Research Report

Toxicity of Three Tropical Plants to Mosquito Larvae, Pupae and Adults  

Owoeye, J. A.1 , Akawa, O. B.2 , Akinneye, J. O.1 , Oladipupo, S. O.1 , Akomolede, O. E.1
1 Department of Biology, Federal University of Technology, P.M.B. 704, Akure, Ondo State, Nigeria
2 Department of Pharmacology, Afe Babalola University, P.M.B 5454, Ado-Ekiti, Ekiti State, Nigeria
Author    Correspondence author
Journal of Mosquito Research, 2016, Vol. 6, No. 16   doi: 10.5376/jmr.2016.06.0016
Received: 10 Apr., 2016    Accepted: 22 Jun., 2016    Published: 28 Jun., 2016
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This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Owoeye J.A., Akawa O.B., Akinneye J.O., Oladipupo S.O., Akomolede O.E., 2016, Toxicity of Three Tropical Plants to Mosquito Larvae, Pupae and Adults, Journal of Mosquito Research, 6(16): 1-6 (doi: 10.5376/jmr.2016.06.0016)


The toxicity of the extracts of three common tropical plants; Tobacco plant (Nicotiana tabacum), West African Black Pepper (Piper guineense) and Jimson weed (Datura stramonium) was evaluated on the larvae and pupae of Anopheles gambiae while the fumigation effect of the extracts was investigated on the adult mosquito. The concentrations of the extract used in the study were 0.1%, 0.2%, 0.4%, 0.8% and 1.0%. The extracts of the leaves of N. tabacum and seeds of P. guineense had significant larvicidal and pupaecidal effect on A. gambiae at 1hour and 24hours observation having LC95 values of 0.67mL and 0.1mL for larvae and 0.19mL and 0.1mL for pupae respectively. However, only N. tabacum at 0.8% and 1.0% had significant adulticidal effect on the adult mosquito under 1hour observation. The extract of D. stramonium leaves had LC95 value of 1.07mL for larvae and 0.91mL for the pupae. It was found to be least toxic to the larvae and pupae after 1hour and 24 hours observation period. It also had no lethal effect on the adult A. gambiae at all concentrations. It is inferred from the results obtained that the extracts used had some bioactive constituents, which resulted in the mortality effects.

Toxicity; Larvicidal; Mortality; Bioassay; Nicotiana tabacum; Piper guineense; Datura stramonium; Anopheles gambiae

1 Background

Mosquitoes have become the most important single group of insects well-known for their public health importance, since they act as the vector for many tropical and subtropical diseases. (Egunyomi et al., 2010). Of the 42 genera of this family; the genus AedesCulexAnopheles and Mansonia are responsible for the transmission of the most common and important diseases causing significant mortality and morbidity in man and animals (Akinkurolere and Zhang, 2007). Mosquitoes are estimated to transmit disease to more than 700 million people annually in Africa, South America, Central America, Mexico and much of Asia with millions of resulting deaths (Akinkurolere et al., 2011). Malaria is the most prevalent of mosquito borne diseases; being endemic in about 109 countries, infecting 190-330 million people and causing about 1 million deaths every year. The species responsible for the transmission of the causative pathogen of malaria is the Anopheles; the control of which has been mainly effected by use of conventional insecticides but these have caused their own problems, such as adverse effects on the environment and the encouragement of pesticide resistance in some mosquitoes (Su and Mulla, 1998). Nigeria is known for high prevalence of malaria and it remains one of the leading causes of childhood cum maternal morbidity and mortality, low productivity and reduced school attendance in the country (Aribodor et al., 2007). The burden of malaria has however been increasing due to development of resistance against anti-malaria drugs and insecticides, complex social structures and rapid environmental changes that have intensified in the last decade (Eliningaya et al., 2008). The increase in the development of mosquito resistance is the primary concern of all control programmes (Pimsamarn et al., 2009). Owing to the fact that application of synthetic insecticide has envenomed the surroundings as well as non-target organisms, these problems stimulated a search for safer alternative anti-mosquito control. This has necessitated the use of other alternatives; and in this case, phytochemicals obtained from plants with proven efficacy against mosquitoes. These phytochemicals have been documented to be environmentally safe, cost effective, biodegradable and benign to man (Egunyomi et al., 2010). Hence in recent years, use of environment friendly and biodegradable natural insecticides of plant origin has received renewed attention as agents for vector control and it has been found that herbal extracts are one safer alternative method of control, especially the extracts of certain medicinal plants (Suwanee et al., 2006). Generally most of these phytochemicals can be grouped into five major chemical categories which are (1) Alkaloids (2) Terpenoids (3) Phenolics (4) Growth regulators and (5) Proteinase inhibitors (Marta and Sarah, 2011). An early report on the use of plant extracts against mosquito larvae was that of Campbell et al(1933) where it was found that plant alkaloids like nicotine, anabasine, methyl anabasine and lumpinin extracted from the Russian weed; Anabasis aphylla, killed the larvae of Culex spMonzon et al. (1994) also reported that some medicinal plants containing natural toxins were effective against mosquito larvae. Accordingly, the efficacies of plant extracts have been extensively researched on (Kovendan et al., 2014; Akinkurolere et al., 2011; Effiom et al., 2012; Tennyson et al., 2012).


