Evaluation of larvicidal potential of Eritrean medicinal plants against Aedes aegypti

Abstract

Larvicidal activity of three Eritrean medicinal plants was evaluated against Aedes aegypti by conducting the bioassay using WHO methods. Efficacy of the plant extracts of O. hadiense, R. officinalis and C. spinarum was evaluated against 3rd instar Aedes aegypti larvae and mortality was recorded. LC₅₀ and LC₉₀ of the various plant extracts were also calculated using probit analysis. The morphological analysis of treated larvae was also performed. Extracts of O. hadiense, C. spinarium and R. officinalis were prepared using different solvents viz chloroform, 70% ethanol and water. Of the screened extracts, the chloroform extracts of O. hadiense exhibited the highest larvicidal activities and has the minimum LC₅₀ and LC₉₀ (24 mg/ml and 198.411 mg/ml respectively). Chloroform extract of C. spinarium exhibited the least larvicidal activity with maximum LC₅₀ and LC₉₀ (736.883 mg/ml and 1188.699 mg/ml respectively). Microscopic analysis confirmed the changes in the Aedes aegypti larvae caused by various plants extracts. An accumulation of dark pigmentation was observed in abdominal region and in the anal papillae after contact and also showed major structural damage such as destruction of the gut.

Introduction

Mosquitoes are the major vector to transmit more diseases than any other group of arthropods and affect millions of people throughout the world. WHO has declared the mosquitoes as “public enemy number one” because of their ability to act as vectors of pathogens causing malaria, dengue, yellow fever, encephalitis and filariasis (Ghosh et al. 2012). Dengue is a potentially fatal mosquito-borne infection with 50 million cases per year and 2.5 billion people vulnerable to the disease especially in countries from subtropical and tropical regions, where they are more prevalent, with consequent social and economic impacts (Rocha et al. 2015). Mosquito-borne diseases contribute significantly to disease burden, death, poverty and social debility all over the world, particularly in tropical countries (Massebo et al. 2009). According to WHO (2009) about two-fifth of the world’s population is now at risk of dengue and the only way to prevent dengue virus transmission is to combat the disease-carrying mosquitoes. To prevent proliferation of mosquito borne diseases, to improve quality of environment and public health, finding an alternative for the chemical insecticides is essential (Govindarajan et al. 2011). Use of chemical insecticides to manage insects is a major cause of destruction of ecosystem and develops resistance. These problems have imposed the need to explore and develop alternative strategies using eco-friendly, environmentally safe, biodegradable plant products which are non-toxic to non-target organisms too (Rocha et al. 2015). Recently, commercial repellent products containing plant-based ingredients have gained increasing popularity among consumers, as these are commonly perceived as “safe” in comparison to long-established synthetic repellents (Mavundza et al. 2017). Therefore, an attempt has been made to search for a novel, complementary and environmental friendly compounds with high larval mortality.

Materials & methods

Materials

Chemicals: 70% Ethyl alcohol, Chloroform, Dimethylsulfoxide (DMSO).

Medicinal plants used: Leaf part of O. hadiense (chomer), leaf part of C. spinarum (Agam), leaf part of R. officinalis (Azmarino).

Vector: 3rd instar larvae of Aedes aegypti.

Collection & identification of plant material

The selected plants (Fig. 1) were collected manually from different localities of Eritrea during January, 2017. R. officinalis was collected from Asmara (15.3229 °N, 38.9251 °E), Eritrea; O. hadiense was collected from Ghinda (15.4453 °N, 39.0895 °E) and Tselot (15.2810 °N, 38.9671 °E), Eritrea and C. spinarum was collected from Semenawi Bahri (Mai nefhi) (15.2521 °N, 38.7835 °E), Asmara, Eritrea. All the plant samples were identified by Mr. Zekarias Andebrhan, Botanist, Department of Biology, Eritrean Institute of Technology (EIT), Mai Nefhi, Eritrea. The voucher specimens were deposited in School of Allied Health Professions (SAHP), Asmara College of Health Sciences (ACHS), Asmara, Eritrea.

