Abstract
Aim: This study aimed to evaluate larvicidal activity of Hypoestes forskaolii R. Br root extract against 3rd instar Anopheles gambiae, Aedes aegypti, and Culex quinquefasciatus.
Methods: A protocol developed by the World Health Organization was adopted, with minor modification using chloroform and methanol extracts with concentrations ranging from 25–750 µg/mL.
Results: The H. forskaolii chloroform extract exhibited very high larvicidal activity after 72 hours of exposure, with LC50 2.0322, 3.8989, 6.0004 μg/mL against A. gambiae, A. aegypti, and C. quinquefasciatus, respectively.
Conclusion: The larvicidal activity of H. forskaolii is reported for the first time in this paper. The effectiveness of H. forskaolii chloroform extract warrants further research to develop botanical mosquito repellants from this source.
Keywords: Hypoestes forskaolii, LC50, Anopheles gambiae, Aedes aegypti, Culex quinquefasciatus
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Introduction
Mosquitoes are a potential vector of several tropical diseases, including numerous viral diseases: of 3,000 species existing, 100 are known to be vectors.1 It has been reported that mosquitoes are more effective in disease transmission than any other arthropods, and thus are regarded public enemy number one, as reported by the World Health Organization (WHO).2 Mosquitoes are known to transmit such diseases as malaria, dengue fever, chikungunya, Rift Valley fever, filariasis, West Nile fever and Japanese encephalitis.3,4 It is estimated that more than 700 million people are infected with mosquito-transmitted diseases annually, which leads to death, poverty, and social and economic disturbances.5 Synthetic chemical pesticides have been used for a long time in controlling mosquitoes, but their arbitrary use has given rise to known and serious problems, including genetic resistance, increasing cost of application, hazards from handling, and environmental pollution.6,7 The search for effective and biodegradable pesticides, including mosquito repellents, is of paramount importance. One of the potential source is plants that are known to be used by communities in the management of insects.
In Tanzania, H. forskaolii is used for the management of houseflies, especially among pastoralist communities. The concoction from the roots of this pant is mixed with milk and placed in an open area. Milk is used, as it attracts houseflies and cockroach especially. Insects that feed on the product die instantly as they feed. The remarkable activity of H. forskaolii against houseflies and cockroaches prompted our research group to investigate the lavicidal activity of H. forskaolii against third-instar Anopheles gambiae, Aedes aegypti, and Culex quinquefasciatus.
H. forskaolii is an annual or perennial herb that grows up to 1 m tall, with its stem and leaves being nearly glabrous. It has pale-pink or white flowers. It is a polymorphic species found in most habitats. It is most common in open woodland and wooded grassland on sandy soils or rocky slopes and in disturbed areas, such as roadsides. It also occurs in riverine areas and open forest. The plant species is widespread in tropical and southern Africa from Senegal to Somalia, south of Namibia, and South Africa. It extends to the Saharan highlands, the Arabian Peninsula, and Madagascar. Musayeib et al reported antiprotozoal activity of methanolic extracts against Plasmodium falciparum, Leishmania infantum, Trypanosoma cruzi, and Trypanosoma brucei.8 This paper thus report the antilarvicidal activity of H. forskaolii chloroform and methanolic extracts.
Methods
Chemicals and mosquito larvae
Chloroform, methanol and dimethyl sulfoxide (DMSO) were purchased from Avantor Performance Materials India. The third-instar larvae of A. gambiae, A. aegypti and C. quinquefasciatus were obtained and reared at the Tropical Pesticides Research Instutute, Arusha, Tanzania.
Collection of plant roots and preparation of extracts
H. forskaolii was identified by Dr Ephraim Njau of the National Herbarium, Tropical Pesticide Research Institute at the collection site (Hanang district in Manyara region, Tanzania). The plant specimen is kept at the Nelson Mandela Institution of Science and Technology, coded as HF1423. Roots were chopped, washed, blended, and sequentially macerated using chloroform (analytical grade) and methanol (analytical grade) for 72 hours. Solvents were removed through vacuum with a rotary evaporator (Heidolph, Germany). Methanolic and chloroform extracts were kept in the refrigerator at −20°C until testing.
Larvicidal activity
The WHO protocol of 1996 year was adopted for larvicidal assays, with minor modifications.9 Larvae of A. aegypti and C. quinquefasciatus were fed with dog biscuits, while those of A. gambiae were fed with tetramine during experiment. Stock solutions for methanol and chloroform extracts (500 mg/mL) were established by dissolving 500 mg crude extract in 5 mL DMSO. With serial dilution, concentrations of 25, 50, 100, 200, and 750 μg/mL were prepared from stock solution. Distilled water was used in the serial dilution. Ten late third–instar mosquito larvae were introduced into the test solution and mortality observed and recorded after 24, 36, and 72 hours. Cups with ten mosquito larvae, distilled water, and 0.5 μg/mL DMSO were taken as negative controls. All experiments were done in triplicate under controlled temperature of 25°C±2°C and relative humidity of 75%−85%. Dead larvae were identified by lack of mobiility and not being able to reach the water's surface.
