Anopheles gambiae larvicidal and adulticidal potential of Phyllanthus amarus(Schumach and Thonn, 1827) obtained from different localities of Nigeria

Kingsley Uchenna Ozioko, Chris Ikem Okoye, Fabian Chukwuemenam Okafor, Rose Nduka Obiezue

Parasitology and Public Health Unit, Department of Zoology and Environmental Biology, University of Nigeria, Nsukka 400241, Nigeria

ABSTRACT

KEYWORDS:Adulticidal; Anopheles gambiae; Different localities;Larvicidal; Phyllanthus amarus

1.Introduction

Anopheles (An.) gambiae is a pest of significant public health importance, responsible for the transmission of malaria in humans.Malaria, which is an infectious disease caused by single-celled protozoan parasites of the genus Plasmodium and transmitted by female Anopheles mosquitoes.In humans, the parasites multiply in the liver to infect red blood cells.Currently, there is no effective vaccine available for the prevention of malaria.One of the methods for reducing and interrupting malaria transmission is by the use of synthetic insecticides[1].Insecticides are used to reduce the population density of hematophagous insects’vectors[2].Despite the effectiveness and benefit of synthetic insecticide against several mosquito vectors, its use has resulted in several issues such as the rising occurrences of insecticide resistance and adverse effects on the ecosystem.The insecticides resistance of mosquito has increased the complexity of malaria elimination and its vectors control programme[3].An.gambiae mosquitoes are the commonly known vectors of seasonal malaria transmission in Nigeria.Several cases of insecticide resistance of An.gambiae have been reported in Nigeria[4,5].Indiscriminate and improper use of insecticides has brought insecticide selection pressure in killing the Anopheles mosquitos, as evidenced by the increased frequency of phenotypic and knockdown resistance[4].Insecticide from plant origin has been known to be biodegradable, target-specific, and cost-effective[6].Plants and their products have been used to control mosquito vectors before the synthetic organic insecticide was discovered[7,8].Abirami et al.[9]reported that the insecticidal activity of plant extract depends upon the plant species, insect species, geographical varieties, plant parts used, extraction method adopted, and the polarity of solvents used during extraction.Phyllanthus (P.) amarus (Euphorbiaceae),also known as stone-breaker, is an herbaceous shrub distributed widely in Nigeria.The plant has been used to treat human-related diseases over the years.It contains bioactive compounds that have behavioral and physiological effects on insects[10].Euphorbiaceae family’s medicinal nature has been attributed to their complex habitat with varying stress factors, as evidenced by the varied assemblage of secondary substances in different species[11].Traditionally, it is called Geeron-tsuntsaayee (Hausa) Eyinolobe (Yoruba), and Enyikwonwa(Igbo) in Nigerian languages[12].To the best of our knowledge,the insecticidal activity of P.amarus has not been demonstrated convincingly, therefore, this study aims to determine the insecticidal effect of extracts of P.amarus from different geographical zones in Nigeria by testing larvicidal and adulticidal activities against An.gambiae.

2.Materials and methods

2.1.Location and sample collection

The selection of the plant for the study was based on previous scientific literature such as widespread distribution and medicinal usage in Nigeria[13-15].Fresh mature shoots and seeds of P.amarus were randomly collected (between April and November, 2018) from each of the three major ethnic districts in Nigeria, namely Yoruba metropolis in the southwest (SW), Hausa metropolis in the northwest(NW), and Igbo land in the southeast (SE) of Nigeria.The samples were collected between latitude 4.57° E and longitude 7.77°N for SW; latitudes 7.43°E and longitudes 10.52°N for NW; latitudes 7.50°E and longitude 7.77°N for SE.Each of the three locations was selected as the representative of a zone with a similar climatic tendency in Nigeria.Southeast receives more than 120 inches (3 000 mm) of rain a year; southwest receives about 70 inches (1 800 mm); while Northwest receives no more than 20 inches (500 mm) a year[16].The ranking order for monthly mean evapotranspiration (mm/day) in the various zones during the peak landscape activity from March to June is SW＜SE＜NW[17].

