Abstract: Background and Objective: Environment pollution and resistance of many pests to the most frequently used chemical insecticides gave the rationale of altering to replace them with natural herbal extracts for pests and vector control. This study investigated the effect of methanolic extract of Annona reticulata on the mortality and development of larvae of Culex quinquefasciatus. Materials and Methods: Methanolic herbal extracts were analyzed using Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC) to identify the phytochemical compounds in them. Ten mosquito larvae were used as replicates and exposed to each of the five concentrations of the plant extract (30, 100, 150, 200 and 250 mg mL–1) and ten larvae were exposed to double distilled water and considered as control. Results: Phytochemical analysis revealed the presence of phenols, steroids, quinones, tannins and saponins. Statistical analysis showed a significant strong correlation and regression between exposure to the different concentrations of the extract and mortality of Culex quinquefasciatus larvae where R2 = 0.982, the Correlation value is 0.991099 (p<0.05). Results also showed that the extracts affect the development of larvae more than mortality. The effect of the extracts on the early larval stages was significantly high compared to the late stages of larvae. Conclusion: According to our knowledge results of this study has been reported for the first time in Saudi Arabia where Annona reticulata is neglected regionally and no study approved its efficacy as a botanical herbal extract against mosquitoes.
INTRODUCTION
Numerous species of mosquitoes cause a real threat to humans and or animals to their vectorial capacity to transmit serious pathogens like filarial warms, malaria parasites, yellow fever and dengue fever viruses1. Many parts of the world were identified as endemic areas for diseases caused by these pathogens2.
Many vector control campaigns focus on reducing densities of larval stages in their breeding habitats because they assume that targeting adults may reduce the density of adults for the short time3. Besides, dispersal of larval stages of mosquitoes is limited compared to the adult stages thus the eradication of a larval population in a breeding site is easier and more applicable4.
Chemical insecticides belonging to various families are regularly used for the control of numerous families of pests and/or vectors to reduce their densities. Many studies recorded that using of these insecticides exponentially correlated with the appearance of insecticide resistance5. To save the environment from the pollution caused by the chemical insecticides as well as to prevent the insecticide resistance problem it’s recommended to use natural products for insects control such as herbal extracts6. The relatively high cost of chemical control compared to herbal extracts as well as the opportunity of affecting non-targeted organisms by chemical insecticides are additional advantages encouraged altering from the chemical vector control to the natural product's control5.
Products of various plants have been used to control either (or both) larval or (and) adult stages of mosquitoes4. Some herbal products are also used as repellents to prevent mosquitoes-human contact7. In Saudi Arabia, few studies reported the efficacy of some plant extracts on mosquitoes larvae such as Solenostemma argel8. Another study approved the efficacy of three herbal extracts on the control of C. quinquefasciatus larvae9.
According to our knowledge, no published study investigated the efficacy of Annona reticulata on the control of Saudi strains of mosquitoes. This gave the rationale to conduct this research.
MATERIALS AND METHODS
Study area: Plants and wild mosquitoes were collected from different localities proximal to Hfouf in Al Ahsa (Eastern Region around 328 km from Riyadh Capital of Saudi Arabia).
Study design and study settings: A prospective study has been conducted in Saudi Arabia between January-June, 2017. Wild populations of C. quinquefasciatus larvae, laboratory-reared larvae of the same species and leaves of wild Annona reticulata trees were used in this study.
Collection and rearing of mosquito larvae: Larvae of C. quinquefasciatus larvae were collected from stagnant water bodies in variant localities in Saudi Arabia distributed in the Eastern, Southern and Central regions. Plastic pipettes and white dishes were used for the manual collection of the larvae. Larvae transferred to the Entomology Research Lab in the Faculty of Science at Princess Nourah Bint Abdurrahman University for morphological identification and rearing. Mosquito larvae anaesthetized using Triethyleamine solution, identified under dissecting microscopes using the classical keys of Knols10 then placed into labelled white dishes containing powder of yeast and biscuit mixture with (1:3) ratio. Pupae were collected via pipettes periodically and transferred to containers containing double distilled water placed inside adult mosquito cages. Each cage was 30×30 cm in size and 30 cm in height.
Temperature and Relative Humidity (RH%) were adjusted to 21±1°C and 77±3% RH during the whole period of the laboratory work in the insectary. Emerged adult male mosquitoes offered 20% Glucose solutions while female adult mosquitoes offered rabbits blood via artificial feeding membranes. Egg rafts were collected daily and replaced with labelled larvae-rearing dishes. Third instars’ larvae were specifically pipetted for bioassay tests11.
Description of Annona reticulata: Annona reticulata is a plant also called Sarifa or sugar apple natively grown in Western Indis but also commonly found in tropical areas. The leaves and branches are used as natural dies due to the blue pigments they contain. Annona reticulata approved significant larvicidal efficacy against mosquito larvae and antimicrobial material against some pathogens infect humans12,13.
