Screening of some weed extracts for ovicidal and larvicidal activities against dengue vector Aedes aegypti

Background: The use of plant-based formulations is an easy and economical method to control insect pests. The objectives of the present study were to evaluate ovicidal and larvicidal potential of ethanolic leaf extracts of four plants, Cyathocline purpurea, Blumea lacera, Neanotis lancifolia, and Neanotis montholonii, and their effects on gut histology of Aedes aegypti (Diptera: Culicidae) larvae. We identified the phytochemicals present in the ethanolic extracts of these plants by LC-MS analysis. The eggs and larvae of A. aegypti were exposed to four concentrations (0.05, 0.1, 0.2, and 0.3 mg/mL) of crude ethanol extracts for different time durations. We observed egg hatching for 48 h, mortality, and gut histology of the 3rd instar larvae after 24, 48, and 72 h of treatments. Results: LC-MS analysis revealed the presence of various active compounds such as alkaloids, tannins, saponins, and flavonoids in the ethanol extracts of all these plants. The extracts of all plants showed ovicidal activity. The extracts of C. purpurea showed the highest larvicidal activity (LC50 at 72 h = 0.088 mg/mL) followed by B. lacera (LC50 at 72 h = 0.758 mg/mL) while N. lancifolia and N. montholonii extracts did not show significant larvicidal activity. All plant extracts affected gut morphology in concentration and treatment duration-dependent manner. The plant extracts used in the present study have ovicidal properties and affected the gut histology of A. aegypti larvae. Conclusion: These results suggest that C. purpurea has the highest larvicidal activity against A. aegypti larvae and can be further evaluated for application purposes.