The collected P.amarus plants were identified in the field by an ethnobotanist and their shoots were transported to BioResources Development and Conservation Centre Nsukka, Enugu State Nigeria for further identification and authentication.A voucher specimen of the dried plant was deposited in the herbarium of the Department of Plant Science and Biotechnology, University of Nigeria, Nsukka,with a voucher number designated as UNH No.96.The seeds were discarded and the leaves were chopped up into small pieces and dried on the laboratory tables at room temperature.Each sample was further ground into fine powders with a grinding machine and sieved to give a particle size of 50-150 mm.The powdered samples were stored at room temperature in three airtight locked polythene bags and labeled as A, B, and C (SW, NW, and SE, respectively).They were later transported to the University of Nigeria, Nsukka, where extraction and analysis were carried out.

2.2.Extraction

The method earlier described with slight modifications was used for preparation of the extracts[18].Powdered samples of each extract were extracted separately with 20 mL/g of ethanol and filtered with filter paper (Whatman No.1) into three fresh conical flasks.The filtrates were subjected to solvent recovery using a rotary evaporator,leaving behind semi-solid extracts, kept in labeled bottles, and preserved in the refrigerator (4 ℃) before use.Standard stock solutions were prepared (at 1%) by dissolving 1 g of the residue in 100 mL of water containing 0.1% ethanol.From the stock solution,different concentrations (200-600 ppm) were prepared with water by serially diluting the stock solution according to WHO protocol[19].The standard Rambo® insecticide powder (0.6% permethrin) at a concentration of 200-600 ppm was prepared separately from 0.5 g of the powder in 100 mL of water.They were conducted simultaneously for larvicidal and adulticidal bioassays.

2.3.Phytochemical analysis of the ethanolic plant extract

The presence of some important phytochemicals such as alkaloids,flavonoids, tannins, saponins, glycosides, terpenes, and phenols was detected by the Sofowora, Trease GE and Evans standard methods described previously[20,21].

2.4.Test organisms

The larvae of mosquitoes were collected from stagnant water within the Nsukka area and identified into different species in the laboratory following the method as described in an earlier publication[22].The An.gambiae larvae were separated from the mixed culture and transferred into mosquito rearing device, fed with yeast, and stabilized to produce adult.The adults were fed with 10% w/v sugar solution and blood-fed with albino rats; the rearing environment was under 12 h light/12 h dark condition at (27±5) ℃.The F2 generations were used for the assay.

2.5.Larvicidal activity

The larvicidal activity of the plant crude extract was conducted according to WHO standard procedure as described by Rohani et al.[19]with little modifications.Each of the treatments was tested in triplicate at various concentrations (200-600 ppm), along with the control experiment dosed up with an equivalent amount of each solution free of the tested treatment.Both treatment and control groups were kept under the same conditions.A total of 480 fourthinstar larvae were used per treatment.The larvae were randomly distributed into 4 groups (1, 2, 3, 4) each of 120 instars larvae and further randomized into 6 replicate experiments (A, B, C, D, E, F)of 20 instars larvae into plastic cups, each containing appropriate extract concentration in 200 mL of water mixed with a small amount of yeast powder.Mortality was observed after 16, 32, and 48 h post-treatment.There were three trials for each treatment from which data were pooled together for the analysis.Dead larvae were identified when they failed to move after tapping their siphons or cervical regions with a needle.Dead larvae were removed as soon as possible to prevent decomposition, which may cause rapid death of the remaining larvae.If more than 2 larvae in each test pupate in the course of the experiment, that particular test is repeated.The mortality of the treated group was corrected using Abbott’s formula as stated by Ashwini et al.[23].