Fig. 1.

Dried Leaves of R. officinalis (Left), O. hadiense (Middle) and C. spinarium (Right)

Processing of plant material

Plant materials (leaves) were collected and washed with tap water. Dried material was coarsely grounded and followed by sieving. The grounded leaves were stored in air tight glass containers (28 °C ± 2 °C) for further use.

Extract preparation

Plant material (100 g each) was placed in conical flask containing aqueous-ethanol (70%), chloroform and distilled water then placed on an electronic shaker for at least 78 h at 45 rpm of speed. The extracts were then filtered using a Buchner’s funnel with Whatman filter paper no 1. The extracts were concentrated using Rota-vapor (Buchi, Inc.) at controlled temperature and pressure. Yield percentage of dry extracts was calculated by using the following formula: %yield = weight of material /weight of extract *100%, All the dry extracts were stored in a glass bottle and refrigerator (4 °C) till further use.

Preparation of various concentrations

2 g of the each crude extract was dissolved in 10 ml of distilled water to prepare a concentration of 200 mg/ml. Desired concentrations of plant extract (200, 100, 50, 25and 12.5 mg/ml) were prepared from the stock solution for exposure the mosquito larva.

Collection of mosquito larva

Larvae of Aedes aegypti (3^rdinstar) were collected from different regions of Eritrea and were reared under the guidance of in-charge, Eilabered Entomology laboratory, Elabered, Asmara, Eritrea. Mosquito were reared at 28 ± 2 °C temperature, 70–80% relative humidity (RH), with photo period of 12 h light and dark cycle. They were provided with commercial fish food.

Larvicidal bioassay

The larval bioassay was carried out by the standard WHO larval bioassay protocol (WHO 2005). Clean plastic cups of 300 ml capacity were used as test containers. Batches of 10 larvae were placed in this 300 ml cup containing 49 ml of test solutions. Three replicates were for each concentration and controls were maintained (Negative control (tap water) and positive control (Transfluthrin 0.88%). Then mortality rates were recorded after 24 h exposure period. Dead and moribund larvae in the triplicates were combined and expressed as a percentage of larval mortality in each concentration. Those larvae which was failed to move after probing with a needle at the terminal segments, siphon or the cervical region were considered as dead larvae and moribund larvae will be those incapable of rising to the surface or not showing the characteristic diving reaction when the water get disturbed.

Determination of LC₅₀ and LC₉₀

LC₅₀ and LC₉₀values (the concentration that cause 50% and 90% mortality) were calculated by using probit analysis. The SPSS software program (version-22) was used for statistical analysis. LC₅₀ and LC₉₀values were determined as significantly different between the extracts with p value < 0.05 (Massebo et al. 2009).

Morphological studies in Aedes aegypti larvae

After the larvicidal bioassay, the dead larvae were separated and probed under light microscope for any aberration in their morphology. Any aberration observed was recorded, photographed and compared with the features observed in the control (water) (Palanikumar et al. 2017).

Statistical analysis

LC₅₀values were determined by probit analysis using the PROBIT software Statistical Package. Analysis of Variance was performed by Probit analysis. Means were compared with Duncan’s and Tukey Multiple Range test (Massebo et al. 2009).

Phytochemical analysis of plant extracts

Phytochemical analysis of the plant extracts was carried out for the presence of various classes of secondary metabolites (qualitative tests) by using standard procedures (Ali et al. 2018).