Statistical analysis
FigP software (Biosoft, Cambridge, UK) was used for analysis, and mean percentage mortality was plotted against logarithms of concentrations. For regression equations, LC50, CIs and regression coefficients were calculated.
Results
Larvicidal bioassay results performed on early-third instars of A. aegypti, A. gambiae, and C. quinquefasciatus with chloroform and methanolic root extracts of H. forskaolii are presented in Tables 1–3, respectively. Mosquito larvae were exposed to extracts prepared in DMSO at 25–750 μg/mL and mortality recorded after 24, 36, and 72 hours' exposure. Since the WHO has not established standard criteria for determining the larvicidal activity of natural products, several authors have developed individual sets of criteria to characterize the potency of mosquito larvicides developed from natural products.10
Table 1.
Larvicidal activity of Hypoestes forskaolii root extracts against Aedes aegypti
| Extract code | Time | LC50 (μg/mL) | 95% CI | R2 | Regression equation |
|---|---|---|---|---|---|
| HFCE | 24 hours | 154.6019 | 1,706.3408–14.0076 | 0.938 | y=10.57logx+26.86 |
| 36 hours | 15.0053 | 110.7175–2.0336 | 0.93 | y=10.44logx+37.72 | |
| 72 hours | 3.8989 | 22.1742–0.6856 | 0.87 | y=12.15logx+42.82 | |
| HFME | 24 hours | 220.4789 | 904.4034–53.7492 | 0.994 | y=18.77logx+6.015 |
| 36 hours | 56.3484 | 226.2731–14.0323 | 0.946 | y=17.02logx+20.20 | |
| 72 hours | 11.5432 | 54.3812–2.4502 | 0.96 | y=14.28logx+34.83 | |
| Control | NM | – | – | – | – |
Abbreviations: HFCE, H. forskaolii chloroform extract; HFME, H. forskaolii methanolic extract; NM, no mortality.
Table 3.
Larvicidal activity of Hypoestes forskaolii root extracts against Culex quinquefasciatus
| Extract code | Time | LC50 (μg/mL) | 95% CI | R2 | Regression equation |
|---|---|---|---|---|---|
| HFCE | 24 hours | 177.5595 | 1661.1320–18.9794 | 0.856 | y=11.43logx+24.29 |
| 36 hours | 18.0962 | 97.3554–3.3637 | 0.933 | y=13.51logx+33.01 | |
| 72 hours | 6.0004 | 27.4143–1.3133 | 0.851 | y=13.93logx+39.16 | |
| HFME | 24 hours | 137.7328 | 530.6684–35.7471 | 0.96 | y=18.70logx+10.55 |
| 36 hours | 31.7442 | 112.8697–8.9271 | 0.97 | y=18.02logx+22.94 | |
| 72 hours | 6.4358 | 23.0496–1.7961 | 0.972 | y=16.51logx+36.65 | |
| Control | NM | – | – | – | – |
Abbreviations: HFCE, H. forskaolii chloroform extract; HFME, H. forskaolii methanolic extract; NM, no mortality.
According to Komalamisra et al, larvicidal activity of the plant extract is consideredinactive when LC50 is >750 μg/mL, weakly effective if LC50 is 200–750 μg/mL, moderate if LC50 is 100–200 μg/mL, effective if LC50 is 50–100 μg/mL, and highly effective if LC50 is <50 μg/mL.11 Results from this study displayed larvicidal activity for H. forskaolii against three species of mosquito tested, giving LC50 values of 220.4789–3.8989 μg/mL for A. aegypti (Table 1), 69.6596–2.0322 μg/mL for A. gambiae (Table 2), and 177.5595–6.0004 μg/mL for C. quinquefasciatus (Table 3). Chloroform and methanolic extracts were highly effective after 72 hours' exposure, and this showed the extracts were remarkably effective in controlling the mosquito larvae tested. The activity was species-specific, which clearly revealed that chloroform extract had higher larvicidal activity: LC50 3.8989 μg/mL against A. aegypti, μg/mL 2.0322 against A. gambiae, and 6.004 μg/mL against C. quinquefasciatus after 72 hours' exposure. The methanolic extract had an LC50 of 11.5432 μg/mL against A. aegypti, 9.5728 μg/mL against A. gambiae, and 6.4358 against C. quinquefasciatus after 72 hours' exposure. The results also showed effective, moderate, and weakly effective larvicidal activity for both chloroform and methanolic extracts after 24 hours' exposure. Chloroform extracts had an LC50 of 154.6019 μg/mL against A. aegypti, 69.6596 μg/mL against A. gambiae, and 177.5595 μg/mL against C. quinquefasciatus. Methanolic extracts also possessed activity: LC50 of 220.4789 μg/mL against A. aegypti, 37.1001 μg/mL against A. gambiae, and 137.7228 against C. quinquefasciatus. Larvicidal effects of H. forskaolii root extract were all <750 μg/mL, which justifies its use in managing the mosquito larvae tested.