Background
Mosquitoes are ecologically and economically important insects as they are the vectors of many life-threatening diseases. Aedes aegypti (Diptera: Culicidae) is a vector of many viral diseases such as dengue, chikungunya, Zika, and yellow fever which are the reason for millions of deaths worldwide and serious health problems (Christophers, 1960;Chapman, 2012;World Health Organization, 2017;Bonica et al., 2019;Kraemer et al., 2019). Despite their economic importance, there are no proper treatments or preventive measures available for these viral diseases. The vectors of these diseases have been major targets for disease eradicating programs (Kumar, Singh, Tomar, and Baijal, 2010). Due to their occurrence in limited space (small pools and puddles), embryonic and larval stages of mosquitoes have been a prime target for the researchers seeking drugs to control mosquito populations (Ignacimuthu and David, 2009;Becker et al., 2010;Day, 2016). Many synthetic pesticides including pyrethroids, carbamates, and organophosphates are available in the market for mosquito control. But, most of them are pollutants, detrimental to the ecosystem, and harmful to non-target animals (Ignacimuthu and David, 2009;Rhind, 2009;Benelli, 2015). Moreover, their repeated use has developed resistance in target animals (Després, David, and Gallet, 2007;Ignacimuthu and David, 2009). For example, larvae of Culex quinquefasciatus and A. aegypti developed resistance when exposed to widely used mosquito controlling chemicals permethrin and temephos, respectively (Muthusamy and Shivakumar, 2015;Ramkumar and Shivakumar, 2015). Therefore, there is a need of screening of novel drugs or combination of drugs to control mosquito populations.
Besides synthetic pesticides, there are several other biological controlling methods including the use of fungal pathogen, predators, traps, and plant-based drugs which are being practiced (Ignacimuthu and David, 2009;Benelli, 2015). Among biological methods used to control mosquitoes, plant-based pesticides are popular due to their low economic cost, easy availability, and ecofriendly nature. Plants of many species contain diverse phytochemicals which can be used to develop drugs against disease-causing insects (Pascual-Villalobos and Robledo, 1998;Benelli, 2015). In fact, chemicals identified in the plant extracts have been reported for their activity against various medically important insects (Rao, Chattopadhyay, and Reddy, 1990;Wachira et al., 2014). Phytochemicals are of diverse chemical nature, and therefore, their application can be the promising opportunity for controlling various insect-borne diseases (Traboulsi et al., 2005;Zhu and Tian, 2011;Wachira et al., 2014). Previously, solvent preparations of the plants or phytochemicals isolated have been reported to have ovicidal or larvicidal activities in several mosquito species (Gupta, Deshpande, Tare, and Sabharwal, 2011;Subramaniam, Kovendan, Kumar, Murugan, and Walton, 2012). In addition, many plant-based extracts/compounds induce changes in morphology, physiology, biochemical processes, and behavior of different life stages of mosquitoes suggesting their importance in controlling mosquito population (Al-Mehmadi and Al-Khalaf, 2010;Gupta et al., 2011;Lija-Escaline et al., 2015;Yu, Wong, Ahmad, and Jantan, 2015).
The aim of the present study is to screen the plants  (Fig. 1). All these plants are common weeds, widely distributed in South Asia, belonging to medicinally important families, and some of them are also known for medicinal properties. C. purpurea is a common weed having importance in traditional medicine. This plant is having various phytochemicals known for medicinal properties including antimicrobial, anticancer, and hypotensive properties (Zu-Qiang, Guo-Yi, Lei, and Xi-Tai, 2006;Ma, Chong, Li, Cheung, and Tattersall, 2009;Joshi, 2013). B. lacera is also a medicinally important plant and shows insecticidal activity (Roy et al., 2005; Hasan, Rahman, Guo, and Hirashima, 2015). The plants N. lancifolia and N. montholonii are native weeds belonging to the family reported to have plant species with medicinal properties, active metabolites, and unpleasant smell (Guerrero-Analco et al., 2007;Karou, Tchacondo, Ilboudo, and Simpore, 2011;Martins and Nunez, 2015). In addition, there are no reports on the anti-mosquito properties of these plants. The objective of the present study was to assess the activity of ethanolic leaf extract of these plants against different life stages of A. aegypti. In the present study, we prepared ethanol extracts of the leaves of these plants and identified phytochemicals present in it using LC-MS. We then studied ovicidal and larvicidal activities of these extracts on A. aegypti. We also studied the effect of these leaf extracts on the gut morphology of A. aegypti larvae.  Fig. 1). The plant materials were brought to the laboratory and cleaned with distilled water. Leaves were separated from the plants and spread on a filter paper for shade drying at room temperature 27 ± 2°C and humidity 75-80%. Dried leaves were grounded into fine powder using Wiley mill. The powder obtained was passed through 2 mm sieve. We mixed 100 g sieved powder in 1000 mL ethanol and kept in a shaker overnight at 25°C and 60 rpm. The sample was then filtered using Whatman filter paper no. 1. The solvent from the filtrate was removed under reduced pressure on a rotary evaporator at 35-40°C. Then, the extract was dissolved in distilled water to make 1 g per 100 mL stock solution. The required amount of stock solution was dissolved further in tap water to make desired concentrations.

LC-MS analysis and identification of compounds
For preliminary screening of phytochemicals, ethanolic leaf extracts of all four plants were subjected to liquid chromatography coupled with mass spectrometry (LC-MS). LC-MS analysis was done by injecting 10 μL filtered samples on a Dionex system composed of P680 pump and ASI-100 autosampler and UVD170U UV detector. This was coupled to a Thermo Finnigan Surveyor MSQ mass spectral detector. Separation was performed on a Doinex Acclaim 120 C18 columns (5 mm, 4.6 × 150 mm) using gradient elution. Compounds were eluted at a 0.7 mL/min flow rate for three minutes at 10% B, a linear gradient to 90% B over 40 min, and held at 90% B for 8 min. UV detection was recorded at 254, 280, and 310 nm. Ionization for MS analysis was performed in both positive and negative ion mode using electro-spray ionization with a nitrogen flow at 80 psi, a cone voltage of 70 V, needle voltage of 3 Kv, and cone temperature of 600°C. Mass data were collected over the range of the gradient program at a rate of one scan per second.