Preparation of herbal extracts: Fresh leaves were collected separately from various locations in Saudi Arabia distributed in the Eastern, Central and Southern localities. The trees were carefully identified and specimens were transferred to the Department of Biology, Faculty of Science, Research laboratory, King Faisal University, Saudi Arabia. Collected leaves were washed using distilled water then let to dry in the laboratory for two weeks (Mean Temperature 21±2°C). Dried leaves were ground using a clean electric grinder. Volumes of methanol and dried powdered were determined according to the method of Mohankumar et al.14 who recommended adding 15 g of the powder to 250 mL of methanol (100% concentration). The crude extract was then filtered using filter papers and concentrated by evaporation until dried in a water bath. Specific concentrations were prepared as follows: 0.6, 0.3 and 0.1% by vortexing the stock of crude extract and diluting volumes of it by the adequate volume of distilled water. Prepared concentrations were eventually then transferred to labelled dark sterile bottles and preserved at a 4 refrigerator. All steps of herbal extracts preparations were conducted according to the method of Awosolu et al.15. Phytochemical analysis was performed using Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC) to identify the chemical compounds in the extracts.
Larvicidal assay: Replicates of larvae (10 larvae/ concentration) were exposed to the five concentrations of the plant extract. About 10 larvae were exposed to double distilled water and considered as control. Proportion mortality of larval mortality due to the exposure to the herbal extract was calculated according to the following Eq.16:
RESULTS
Phytochemical analysis showed that the methanolic extract of Annona reticulata is positive for phenols, steroids, quinones, tannins and saponins. Results of phytochemical analysis also showed variations in the levels of these elements in the extracts. Phenols and Tannins were existing at high levels while Steroids were present at moderate levels. In contrast to this, Quinones and Saponins were present in low levels (Table 1).
Five concentrations 30, 100, 150, 200 and 250 μg mL–1 of the methanolic plant extracts were used for bioassay tests. Statistical analysis showed a significant strong correlation and regression between exposing to all prepared concentrations of Annona reticulata methanolic extract and mortality of Culex quinquefasciatus larvae where R2 = 0.982, the Correlation value is 0.991099 (p<0.05) (Table 2, Fig. 1).
| Fig. 1: | Linear regression and correlation between mortality of larvae and concentrations of the herbal extract |
| Table 1: Phytochemical analysis of Annona reticulata methanolic extract | |
| Components | Results |
| Amino acids | - |
| Proteins | - |
| Carbohydrates | - |
| Phenols | +++ |
| Steroids | ++ |
| Tannins | - |
| Quinones | + |
| Acidis | - |
| Resins | - |
| Oils and fats | - |
| Tannins | +++ |
| Saponins | + |
| Resins | - |
| -: Negative, +: Positive with low level, ++: Positive with a moderate level and +++: Positive with high level | |
Results of the cumulative mortality of larvae after exposure to different concentrations of the methanolic extract showed the median concentration of 100 μg mL–1 induced high mortality to approximately near half of the population of mosquito larvae (40% cumulative mortality) where the lethal concentration (LC90) was 160.03 μg mL–1 while the maximum concentration 250 μg mL–1 caused 96% cumulative mean mortality of the population of mosquito larvae and the (LC90) was 262.64 μg mL–1) (Table 3, Fig. 2).
Statistical analysis also showed that the effect of exposure to double LC50 concentration of Annona reticulata methanolic extract for 24 hrs significantly affected the development to the next developmental stage compared to the effect on mortality where Chi2 values were 5.5515 and 1.76986 (p<0.05), respectively. Besides, the extract significantly affected the earliest larval stages compared to the latest stages of larvae in their mortality and development.
| Fig. 2: | Mortality and development of larvae after 24 hrs of exposure to double LC50 concentration of the extract *Chi2 values (p<0.05) = 1.76986 and 5.5515 for mortality and larval development, respectively |
| Table 2: Effect of exposure periode to Annona reticulata methanolic extracts on the average mortality of C. quinquefasciatus larvae | |||||||||||
| Average mortality (%)/concentration | |||||||||||
| Exposure periode (hrs) | Mortality (%) in control |
30 (μg mL–1) |
p-value |
100 (μg mL–1) |
p-value |
150 (μg mL–1) |
p-value |
200 (μg mL–1) |
p-value |
250 (μg mL–1) |
p-value |
| 6 | 0 |
1 |
8.46 |
3.6 |
4.1 |
15.4 |
0.02 |
19.6 |
0.4 |
23 |
0.9 |
| 12 | 0 |
2.1 |
6.3 |
16.7 |
20.1 |
23.9 |
|||||
| 24 | 0 |
3.1 |
10.4 |
17.5 |
21.9 |
24.4 |
|||||
| 48 | 0 |
5.8 |
19.7 |
18.4 |
22.4 |
24.7 |
|||||
| Table 3: Cumulative mortality of Culex quinquefasciatus larvae after exposure to different concentrations of Annona reticulata methanolic extract | |||||
| *Conc. (μg mL–1) | Mortality (%) |
LC50 (μg mL–1) |
LC90 (μg mL–1) |
95% FL |
Slope±SE |
| 30 | 12 |
31.2 |
139.4 |
84.9–179.1 |
2.49±33.94 |
| 100 | 40 |
52.3 |
160.03 |
101.48–298.2 |
2.49±14.14 |
| 150 | 68 |
71.5 |
199.7 |
128.7–496.3 |
2.49±5.66 |
| 200 | 84 |
53.17 |
236.8 |
151.48–638.5 |
2.49±16.97 |
| 250 | 96 |
95.24 |
262.64 |
172.23–931.30 |
2.49±25.46 |
| *Conc: Concentration (μg mL–1), *LC50: Median lethal concentration, LC90: Lethal concentration, FL: Fiducial limits and DF: Degree of freedom | |||||
DISCUSSION
A piece of scientific evidence for the effectiveness of Annona reticulata in the control of C. quinquefasciatus larvae have been provided from this study. This evidence is valuable because searching for alternatives to control pests and vectors became an essential issue during the last decades due to the devastating hazard of chemical control to both environment and non-targeting organisms17. Some herbal extracts are found to be potentially effective to replace synthetic chemical insecticides in the control of numerous pests (and vectors) due to their high pests (or vectors) specificity and their relative minimum negative impact on the environment and the vertebrate and invertebrate organisms living there18. In this study, concentration was given to C. quinquefasciatus mosquitoes because it’s the most dominant domestic species of mosquitoes in Saudi Arabia which makes it necessary to select a friendly insecticide to control the larvae of this species in their breeding habitats which are mostly proximal to human dwellings19. International studies approved the potentiality of the neglected herbal extracts of Annona reticulata on the control of C. quinquefasciatus larvae in some countries20,21.