Result

Percentage yield

The percentage yield of the three plant extracts (R.officinalis, O.hadiense and C. spinarium) in different solvents (Chloroform, Ethanol and water) was calculated. The total yield for 70% ethanol extract of O. hadiense was 11.33 g, 22.61 g for C. spinarum and 24.48 g for R. officinalis and the maximum yield was obtained from R. officinalis (24.48%) and the minimum was obtained from O. hadiense (11.33%).The total yield for chloroform extract of O. hadiense was 2.09 g, 6.20 g for C. spinarum and 20.45 g for R. officinalis and the maximum yield was obtained from R. officinalis (20.45%) and the minimum was obtained from O. hadiense (2.09%). And also the total yield for water extract of O. hadiense was 10.52 g, 13.16 g for C. spinarum and 9.31 g for R. officinalis and the maximum and the minimum yield was obtained from C. spinarum (13.16%) and R. officinalis (9.31%) respectively.

Phytochemical analysis

Phytochemical analysis of the plant extracts confirms the presence of Alkaloids, Saponins, Phenols, Tannins and Flavonoids. The leaf part extract of R. officinalis contains phenols and flavonoids as it forms bluish black colour and white precipitate when detected by Ferric chloride test and Gelatine test respectively. O.hadiense also has Saponin, Alkaloids, Phenols and Flavonoids, foams which persists for 10 min, reddish precipitate, bluish black and yellow colour precipitate were formed when detected by Foam test, Wagner’s Test, Ferric Chloride Test and Lead acetate test respectively. Moreover C. spinarium contains Saponins, Phenols, Flavonoids and Tannins and exhibit colours and precipitates as mentioned above when detected by the different tests.

Larvicidal potential of plant extracts

The maximum larvicidal potential was obtained from O. hadiense in chloroform at concentrations of 200 mg/ml with average larvae death of ten whereas in 70% ethanol eight larvae were found dead after 24 h of exposure at 200 mg/ml concentration where as in water extract only two larvae were found dead at highest concentration used. In the case of R. officinalis, the best effect (90% mortality) was observed with chloroform extract at 200 mg/ml. While C. spinarium showed 50% mortality was observed with water extract at 200 mg/ml (Figs. 2, 3).

Fig. 2.

Average larvicidal potential of O. hadiense

Fig. 3.

Average larvicidal potential of C. spinarium

LC₅₀ and LC ₉₀ of the various plant extracts

LC₅₀ and LC ₉₀ of each plant extract was calculated using Probit analysis and the result obtained is summarized and tabulated (Table 1). Results shows the LC₅₀ and LC₉₀ values of the extract of different plants tested against 3rd Aedes aegypti larvae in the laboratory. LC₅₀ of R. officinalis, in chloroform, 70% ethanol and distilled water was found to be 111.735 mg/ml, 178.500 mg/ml and 87.381 mg/ml respectively. And LC₅₀ of O. hadiense and C. spinarium in chloroform, 70% ethanol and water was 24.561 mg/ml, 651.384 mg/ml, 335.317 mg/ml, 736.883 mg/ml, 412.240 mg/ml and 253.457 respectively. Moreover, the LC₉₀ of R. officinalis, O. hadiense and C. spinarium in Chloroform, Ethanol and water was 204.382 mg/ml, 315.899 mg/ml, 279.229 mg/ml, 198.411 mg/ml, 2161.286 mg/ml, 526.317 mg/ml, 1188.699 mg/ml, 412.240 mg/ml and 456.217 mg/ml respectively.

Table 1.

LC₅₀ and LC ₉₀ of each plant extracts

Plant type	Plant extracts	LC50 (mg/ml)	LC90 (mg/ml)
Rosmarinus officinalis	Chloroform	111.735	204.382
	70% Ethanol	178.500	315.899
	Water	87.381	279.229
Ocimum hadiense	Chloroform	24.561	198.411
	70% Ethanol	651.384	2161.286
	Water	335.427	526.317
Carrisa spinarium	Chloroform	736.883	1188.699
	70% Ethanol	412.240	777.452
	Water	253.457	456.217

