. For instance, hatchability of An. gambiae eggs to first larval instar (L1) and subsequent transformation to the various larval stages to pupae and then adult reduced greatly. That is fewer eggs hatched from experimental groups as opposed to control groups. Hatchability rate was also dose dependent with the higher the dose of treatment used the fewer the hatchlings. Additionally a small number of larvae hatching from treatment groups survived to adulthood with survival being greatly influenced by the dose to which the eggs the hatchlings emerged from were exposed.

Anopheles gambiae eggs are embryonated when laid. Indeed it had earlier been reported that An. gambiae eggs laid on damp or moist substrates hatch spontaneously even at a level of dampness as the first-instar larvae was observed to wait inside the cap-opened chorions, with only the head protruding, for water to arrive (Huang et al., 2006). If this is the case then in the present study it is apparent that the treatments adduced stress to the developing embryo or the emerging larvae. This then either killed or affected the developing exposed embryo and explained the reasons for some eggs remaining unhatched (dead) or the larvae dying immediately thereafter or later on. However, when the embryo survived the onslaught, the period over which the larvae emerged into adults was greatly protracted.

Stress element introduced by treatments to the exposed An. gambiae eggs in the present study indeed enhanced development time of the strain. It is speculated that if extracts of P. dodecandra are to be used in nature, exposed An. gambiae mosquitoes are likely to suffer the consequences of running the risk of exposure to potential predators and human interference as observed in Aedes aegypti (Padmanabha et al., 2011). This is likely to alter the female mosquito fertility and fecundity (Zuharah et al., 2016), crucial vectorial traits necessary for survival. It is also likely to affect epidemiologically relevant mosquito disease transmission traits such as longevity or vector competence (Beldomenico and Begon, 2010; Muturi et al., 2011; Takken et al., 2013; Moller-Jacobs et al., 2014). Indeed it was reported that when exposed to insecticides containing temephos and propoxur, Anopheles stephensi females produced less viable eggs (Sanil and Shetty, 2012). 

It is speculated that chemical constituents of P. dodecandra could have caused abnormalities in the developing embryo and this had a direct effect on the larvae and some fundamental aspects related to the larval development (Promsiri et al., 2006). Similar findings were reported in fertility studies of Stegomyia albopicta, in which the percentage of their egg hatching was reduced after sublethal exposure of females to boric acid sugar bait (Ali et al.,2006) and Cx. quinquefasciatus and Ae aegypti, after exposure to leaf extracts of Calophyllum inophyllum and Rhinocanthus nasutus (Muthukrishnan and Pushpalatha, 2001).

It has been observed that an insect’s immune system is susceptible to different factors including but not limited to insecticides or plant-derived compounds (Zibaee and Bandani, 2010; James and Xub, 2012). The suppression of this system upon exposure to biopesticides arrests the insect’s cell cycle and induces apoptotic effects in insect cell lines (Huang et al., 2011; Shu et al., 2015) and also makes the insects more susceptible to infection (Zibaee et al., 2012) and other negative environmental factors. Although the physiological effect of exposure to extracts of P. dodecandra on exposed An. gambiae mosquitoes was not assessed in the present study, the fact that the embryos died and larvae either died immediately after hatching or had a protracted larval development period suggests that exposure to extracts of P. dodecandra could have impaired the exposed An. gambiae embryo’s immune system interfering with their humoral and cellular immunity responses. This then could have made them vulnerable to infections and other environmental stressors that could have interfered with their development patterns. In this regard, the finding of this study is similar to an observation reported for Rhodnius prolixus Stal (Hemiptera: Reduviidae) (Figueiredo et al., 2006), Spodoptera litura Fabricius (Lepidoptera: Noctuidae) (Sharma et al., 2003) and Spodoptera littoralis Boisduval (Lepidoptera: Noctuidae) (Shaurub et al., 2014) when they were exposed to azadirachtin an active chemical present in extracts of Azadirachta indica leaves.