Since these chemicals are taken from medicinal plants, they are expected to have low human toxicity, a high degree of biodegradation, reduce the risk of adverse ecological effects and should not induce pesticide resistance in mosquitoes (Choochote et al., 1999). As such, the toxicity of the extracts of Tobacco plant (Nicotiana tabacum), West African Black Pepper (Piper guineense) and Jimson weed (Datura stramonium) was evaluated on the larvae and pupae of A. gambiae while the fumigation effect of the extract was investigated on the adult mosquito. This paper reports on the study to;

i.Evaluate the toxic effect of plant extracts on the larva and pupae stages of A. gambiae.

ii.Evaluate the fumigant effect of the plant extract on adult A. gambiae.

iii.Estimate the lethal concentration of each extract to the larval and pupal stages of the A. gambiae.

iv.Determine the most potent plant extract by comparison of their total effect.


2 Materials and Method

2.1 Extraction of oils from plant materials

The extraction of plant materials were done based on the procedures of Hidayatulfathi et al. (2004) with slight modifications. 100g of each plant (sun dried) was blended and filtered using a sieve. The powder was macerated with 500mL of n-hexane, petroleum ether and ethanol solution and left to stand at room temperature for 72 hours. The mixture was filtered through a muslin cloth and glass funnel. The filtrate was then concentrated using the Rotary evaporator in order to get the plant extracts. The extract was collected using a plastic bottle and this was exposed to the air for 24 hours in order to remove the vapour and traces of the extraction solvents that might remain. It was then stored in the refrigerator at 4°C until needed for tests.


2.2 Breeding of mosquitoes

Water, sand and dried leaves were collected in plastic bowl and placed in a shady area of a residential building. After a few days mosquito larvae were observed to be emerging from eggs which have been laid by the adult mosquitoes and the set up was transferred to the laboratory. In the laboratory, the larvae were transferred into plastic containers with water; these were in turn transferred into a cage (30cm x 30cmx 20cm). The cage had net with minute mesh sizes that will not allow the passage of adult mosquitoes. It had a sliding screens and inlets by the side through which the experimenter could put a hand in order to operate within the cage yet preventing the escape of the mosquitoes. The larvae in the beaker were fed with yeast and after a few days, adult mosquitoes were observed to be emerging. Sucrose solution was provided for the male mosquitoes to feed on while a live rat (Rattus norvegicus) was provided for the females to obtain blood meal from.


2.3 Bioassay of extracts

Different concentrations; 0.1%, 0.2%, 0.4%, 0.8% and 1.0% each of the plant extracts were prepared in beakers for the larvicidal and pupaecidal effect. 10 Larvae and pupae of A. gambiae were then introduced into the assays and were afterwards observed every 10 minutes for 1 hour for mortality effect of each extract. The lethal effect of the extracts was also observed after 24hours. The experiment was replicated four times for each concentration of each plant extract. The fumigant effect of the plant extracts on adult A. gambiae was tested by depositing 0.1mL, 0.2mL, 0.4mL, 0.8mL and 1.0mL of each extract on 2cm x 2cm Whatman’s no.1 filter paper strips (Hidayatulfathi et al., 2004). These strips of filter paper were then introduced into the test tubes containing the mosquitoes to be fumigated. The lethal action of each extract was observed and recorded every 10 minutes for 1 hour. Each of the treatment was replicated four times per concentration of each plant extract.


2.4 Statistical analysis

All data were subjected to Analysis of Variance (ANOVA) at 95% confidence level using the Statistical Package for Social Sciences (SPSS) for windows version 16.0. This is done to determine if there are significant differences among the effects of the different concentrations of treatments using same plant extract and to determine if there are significant differences in the effect of the different plant extracts at the same concentration. Tukey’s Post Hoc test was used to separate the means at P<0.05 for significant data after ANOVA analysis. 