Comparative evaluation of larvicidal potential of plants

Results shows the LC₅₀ and LC ₉₀ of the chloroform, ethanol and water extracts of selected plants and the comparative evaluation of plant extracts with maximum and minimum efficacy can be observed. The plant extract with the lowest LC₅₀ and LC₉₀ value was found to be the most effective and plant extract with the highest LC₅₀ and LC₉₀ was found to be the least effective. The chloroform extract of O. hadiense with LC₅₀ and LC ₉₀ of 24.561 mg/ml and 198.411 mg/ml respectively, was found to be the most effective followed by water and chloroform extract of R. officinalis with LC₅₀ of 87.381 mg/ml and 111.735 mg/ml respectively. Chloroform extract of C. spinarium with LC₅₀ of 736.883 mg/ml and 70% ethanol extract of O. hadiense with LC₉₀ of 2161.286 mg/ml respectively, were found to be the least effective.

Morphological deformity of treated larvae

Morphological abnormalities and behavioral changes were recorded among the larvae out to different concentrations of various plant extracts. These mosquito larvae showed signs of unnatural restlessness, squirming movement and frequent sinking followed by floating, after 1–5 h of treatment. Such behavior persisted and observed until the larvae became sluggish, paralyzed and eventually sank to the bottom of the container. Further investigation under the light microscope revealed that the treated larvae had damaged inner structures like gut. Similar observations were noted for all larvae treated with different extracts. Such deformity was not observed in the control group (water) (Figs. 4, 5).

Fig. 4.

Larvae of Aedes aegypti A and B treated with chloroform extract of O. hadiensie showing darkened body parts and anal papillae

Fig. 5.

Morphological changes of treated larvae: A with water extract R. Officinalis showing highly pigmented granules in the digestive tract and high pigmentation in abdominal body segments and the anal part B Morphological changes of treated larvae: with water extract of C. spinarium showing darkened body parts and anal papilla

Discussion

The present study showed that most of the tested extracts demonstrated significant larvicidal activity against larvae of Aedes aegypti. Among the three selected plants the maximum larvicidal activity was found in the chloroform extract of O. hadiense, followed by chloroform extract of R. officinalis and water extract of C. spinarum. The maximum larvicidal potential was obtained from O. hadiense in chloroform at concentrations of 200 mg/ml with average larvae death of 10 whereas in 70% ethanol eight larvae were found dead after 24 h of exposure at 200 mg/ml concentration where as in water extract only two larvae were found dead at highest concentration used. In the case of R. officinalis, the best effect (90% mortality) was observed with chloroform extract at 200 mg/ml. While C. spinarium showed 50% mortality was observed with water extract at 200 mg/ml. In a study done by Manzoor et al. (2013) five essential oils from various parts of five plant species i.e. Acorus calamus, Mentha arvensis, O. basilicum, Saussrea lappa and Cymbopogan citrates were investigated for their larvicidal property against Aedes aegypti and they reported maximum larvicidal potential in O. basilicum oil (Saranya et al. 2013). Furthermore, Saranya et al. (2013) reported Larvicidal, pupicidal activities and morphological deformities of Spathodea campanulata aqueous leaf extract against the dengue vector Aedes aegypti and reported that aqueous extract was effective against larvae but pupae showed the resistance to the aqueous leaf extract of Spathodea campanulata. LC₅₀ and LC₉₀ values are the concentration that causes 50% and 90% mortality. The present study discusses that in O. hadiense the LC₅₀/24 h was obtained 24.561 mg/ml, 651.384 mg/ml, 335.317 mg/ml in chloroform, 70% ethanol and distilled water respectively and LC₉₀ values of O. hadiense found to be 198.411 mg/ml, 2161.286 mg/ml and 526.317 mg/ml in chloroform,70% ethanol and distilled water respectively. LC₅₀ of R. officinalis, in chloroform, 70% ethanol and distilled water was found to be 111.735 mg/ml, 178.500 mg/ml and 87.381 mg/ml respectively. And LC₅₀ of C. spinarium in chloroform, 70% ethanol and water was 736.883 mg/ml, 412.240 mg/ml and 253.457 respectively.