2.6.Adulticidal activity

An adulticidal bioassay was performed according to a previous study[24]with slight modifications.The adulticidal activities of the treatments were evaluated at various concentrations and control groups.Each treatment was tested in triplicate at various concentrations (200-600 ppm), along with the control experiment dosed up with the equivalent amount of each solution free of the tested treatment.Both treatment and control groups were kept under the same conditions.The bioassay was conducted in experimental kits consisting of transparent plastic tubes.Each tube was sealed on top with a cover quaze.Some tubes were used to expose the mosquitoes to treatments, while some were used to hold them after the treatments.A total of 360 An.gambiae adults were used per treatment.The adults were randomly distributed into 4 groups (1, 2,3, 4) each of 90 An.gambiae adults and further randomized into 6 replicate experiments (A, B, C, D, E, F ) of 15 adults into each of the plastic holding tubes and allowed to acclimatize for 1 h.Subsequently,filter papers impregnated with various concentrations of the treatments were placed on top of the cover quaze and knockdown rate was recorded from the average of three replicates after 1 h post-treatment period.At the end of the exposure, the mosquitoes were transferred back to the holding tubes where pads of cotton wool soaked with 10 percent glucose solution were introduced.The holding tubes were then placed inside the mosquitoes rearing chamber and mortality was observed after 24 hours post-treatment recovery period.There were three trials for each treatment from which data were pooled together for the analysis.The adulticidal activity was calculated by counting dead mosquito from the introduced mosquito.Mosquito was considered dead if it was lying on its back or side and was unable to maintain flight after a gentle tap with a soft brush.The control mortalities were corrected using Abbott’s formula as shown below:

2.7.Statistical analysis

The data were subjected to log-probit analysis to calculate LCand LCwith a 95% confidence limit.χgoodness of fit tests, and regression co-efficient, were recorded.Whenever χvalue was found significant (P＜0.05).A heterogeneity correction factor was used in the calculation of confidence limits.The effect of treatments at various concentrations on mosquito mortality was evaluated by one-way analysis of variance followed by Tukey test.Mortality data were tested for normality before analysis and log-transformed due to violations of homogeneity of variance.The collected data were analyzed statistically using MS Excel® 07 and SPSS software version 20.0.P＜0.05 were considered to be statistically significant.

3.Results

3.1.Phytochemicals screening

Each of the plant samples revealed the presence of alkaloids,flavonoids, saponins glycosides, terpenes, phenols, and tannins(Table 1).

Table 1.Phytochemicals present in Phyllanthus amarus from three different geographical zones.

3.2.Mortality of 4th instar of An.gambiae larvae at 48 h post-treatment with various extracts in comparison with a commercial insecticide

Table 2 shows the mean mortality of An.gambiae larvae after 48 h exposure to different concentrations of P.amarus plant extracts and standard insecticide.The mosquito mortality occurred in a dosedependent manner.The highest mortality was observed in NW extract (LC=263.02 ppm; LC=512.86 ppm), followed by SE extract (LC=288.40 ppm; LC=562.34 ppm), and SW extract(LC=295.12 ppm; LC=602.55 ppm).No mosquito mortality was observed in the concentration control group.A significant difference in mortality (P＜0.05) existed among SW extract, SE extract, NW extract, and standard insecticide at various concentrations, apart from 300 ppm concentration of SE extract that was not significantly different from that of SW extract.

3.3.Larvicidal-effect of ethanolic extracts of P.amarus against the 4th instar of An.gambiae larvae

The effect of the various extracts of P.amarus on the fourth instar larvae of An.gambiae is shown in Table 3.Mortality of the larvae wasobserved at different concentrations of each sampled extract.The larvae mortality of various extracts was found to increase with concentration and duration of exposure.All the extracts treated groups showed good larval mortality against An.gambiae, while concentration control groups showed no mortality after 48 h post-treatment.The LCvalues at 16, 32, and 48 h were 1 819.70 ppm, 1 202.26 ppm, and 912.01 ppm for SW extract, 3 715.35 ppm, 1 862.08 ppm, and 588.84 ppm for NW extract, 1 778.27 ppm, 1 548.81 ppm, and 616.59 ppm for SE extract, respectively.Apart from NW extract at 600 ppm that exhibited the highest mortality at 500 ppm after 32 h(16 h time interval), all other extracts caused the highest mortality against An.gambiae larvae at 600 ppm.Both the NW and SE extracts at 600 ppm exhibited around 50% mortality after 48 h (at 16 h time interval) post-treatment.

Table 2.Mortality of 4th instar of Anopheles gambiae larvae after 48 h post-treatment at different concentrations of various extracts of Phyllanthus amarus in comparison with a commercial insecticide.

Table 3.Effect of the extracts against the 4th instar of Anopheles gambiae larvae at three time interval of 16 h (0-16 h, 16-32 h and 32-48 h).