3 Results
Piper guineense and Nicotiana tabacum were effective within 1 hour, achieving more than 50% mortality at 0.8% and 1.0%. There was no significant difference in the effect of Piper guineense (F= 18.48; Df = 23) and Nicotiana tabacum (F= 49.54; Df = 23) at 0.1%, 0.2% and 1.0% but at 0.4% and 0.8%, there was significant difference in their effects as Nicotiana tabacum (F= 49.54; Df = 23) effected more death of larvae than Piper guineense (F= 18.48; Df = 23) at those concentrations. Datura stramonium (F= 0.0; Df = 23) is the least effective extract within the hour as no mortality of larvae was recorded in any of its treatment within the hour. Nicotiana tabacum on the other hand is the most effective as 100% mortality was achieved at its treatments’ 0.8% and 1.0% within 1 hour (Table 1).



Table 1 Table of larvae mortality per concentration of plant extract in 1 hour


Although all the extracts effected larvae mortality at all concentrations after 24 hours, there was significant difference at P<0.05 in the percentage mortality of Datura stramonium compared with those of Piper guineense and Nicotiana tabacumPiper guineense achieved 100% larvae mortality at all concentrations after 24hours but its effect was only significantly different from that of Nicotiana tabacum at 0.1% and 0.2% concentrations. There were no significant differences at P<0.05 in their effects at the remaining concentrations(Table 2).



Table 2 Table of larvae mortality per concentration of plant extract in 24 hours


Table 3 shows the toxicity of the plant extracts to the pupae after 1 hour of application. N. tabacum and P. guineense were effective within the hour, achieving about 100% mortality effect. D. stramonium had little or no significant effect on the pupae of the A. gambiaeTable 4 shows the effect of the plant extracts on the pupae after 24 hours. P. guineense was able to attain 100% mortality at all concentrations after the 24 hours period. D. stramonium treatments achieved significant mortality rates at 0.8% and 1.0% but still remained the least active extract in the set up.



Table 3 Table of pupae mortality per concentration of plant extract in 1 hour



Table 4 Table of pupae mortality per concentration of plant extract in 24 hours


Table 5 shows the fumigant effect of the extracts on the adult A. gambiaeD. stramonium and P. guineense were both ineffective at all concentrations while N. tabacum only produced tangible results at 0.8% and 1.0% concentrations within the 1 hour observation period. Effectiveness of extract can be assumed to be concentration dependent as higher mortality was recorded at 1.0% concentration than at 0.8% concentration of the N. tabacum treatment.



Table 5 Table of adult mortality per concentration of plant extract in 1 hour


4 Discussion

According to Saxena et al. (1993), exposure of larvae of Anopheles stephensi to plant alkaloids resulted in a significant loss in fecundity, fertility and caused mortality in the adult species of the mosquitoes. In the same vein, the ethanol, petroleum ether and n-hexane extracts of the leaves of D. stramonium, seeds of P. guineense and leaves of N. tabacum respectively showed toxicity to the pupal and larval stages of the mosquito. However it was only the extract of N. tabacum that exhibited adulticidal activity against the mosquito at the treatment concentrations studied.


Out of the three plant extracts, N. tabacum and P. guineense produced the most effective larvicidal activity achieving close to 100% larvae mortality within 1 hour period at high concentrations. At 24 hours, the extracts of these two plants ultimately achieved 100% larval mortality at all treatment concentration. The LD95 values for larvae are; 0.10mL, 0.67mL and 1.07mL for P. guineense, N. tabacum and D. stramonium respectively.


In a similar experiment by Khalequzzaman et al. (2010), P. nigrum; a close relative of P. guineense was reported to have been effective exhibiting larvicidal activity at LD50 values of 0.56 ppm and 0.65 ppm accordingly. This effect was related to the presence of plant alkaloid which is suspected to have toxic effect on mosquito larvae.