Moreover the LC₉₀ of R. officinalis, and C.spinarium in chloroform, 70% ethanol and water was 204.382 mg/ml, 315.899 mg/ml, 279.229 mg/ml, 1188.699 mg/ml, 412.240 mg/ml and 456.217 mg/ml respectively. Rocha et al. 2015, carried out the bioassays for larvicidal activity and revealed that 3rd instar larvae of Aedes aegypti are susceptible to EO of F. vulgare, with LC50 of 28.2 mg/mL and LC90 of 39.6 mg/mL respectively (Rocha et al. 2015). In fact many researchers have reported the effectiveness of plant extract against Aedes aegypti. One study done in Ethiopia, P. nigrum oil with LC₅₀ and LC₉₀ 9.1 ppm and 13.5 ppm respectively and O. lamiifolium hochst oil with LC₅₀ of 8.6 ppm and LC₉₀ 13.4 ppm respectively against Aedes aegypti, were the most effective oils and E. globulus and M. spicata with these LC₅₀ 52.9 ppm and 67.8 ppm and LC₉₀ values of 102 ppm 96.4 ppm respectively was the least effective (Massebo et al. 2009).

In the present study the Aedes aegypti larvae treated by different doses of the different extracts of R. officinalis, O. hadiense and C. spinarium were showed various morphological abnormalities and behavioral changes were also observed among the larvae. These mosquito larvae showed signs of unnatural restlessness, wriggling movement and frequent sinking followed by floating, after 1–5 h of treatment. Such behavior persisted until the larvae became sluggish, paralyzed and eventually sank to the bottom of the container. Similar observations were reported by Palanikumar et al. (2017) in methanol extracts of Callistemon citrinus against Aedes aegypti larvae. The larvae resorted to swift wriggling movements which persisted for approximately after 30 min, 25%larvae started sinking at the bottom of plastic jars. In contrast, remaining larvae exhibited severe restlessness behavior with aggressive self-biting of their anal papillae with their mouthparts (Palanikumar et al. 2017). In addition, some of the visible malformations in larvae under the treatment of various plant extracts were observed in the present study like darkened body segments and anal papillae as compared to the normal larvae. Investigation under the light microscope revealed that the treated larvae had damaged gut. Similar observations were noted for all larvae treated with different extracts. Such modification was not observed in the control group (water).

Conclusion

About two-fifth of the world’s population is now at risk of dengue, chikungunya, zika and yellow fever. The only way to prevent various virus transmissions is to combat the disease carrying mosquitoes. Larviciding can be a successful way of reducing mosquito population in their breeding sites before they emerge into adults. The larval stage is the weakest link in the life cycle of mosquito, as they are immobile and more concentrated at this stage than the adult stage. Therefore, larvicides can be used as an effective method to control mosquito breeding. The finding of the present study proposes that the chloroform extract of O. hadiense was found to be the most effective followed by water and chloroform extract of R. officinalis. This knowledge may help in designing and implementing an effective strategy from a resistance management perspective against Aedes agypti. On the basis of the findings these plants have very high potential as larvicide. Extract can be purified and active ingredients can be identified by various phytochemical & spectroscopic techniques for further development of a green and safe larvicidal agent.

Author contributions

All the authors contributed to the study conception and design. Work supervision, Material preparation, results analysis was performed by JJK, AK and DM. The laboratory work was done by RA, HK, KW, EB, EE. All authors read and approved the final manuscript.

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Declarations

Conflict of interest

The authors have no relevant financial or non-financial interests to disclose.

Ethical approval

This is an in vitro biological screening. The college Research Ethics Committee has confirmed that no ethical approval is required.

Human participants and/or animals

No.

Informed consent

Not applicable.