Table 2.
Larvicidal activity of Hypoestes forskaolii root extracts against Anopheles gambiae
| Extract code | Time | LC50 (μg/mL) | 95% CI | R2 | Regression equation |
|---|---|---|---|---|---|
| HFCE | 24 hours | 69.6596 | 330.1227–14.6989 | 0.853 | y=15.03logx+22.30 |
| 36 hours | 8.8111 | 33.1905–2.3391 | 0.954 | y=16.19logx+34.70 | |
| 72 hours | 2.0322 | 6.6260–0.6233 | 0.866 | y=16.82logx+44.82 | |
| HFME | 24 hours | 37.1001 | 159.9947–8.6029 | 0.984 | y=16.00logx+24.89 |
| 36 hours | 7.4977 | 25.1042–2.2393 | 0.940 | y=17.43logx+34.75 | |
| 72 hours | 9.5728 | 15.22–1.5400 | 0.973 | y=8.965logx+68.02 | |
| Control | NM | – | – | – | – |
Abbreviations: HFCE, H. forskaolii chloroform extract; HFME, H. forskaolii methanolic extract; NM, no mortality.
Discussion
Mosquitoes in the larval stage are attractive targets for pesticides, because their breeding sites in stagnant water can be easily accessed, but the use of chemical pesticides in water sources introduces more risks to humans and the environment.12,13 Natural pesticides derived from plants are thus promising tools, especially for managing mosquito larvae.14–16 H. forskaolii has been used to manage insect vectors and handling insect vector–borne diseases in Tanzania.17 The latter study revealed larvicidal activity of H. forskaolii against A. aegypti, A. gambiae, and C. quinquefasciatus. Mortality was up to 50%, with LC50 of 220.478–2.0322 μg/mL. H. forskaolii chloroform extract demonstrated the highest larvicidal activity, with LC50 of 3.8989, 2.0322. and 6.004 μg/mL against A. aegypti, A. gambiae, and C. quinquefasciatus, respectively. These findings of the lavicidal activity of H. forskaolii root extracts suggest the use of this plant in the management of mosquitoe larvae.
Winisia et al reported on larvicidal activity of fruits and leaves of Clausena anisata growing in Tanzania. C. anisata ethyl acetate and methanolic leaf extracts exhibited remarkable larvicidal activity, with LC50 of 0.0977 and 0.9362 μg/mL.18 This information qualifies the potential of Tanzania plants in the management of mosquitoes and thus mosquito-borne diseases.
Gas chromatography–mass spectrometry was used to analyze H. forskaolii chloroform root extract, wherein 102 secondary metabolites belonging to sesquiterpenes, diertepenes, fatty acids and alkane were identified. Of the phytochemical compounds identified caryophyllene and caryophyllene oxide have been reported to exert larvicidal activity.19,20
Mosquito management has been exercised through tremoval of mosquito habitats, use of structural barriers, control of mosquitoes at the larval stage, and control of adult mosquitoes. In some cases, an integrated mosquito-control strategy has been used. Each tactic has its own advantages and disadvantages. Focusing mosquito-reduction efforts on the larval stage has the advantage of controlling the vector prior to dispersal or acquisition of the disease and interrupting the life cycle before it can cause harm. Although a botanical larvicidal agent has not yet impacted the market, there are a number of chemicals available to target mosquito larvae, including such organophophates as temephos and insect growth regulators like methoprene,21 although resistance has been found to each of these in the field.22,23 Resistance of mosquito larvae to available larvicidal agents has prompted the intensity of the search for agents. The findings presented in this paper throw light on the possibility of developing botanical larvicidal agents from H. forskaolii.
Conclusion
The results clearly reveal that both chloroform and methanol root extract of H. forskaolii are potential sources of larvicidal agents against A. aegypti, A. gambiae, and C. quinquefasciatus.
Acknowledgments
The authors are grateful to the Africa Center for Excellence in Research, Agriculture Advancement, Teaching Excellence, and Sustainability (CREATES) through the Nelson Mandela African Institution of Science and Technology for funding this study, Cornelius T Nary the herbalist from Endasak–Hanang for availing us of ethinomedicinal use of the plant, and Dr Ephraim Njau the botanist from Tropical Pesticide Research Institute for the identification of the plant.
Disclosure
The authors report no conflicts of interest in this work.
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