Table 4.Mortality of Anopheles gambiae adults after 24 h post-treatment at different concentrations of various extracts of Phyllanthus amarus in comparison with a commercial insecticide.

3.4.Mortality of An.gambiae adults after 24 h posttreatment with various extracts in comparison with a commercial insecticide

Table 4 indicated the number of adult death was proportional to the level of concentration in each plant extract.Generally, as the concentration increases, the rate of adult mosquito mortality increases.The LCand LCvalues of SW, NW, and SE extracts of P.amarus against An.gambiae were 275.42 & 588.84 ppm,301.99 & 602.55 ppm, and 316.22 & 660.69 ppm, respectively.The exposure of ethanol extract of P.amarus at the concentration of 200 ppm for 24 h to An.gambiae caused no significant decrease (P＞0.05)in the mosquito mortality as compared to the treatment control.The SW extract showed maximum adulticidal effect against An.gambiae with LCand LCvalues (275.42 ppm and 588.84 ppm,after 24 h).No mortality was detected in any of the concentrationcontrol groups.The mortality of the SW extract was almost equal to that of the Rambo® insect powder at 600 ppm, and there was no statistically significant difference between the two groups.An.gambiae adults initially flew slowly and then ceased to fly when disturbed (knockdown) before death during the exposure period.

Table 5.Median lethal concentration and knockdown rate of Anopheles gambiae adults to ethanolic leaf extract of Phyllanthus amarus and a commercial insecticide within 1 h exposure period.

3.5.Median lethal concentration and knockdown rate of An.gambiae adults to ethanolic leaf extract of P.amarus after 1 h post-treatment

The knockdown rate of An.gambiae adults to various extracts of P.amarus using impregnated papers was shown in Table 5.The knockdown rate was recorded at various concentrations ranging from 200 ppm to 600 ppm.All extracts showed knockdown increases with concentrations from 200-600 ppm except for SE and SW extracts at 500 ppm where it slightly decreased.No knockdown was detected in any of the concentration control groups.The statistical data LC, 95% confidence limits and slope of regression lines were also calculated.The LCvalues were expressed as follows:LCvalue of SW extract was 562.34 ppm, NW extract was 645.65 ppm, SE extract was 630.95 ppm, and Rambo® insecticide was 389.04 ppm.