Culture of Aedes aegypti
A mother culture of A. aegypti was obtained from Ross Life Sciences Pvt. Ltd., Pune, India, and maintained in a laboratory following established guidelines (Das, Garver, and Dimopoulos, 2007;World Health Organization, 2005). The larvae were maintained in steel trays (35 × 26 × 6 cm) with 600 mL de-chlorinated tap water from a public water supply at 27 ± 2°C, relative humidity 75-85% under 12:12 h light and dark condition. A pinch of finely powdered fish food was provided daily as food for larvae. Adult mosquito colonies were maintained in rearing cages (60 cm × 60 cm × 60 cm) with 50 μm mesh size. Adult mosquitoes were maintained on 10% sugar solution, and female mosquitoes were fed on chicken for blood meal once in a week at least for 6 h. After 72 h of blood feeding, female mosquitoes were allowed to lay eggs in a container (height 6 cm, base diameter 9.5 cm). Fresh dried egg stripes were used for ovicidal assays. Third instar larvae were used for larvicidal assays and histopathological study. Ovicidal, larvicidal assays and histopathological study were carried out in the Department of Zoology, SPPU.

Ovicidal activity
Ovicidal activity of leaf extracts was studied following the modified method described previously (Su and Mulla, 1998). The whole egg strips were kept in the water containing desired concentrations (0.05, 0.1, 0.2, and 0.3 mg/mL) of each plant extracts. The assay was carried out in four replicates for each concentration.
Ovicidal activity was carried out in plastic containers (height 6 cm, base diameter 9.5 cm with water level 2.5 cm). Hatching success in each replicate of each concentration was monitored up to 48 h. After 48 h, hatched and unhatched eggs were counted under Leica stereozoom microscope (Leica-MZ75). We combined the hatching data of all four replicates to calculate the total number of hatched and unhatched eggs from each concentration. The ovicidal activity of each concentration was expressed in percent mortality (number of unhatched eggs).

Larvicidal activity
Larval susceptibility tests were carried out by following the protocol recommended by WHO (World Health Organization, 2005). Based on preliminary tests, we treated third instar larvae with 0.05, 0.1, 0.2, and 0.3 mg/ mL concentrations in triplicate (25 larvae in each replicate). Untreated larvae were considered as a control. The larvae were reared in 500 mL beakers with 250 mL water containing desired concentrations of extracts. We recorded mortality of larvae treated with all concentrations at 24, 48, and 72 h. Larvae were considered dead when they stopped activity for long period. We confirmed the death of the larvae using a spatula as a probe.
If the larva does not move even after gently probing, it was considered as dead. We combined the data from all the replicate of each concentration and calculated LC 50 and LC 90 . LC 50 and LC 90 values were calculated for each concentration at 24, 48, and 72 h using Probit analysis in SPSS 20.

Histopathological study
To study the effects of leaf extracts on gut morphology of A. aegypti larvae, third instar larvae were treated with 0.1 and 0.3 mg/mL (N. montholonii N. lancifolia, and B. lacera) or 0.05 and 0.2 mg/mL (C. purpurea) concentrations using the same protocol described above. Six live larvae from each treatment were sampled at 24 h, 48 h, and 72 h and fixed in Bouin's fluid for 24 h. Then, they were passed through the series of ascending grades of ethanol (30, 50, 70, 90, and 100%) for dehydration, passed through xylene, and embedded in paraffin wax at 58-60°C (MERCK). Tissues (anterior part of the midgut of larvae) were sectioned at 7 μm using Leica RM 223 rotary microtome and stained with hematoxylin and eosin. Stained sections were observed and photographed under a microscope (Leica-DF450) to analyze the changes in the gut morphology.

Ovicidal activity
Results showed that the leaf extracts of all plants have ovicidal activity. Extract of all the plants caused 70-90% mortality at higher concentrations (Fig. 2).

Larvicidal activity
Among four leaf extracts used in the present study, C. purpurea had the highest larvicidal activity. LC 50 values for C. purpurea extract at 24, 48, and 72 h were 0.72, 0.18, and 0.088 mg/mL while LC 50 values for B. lacera leaf extract were 7.349, 2.457, and 0.758 mg/mL, respectively (Table 1). The leaf extracts of N. montholonii and N. lancifolia did not show significant larvicidal activities ( Table 1). The larvae from the control group and the groups treated with different concentrations of N. montholonii and N. lancifolia started pupation generally after 48 h.