Results of phytochemical analysis exhibited the presence of some bioactive compounds like Phenols, Tannins, Steroids, Quinones and Saponins. These results coincide with the results of previous similar published studies that recorded the same compounds in the extracts of A. reticulate leaves22,23. The presence of these components indicates the possibility of utilizing this herbal extract as a potential insecticide24. Alkaloid compounds such as Steroids have been found to reduce the combining site at the acetylcholine enzyme in more than one living organism25 which also adds to the assumption that A. reticulate herbal extracts can be recommended as a potential insecticide.
The findings of this study indicated a significant effect of the methanolic extract of A. reticulate on the mortality and development of the native Saudi strands of C. quinquefasciatus larvae. Larvicidal tests specifically revealed that the herbal extracts of A. reticulate are highly toxic against C. quinquefasciatus larvae especially the first two instars. Many studies are in agreement with these findings26,27 thus, using these herbal extracts against the early developmental stages of C. quinquefasciatus mosquitoes would be more valuable rather than the late stages. Toxicity of A. reticulate indicated in this study by the ability of the extract to induce a high mortality rate and to reduce the number of larvae succeeded to complete their development to the next stage in the life cycle. More than one study has already reported significant variations in the susceptibility of mosquitoes during their different stages to the phytochemical compounds found in the herbal extracts28. The key result of this study is the determination of the recommended lethal dose (100 μg mL–1) that induced high mortality to approximately 40% of mosquito larvae. A previous study also recommended this concentration as the lethal dose to control mosquito larvae29. Another golden outcome of this study is the finding that A. reticulate methanolic extract retard the development of the mosquito larvae more than killing them. This indicates the relatively slow but less toxic effect of this herbal extract on invertebrate organisms in the environment. Coinciding to this result, a study reported that the best herbal extracts recommended to be used as pesticides should induce acute toxicity to the targeted pests as well as less residual capacity28. Finally, despite the advantages already found and discussed in this study of using A. reticulate as effective pesticides still some disadvantages of using herbal extracts in general as alternatives to synthetic chemical insecticides like unknown properties of active ingredients of many herbal pesticides were reported in some studies21. Further studies are essential to evaluate the sustainability of A. reticulate in the environment and to ensure its safety to non-targeting organisms before recommending it as a botanical pesticide for C. quinquefasciatus larvae. The effect of this herbal extract on other species of mosquitoes as well as other developmental stages such as pupae and adults should be investigated.
CONCLUSION
Methanolic extracts of A. reticulate retarded the development of C. quinquefasciatus larval development and induced significant mortality especially in the early stages of larvae and didn’t induce adverse effects at LC50 and LC90 values. Further studies should be conducted to ensure the safety of these extracts on the non-targeted organisms before they can be recommended as potential pesticides against C. quinquefasciatus larvae. According to our knowledge results of this study are the first records in Saudi Arabia.
SIGNIFICANCE STATEMENT
This study discovered the potential efficacy of A. reticulate in the control of the domestic mosquito species C. quinquefasciatus that can be beneficial for using natural products as insecticides replacing the chemical control which has disadvantages such as environmental pollution and the resistance of insects to chemical insecticides. This study will help the researchers to uncover the critical areas of the suitability of using A. reticulate to control mosquito larvae that many researchers were not able to explore in the Middle East. Thus, a new theory on these herbal extracts as potential natural insecticides against C. quinquefasciatus mosquitoes may be arrived at.
ACKNOWLEDGMENT
This research project was funded by the Deanship of Scientific Research, Princess Nourah bint Abdulrahman University, through the Research Funding Program (Grand No. 208-S-38).