4.Discussion

Nigeria has various geographical distribution and climate types with unique biodiversity that provide supports to millions of people for centuries.The country also has numerous mountains, plains,and a desert that result in distinct weather and climate ranging from equatorial in south, tropical in center, and arid in north.Each of the geographical location is covered with plants of various species;many have been published in ethnobotanical data, and culturally characterized in literature, including traditional herbal insecticides for controlling insect pest.The present study has demonstrated that P.amarus plant extracts possess insecticidal properties against An.gambiae that transmit malaria parasites.Similarly, Okonkwo and Onyeji[27]have reported a considerable insecticidal effect of P.amarus on An.gambiae adults, where it caused 100% mortalities in 12 h exposure period.Several studies have also demonstrated the insecticidal effect of P.amarus plant extract on several mosquitoes species, including Aedes aegypti and Culex tritaeniorhynchus[25],and other disease causing organisms such as fungi and bacteria[26].Insecticidal activities of other plant extracts against An.gambiae mosquitoes have also been previously reported.For instance, a study performed[28]with each of the Ocimum gratissimum, Chromoleana odorata, Terminalia catappa, Carica papaya, Vernonia amygdalina,and Cymbopogon citrates showed exposure dependent LCand lethal concentration values, which are consistent with our results on the mortality of An.gambiae at different concentrations and varying levels of LCvalues.Similar active compounds such as flavonoids,phenols, alkaloids, tannins, saponins, terpenes, and glycosides were identified from each sampled extract which is in line with the study reported previously[12,29].Therefore, confirming the presence of similar active compounds in each plant extract probably indicates that they are basic compounds which are common to all the samples independent of the localities of collection.Secondly, the tested samples possess important bioactive compounds with insecticidal potential, which can act as natural candidates for the discovery of new products to combat Anopheles.The insecticidal activities observed can be attributed to the secondary metabolites presented in P.amarus that are readily extracted in ethanol[30].Although the active compounds were common in all the sampled extracts, their chemical compositions vary from location to location, which is in line with the report of Boko-Haya et al.[31]that P.amarus extracts contain large families of chemical groups whose number and nature vary according to ecological zones.Variation in activities may be due to differences in the phytochemical composition of the various extracts.The sampled extracts were from three different geo-climatic zones of Nigeria.Each sampled extract contains flavonoids, saponins, and tannins that play a role in plant defense against insects[32]and might have been responsible for larval and adult deaths.Saponins possess insecticidal properties that affect insect physiology in many different ways[33], likewise flavonoids.Glycosides can cause physiological disruption and inhibition of insect vital respiratory enzymes[34].Plant secondary metabolites can penetrate mosquito integument to disrupt its important metabolic reactions[35], hence deprive adult mosquitoes of oxygen and lead to suffocation[36].The present study has indecated that the highest content of flavonoids in NW extract showed the best larvicidal activity, while the lowest content of glycosides in SW extract demonstrated the least larvicidal activities.LCgreater than 200 ppm was recorded in the ethanol extract of P.amarus against 4th instars larvae of An.gambiae, likewise previously reported in petroleum ether extract of the same plant species against 4th instars larvae of mosquito vectors[37].The insecticidal activities expressed by each of the extracts reflect their differences in chemical composition and climatic condition[38].The concentration-dependentmortality results of P.amarus plant against An.gambiae have shown that the concentration may be a factor in the method for toxicity tests on mosquitoes[30]as well as the climatic condition, which is in line with the work of Hada et al.[39].The sequential mortality in correlation with an increase in exposure of plant extracts did not occur in the study as most of the test organisms could resist the effect of the treatments at various concentrations.However, non significant larva percent mortality effects (P＜0.05), observed after 48 h at a higher concentration between 500 and 600 ppm might be to resist the effect of the extract by the larva.NW extract exhibited lesser saponins content than other extracts but still had the highest larvicidal activity.These might be due to the wrong proportion of bioactive compounds or different active compounds with a synergic effect[40].The studied extracts demonstrated remarkable knockdown effects on An.gambiae, hence they can rapidly disrupt the mosquito life cycle at adult stages and effectively reduce its population.Additionally, the efficacy of each sampled extracts on 4th instar larvae of An.gambiae is directly comparable to that in the positive control.

In conclusion, our results indicated that extracts of P.amarus obtained from different geo-climatic zones in Nigeria would be effective against An.gambiae at larva and adult stages of its life cycle.The analysis of the mosquito mortality showed considerable variability in the activities of NW, SW, and SE plant extracts.The variability in insecticidal activities depends on the time of exposure and concentration of the extracts[41,42]as well as ecologic conditions(geographical regions, climate conditions and altitude).Although all extracts showed remarkable insecticidal effects on both An.gambiae larvae and adults, the intensity of these activities remained below that of the standard insecticide.Thus this plant extract provides the bases to act as an alternative to synthetic insecticide in the control programme of mosquito, especially in high prepared stock solutions.It constitutes a potential source of bioactive ingredients, cheaper than synthetic insecticides, and may pose no harmful effects to the ecosystem.Bioactive compounds analysis of extracts in different locations can help in selecting areas for mass production of this plant species to enhance its pharmaceutical and marketing values.The extract can as well be used directly as an insecticidal agent in small volume aquatic habitats or breeding sites of limited size around human dwellings, thereby controlling malaria transmission.

Conflict of interest statement

The authors report no declarations of interest.

Acknowledgements

The authors are grateful to Department of Zoology and Environmental Biology University of Nigeria, Nsukka Postgraduate Board for their lecturers specialist input to the discussion during the proposal presentation and progress of this research and BioResources Development and Conservation Centre, Nsukka for all the help,guidance and facilities provided to accomplish this work and also to the field workers that participated in the sample collection.

Authors’contributions

KUO, CIO, and FCO conceived of the presented idea.KUO,RNO and CIO wrote the protocol, KUO prepared the first draft of the manuscript and conducted the laboratory experiments while CIO supervised the work.KUO and FCO performed the statistical analysis.All the authors read, interpret, and approved the final manuscript.