Histopathological study
All plant extract treatments showed a similar pattern of larval gut damage. Therefore, we categorized the deterioration of midgut epithelium/cells integrity into four stages/groups based on the damage severity ( Fig. 3 and Table 2). In the first stage (control), gut epithelial cells (cuboidal/flattened cells) with round nucleus and clear cytoplasm were observed (Fig. 3A). In the midgut of stage 2, swollen cells or vesicles of different sizes were visualized (Fig. 3B). The third stage consists of a midgut with lost cell integrity, damaged nucleus and cell membrane, cells detached from basal lamina, and loss of nuclear and cytoplasmic materials (Fig. 3C). In the fourth stage, complete damage of mid-gut epithelium/cells could be seen. It included the cells detached from basal lamina as well as loss of cell-cell contact and cellular integrity (Fig. 3D).
All the four leaf extracts used in the study affected gut morphology in treatment duration and concentrationdependent manner. Among all the plant extracts used, C. purpurea leaf extract induced severe gut damage followed by N. montholonii, N. lancifolia, and B. lacera (Table 2; Fig. 3).

Discussion
The discovery of pharmaceutical drugs is the first step towards the control of parasitic vector insects. Plantbased formulations provide an easy and economical method to screen novel paste controlling drugs. Moreover, mosquito eggs and larvae are the ideal systems for screening novel insecticides present in the plant-based formulations (Rawani, Haldar, Ghosh, and Chandra, 2009;Subramaniam et al., 2012;Benelli et al., 2017). In the present study, we used ethanolic leaf extracts of four common weeds and showed their stage and concentration-dependent effects on A. aegypti.
The results revealed that the plant extracts used in the present study contain diverse phytochemicals. Many of the compounds having similar chemical nature to these phytochemicals have been reported to be present in other plant extracts and found to have activity against various disease-causing insects (Lee, 2000;Senthilkumar, Kannathasan, and Venkatesalu, 2008;Rawani et al., 2009;Adhikari, Singha, and Chandra, 2012). The results also showed that the leaf extracts of C. purpurea and B. lacera have the highest ovicidal and larvicidal activities among the extracts used in the present study whereas all plant extracts affected gut morphology at different concentrations. C. purpurea and B. lacera are known to have medicinal properties such as anti-cancer, antiinflammatory, antibacterial, hypoglycemic, analgesic, anti-arthritic, cytotoxic, and antiviral (Chiang, Cheng, Chen, and Lin, 2004;Ma et al., 2009;Joshi, 2013;Bihani, Rojatkar, and Bodhankar, 2014;Hasan et al., 2015;Tambewagh et al., 2017). Ethanolic leaf extracts of these  plants possess various types of phytochemicals including essential oils, terpenoid, alkaloids, phenols, flavonoids, anthraquinones, cardiac glycosides, cyanogenetic compound, steroids, and terpenes. Similar to these phytochemicals, compounds present in the other plant extracts have been reported for their larvicidal activities in insects (Liu, Liu, Du, and Deng, 2012;Gautam, Kumar, and Poonia, 2013;Govindarajan, Sivakumar, Rajeswary, and Veerakumar, 2013;Benelli et al., 2017). Previous studies reported the presence of various phytochemicals in the essential oils obtained from hydrodistillation of C. purpurea (Joshi, 2013). In addition, we first time identified the phytochemicals present in B. lacera, N. montholoni, and N. lancifolia. Further studies need to direct the focus on the isolation, characterization, and insecticidal properties of the phytochemicals present in these plant extracts and evaluate their insecticidal activities and effects on non-target animals as well.
In the present study, we observed considerable differences in the ovicidal and larvicidal activities of the leaf extracts of these plants. For instance, C. purpurea and B. lacera show high larvicidal activity while N. montholonii and N. lancifolia did not show significant larvicidal activity. Among these plants, B. lacera extract reported to have insect repellent and toxic properties. Petroleum ether extract of B. lacera is a strong repellent and toxicant for Lesser Grain Borer and Rice Weevil (Roy et al., 2005). The plants C. purpurea, N. montholonii, and N. lancifolia have never been explored for insecticidal   (Autran et al., 2009;Gupta et al., 2011). For example, a-amylase inhibitor isolated from Macrotyloma uniflorum possesses larvicidal activity but not ovicidal activity (Gupta et al., 2011). The differences in the activity patterns of the plant extracts in mosquitoes can be partially attributed to the chemical composition of the extracts and the chemical nature of the compounds (AhbiRami, Zuharah, Thiagaletchumi, Subramaniam, and Sundarasekar, 2014;Porto et al., 2017). Plant extracts (combination of chemicals) may have a variety of effects and show solvent-dependent larvicidal or ovicidal activities (Traboulsi et al., 2005;Talontsi, Matasyoh, Ngoumfo, and Chepkorir, 2011;Zhu and Tian, 2011;Chapman, 2012;Lija-Escaline et al., 2015). For instance, acetone extract of Ipomoea carica showed high mortality as compared to those of the methanol extract (AhbiRami et al., 2014). Phytochemicals are also known to affect different systems insects including physiology, behavior, and morphology (Broadway and Duffey, 1988;Lija-Escaline et al., 2015;Procópio et al., 2015;Yu et al., 2015). Gut histology of mosquito larvae has been one of the most promising methods to evaluate insecticidal effects of plant extracts (Al-Mehmadi and Al-Khalaf, 2010; Lija-Escaline et al., 2015;Procópio et al., 2015). In the present study, all plant extracts affected gut morphology of A. aegypti larvae. Higher concentrations of N. lancifolia and N. montholonii affected gut morphology while low concentrations of B. lacera and C. purpurea were sufficient to induce gut damage. The susceptibility of A. aegypti larvae gut to different plant extracts has been reported in some previous studies. The extracts of some plants affect gut morphology at high concentrations while some plant extracts show their effects at very low concentrations (Lija-Escaline et al., 2015;Procópio et al., 2015;Yu et al., 2015). The concentrations of green seaweed extracts used in the previous study (Yu et al., 2015) were within the range of the concentration used in the present study. Aqueous preparations of the pesticides can be made available easily and applied for the target animals in the water bodies. Since mosquitoes breed and their embryonic and larval stages occur in pools and puddles, the application of phytochemicals for their control is manageable (Becker et al., 2010;World Health Organization, 2017). Many studies have reported the larvicidal activities of plant extracts, and their direct applications seem to be promising (Ghosh, Chowdhury, and Chandra, 2012;Vivekanandhan, Senthil-Nathan, and Shivakumar, 2018a;Vivekanandhan, Usha-Raja-Nanthini, Valli, and Subramaniam Shivakumar, 2018b). The present study reports the effects of some weed extracts on different life aspects of A. aegypti and demands further applicationoriented studies considering less harm to non-target species. Additionally, the carryover effects of sub-lethal concentrations of plant extracts (N. montholoni and N. lancifolia) on pupae and adult mosquitos need to be further evaluated. The plants used in the present study can be a valuable source of various medicinally important phytochemicals as these plants are widely distributed as common weeds (Almeida, 1996;Singh, Lakshminarasimhan, Karthieyan, and Prasanna, 2001).