The overall effectiveness of plant extracts on larvae was attributed to the properties of the alkaloids they contain. The main phytochemical contained in the seed of P. guineense is Piperine and the effectiveness of P. guineense could be directly attributed to its action. However, Dillapiol, a phenylpropanoid isolate from essential oils of leaves of Piper sp could also be involved. It has been by shown by Rafael et al., (2008), that dillapiol affects the formation of micronuclei during mitosis leading to chromosomal aberrations. It also affected oviposition and caused mortality when extracted from Piper aduncum and tested in vivo on the larvae and pupae of Aedes aegypti. For N. tabacum, the active phytochemical is suspected to be Nicotine which acts as Synaptic poison (Acetylcholine receptor agonist). It blocks nicotinergic acetylcholine receptors leading to the accumulation of acetylcholine, resulting in the insect's paralysis, and eventually death.


The extract of D. stramonium was the least toxic to the larvae of the A. gambiae. It had no lethal effect on the larvae within 1 hour observation period but produced considerable mortality at the 24 hours observation period, having a LC95 value of 1.07mL. The active phytochemicals in D.stramonium are atropine, hyoscyamine and scopolamine which are classified as deliriants.


Sharma and Saxena (1994) also found that the petroleum ether extract of Tagetes erectes had toxic effect on larvae of Anopheles stephensi and on its significant growth index. Thus the physiology and morphology of the larvae may also be considered as factors predisposing them to effectiveness of plant extracts as larvae cuticle may probably selectively allow the permeability of substances.


Effect of the plant extracts on the pupae of the mosquito was more tenacious as mortality was achieved faster than that of the larvae within 1 hour. This may be due to the physiological process of moulting that is imminent to the pupae. There were no significant differences at P<0.05 in the treatments of N. tabacum and P. guineense except at the 0.1% concentration. That of D. stramonium was significantly different at P<0.05 from the two, only the 8% concentration of its extract recorded a 2.5% mortality which is negligible as the same percentage of mortality was recorded in the control experiment. Some larvae in the lower concentrations of D. stramonium treatment were still able to moult and metamorphose into adult while some others moulted and drowned.


N. tabacum achieved 100% mortality at all concentrations and is considered the most toxic to the pupal stage of the A. gambiae mosquito. Mortality rate might also be considered to be concentration dependent as was observed in the treatments of Piper guineense extracts.


Against the adult, D. stramonium and P. guineense were both ineffective at all concentrations within the observation period. The ineffectiveness of P. guineense contradicts a result of a similar experiment on Stegomyia aegypti where the adulticidal effect of some close relatives of P. guineense was established. In the experiment, P. sarmentosum, P. ribesoides and P. longum had LD50 values of 0.14, 0.15 and 0.26 microg/female, respectively (Choocote et al.; 2006). In a similar experiment by Hidayatulfathi et. al. (2004), it was found that hexane fraction of the P. aduncum crude extract was effective showing LC50 and LC90 values of 0.20 mgcm-2 and 5.32 mgcm-2N. tabacum only produced tangible results at 0.8% and 1.0% concentrations within the 1 hour observation period. Effectiveness of extract can be assumed to be concentration dependent as higher mortality was recorded at 1.0% concentration than at 0.8% concentration of the N. tabacum treatment. Volatility, stability and pungency of plant alkaloids may also be an important factor to be considered in the efficiency of the plant extracts as adulticides.


Generally, effectiveness of plant extract is related to concentration and exposure period of test populations of insect life stages to extracts of plants as mortality was higher in higher concentrations and more mortality observed at longer exposure time.


Comparatively, N. tabacum extract is the most toxic extract on all the stages of the A. gambiae mosquito whose susceptibility was investigated. However, P. guineense was the most effective against the aquatic life stages of A. gambiae. D. stramonium was the least toxic of the utilized plant extracts. It achieved the lowest mortality rate in all the stages of A. gambiae that was investigated. This might probably be due to the part of plant that was used.


5 Conclusion and Recommendation

It was inferred from the experiments conducted on the effectiveness of the extracts of N. tabacum, P. guineense and D. stramonium as mosquitocides that they all have bio constituents that are toxic to the larvae and pupae of the A. gambiae mosquito. Only N. tabacum had considerable adulticidal effect. Further investigations on the fumigant and repellence effect of these extracts at higher concentrations are recommended. Findings like this are useful in promoting research aimed at the development of new agents for mosquito control based on bioactive natural chemical compounds from indigenous plant sources. The potential of these plant species, as possible mosquitocides, established convincing activity for further researches to develop natural substances for combat against adult mosquitoes. Hence, providing a great stride which will help cover large grounds in the step to controlling mosquitoes and the pathogens they transmit.


However with respect to availability, efficiency, ease of extraction and quantity of active ingredient extractable, N. tabacum is considered the best to be used as mosquitocide.



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