3 Material and Methods

3.1 Study area and experimental mosquitoes

The experiments were conducted in the Entomology laboratory at the Centre for Global Health Research/Kenya Medical Research Institute (CGHR/KEMRI). An. gambiae mosquitoes maintained at the laboratories and reared following standard techniques (Das et al., 2007; Parekh et al., 2005) were used. Climatic conditions within insectaries were 29°C~30°C, 70%~80% relative humidity, and 12:12, L:D photoperiod (Yugi et al., 2014).

3.2 Plant extracts preparation

Fresh leaves (shoot and midsection) and mature green fruits of P. dodecandra were collected from the field near Moi Girls High School, Eldoret [+0.518829°N, 35.284927°E], and Kanyagwal, Nyando [-0.250393°N, 34.870190°E]. Fresh leaves of Azadirachta indica (Neem) were also collected from Kanyagwal, Nyando [-0.250393°N, 34.870190°E]. The plant parts were identified by Mr. Patrick Mutiso of the School of Biological Sciences, University of Nairobi, and voucher specimen number JOY2012/001 for P. dodecandra and JOY2012/002 for Azadirachta indica issued and thereafter deposited in the herbarium at the School. The plants were prepared and extracted using standard methods (Das et al., 2007) and the extract kept in airtight glass bottles to serve as stock quantity.

3.3 An. gambiae life history bioassay

An informal ‘after-only with control’ experimental research design (Kothari, 2004) was used to investigate influence of ethanol and water extracts of mature green fruit and leaves of P. dodecandra as environmental stressor on the development time and survival rate of An. gambiae mosquito life stages to adulthood. Eighty milligrams of stock’s crude ethanol extracts of mature green fruits of P. dodecandra was weighed and dissolved in 100 millilitres of rain water and the solution serially diluted to different concentrations of 80, 40, 20, 10, 5, and 2.5 mg/100mL and then added into five groups of six sets of larval rearing trays. Each tray measured 21 cm × 15 cm × 8 cm and contained 100mL of a particular concentration. The procedure was repeated for ethanol extracts of leaves of P. dodecandra, and Azadirachta indica and for water extracts of similar parts of P. dodecandra and Azadirachta indica. Similar concentrations of Deltamethrin were used together with ethanol and water extracts of the plant parts as comparison. Concentrations of extracts of Azadirachta indica and deltamethrin and rain water only were used as positive and negative control respectively. To each of the trays was added 100 freshly laid An. gambiae eggs and the set ups left to stand in the laboratory at temperatures of 29°C~30°C. An. gambiae mosquitoes were exposed to the treatments at the egg stage only. The experiments were replicated four times.

The eggs were left to incubate in the treated solutions and observed on the 3^rd, 5^th, 7^th, 9^th, 11^th, and 13^th day to access and count emerged larval instars 1 (L1s), larval instars 2 (L2s), larval instars 3 (L3s), larval instars 4 (L4s), pupae and emerged adults respectively. Periodic observations were made to determine the number of individuals of each An. gambiae immature stage that died or survived. Rearing water was changed twice; at the L2s, and the L4 stage respectively. Feeding was done once every two days at a rate of 0.03 milligrams of TetraMin^® Baby fish food per larva. Pupae developing from L4s were collected put in collection plastic cups and placed inside adult mosquito holding paper cups with mouths covered in mosquito netting to prevent the emerged adults from escape. The paper cups were labeled with the type of treatment used against the larvae and provided with 10% sugar solution soaked in cotton wool placed on the netting cover to feed the emerged adults. The emerged adults were counted and noted for each treatment and concentration and thereafter the experiment stopped.

Toxicological activities of the extracts were tested in accordance with the WHO procedure (WHO, 1997). Records of the immature mortalities were taken at the end of observation activities and for every set up, moribund, and dead larvae were collected and disposed off in a septic tank. Mortality rate was 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).

3.4 Data analysis

Data was entered in excel spreadsheets and the relationship between larvicidal 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.

Acknowledgements

The author wishes to sincerely thank Richard Amito, Charles Owaga, Trevor Omondi, and Harnel Owiti for culturing the experimental mosquitoes, Centre for Global Health Research (CGHR) for laboratory space and mosquitoes. This study was funded by the National Commission for Science Technology and Innovation [(NACOSTI) Grant contract # NCST/5/003/3^rd Call PhD/056].

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