Conclusions
The leaf extracts of all the studied plants have ovicidal activity against A. aegypti. The leaf extracts of the weeds C. purpurea and B. lacera have lervicidal properties and induce gut damage. The leaf extracts of N. montholonii and N. lancifolia affected gut morphology although they do not have larvicidal activities. All four extracts affected gut morphology of A. aegypti larvae in a concentrationdependent manner and their severity increased with treatment duration. The weeds C. purpurea and B. lacera can be further evaluated for immediate application purposes.
Additional file 1: Figure S1. LC-MS profile of the ethanol extract of C. purpurea. Table S1 Chemical compounds identified in the ethanol extracts of C. purpurea using LC-MS (*Concentration of the constituents was derived from the calibration curve of the authentic compound)). Figure S2. LC-MS profile of the ethanol extract of B. lacera. Table S2 Chemical compounds identified in the ethanol extracts of B. lacera using LC-MS (*Concentration of the constituents was derived from the calibration curve of the authentic compound). Figure S3. LC-MS profile of the ethanol extract of N. montholonii. Table S3 Chemical compounds identified in the ethanol extracts of N. montholonii using LC-MS (*Concentration of the constituents was derived from the calibration curve of the authentic compound). Figure S4. LC-MS profile of the ethanol extract of N. lancifolia. Table S4 Chemical compounds identified in the ethanol extracts of N. lancifolia using LC-MS (*Concentration of the constituents was derived from the calibration curve of the authentic compound).