Development of Plant-Based Larvicide and Herbal Mosquito Repellent Fast Card with Reference to Identification of the Functional Bioactive Compounds Effective Against Culex Mosquito

Warm and humid climate creates ideal conditions for mosquito breeding. The ability of these vectors to spread a number of diseases to humans causes millions of deaths every year. Indiscriminate use of synthetic insecticides leads to the development of resistance in vector mosquitoes and along with this, these pesticides cause biological magnification of toxic components and affects adversely the non-target organisms including human being. Commercially available, chemically manufactured mosquito repellent fast cards are convenient to use and quite effective but burning of such card generates a lot of smoke and might be hazardous to human health in the long run. Thus, alternative approaches are to be adopted to control the population load of vector mosquito. Like that, the present study also reveals the larvicidal effect of Duranta leaf extract against Culex mosquito. In the present study, mosquito repellent fast card has been developed by Duranta-algal mixture which has shown better result than commercially available fast card on the basis of mosquito mortality as well as the amount of gases emitted. Again, the ethanolic crude extract of Duranta leaves leads to 100% mortality of all instars (Culex pipiens) larvae at both 1000 ppm and 500 ppm concentration. Therefore, the active component of Duranta has also been investigated. In Duranta, highest area percentage and peak have been shown by propionic acid in the retention time 18.086 by GC–MS. So, it can be confirmed that the major active ingredient is propionic acid in Duranta which is responsible for the mosquitocidal properties. Occurrence of propionic acid in Duranta has also been confirmed by the HPLC analysis.


Introduction
A number of diseases like dengue, malaria, filariasis, Japanese encephalitis, West Nile fever are transmitted among humans and animals mainly by 4 genera of mosquitoes, viz. Culex sp., Anopheles sp., Aedes sp., and Mansonia sp. [1,2]. Hence, both vector and vector borne diseases have become challenging issues that have socioeconomic impact [3]. At present, 120 million people are estimated to be infected with lymphatic filariasis caused by Culex sp. and approximately 80% of these populations are living in the tropical countries like Bangladesh, India, Indonesia, Mayanner, Nigeria, and Philippines.
One of the major strategies is to use larvicides and adulticides to control the mosquito population. In general, chemical pesticides are recognized as the most worthwhile strategy for regulating mosquitoes. However, public concern has increased significantly regarding their negative impacts such as potential health hazards, water contamination, environmental pollution, toxicity to non-target organism including man, and the development of resistance in mosquito [4,5]. The Environmental Act (1960) has made rules and regulation to check the application of such chemical insecticides in nature. It has motivated the researchers to investigate for alternative approaches that would be environmentally sound, cost effective, and target specific. A number of scientists have reported that plants contain a wide variety of potential phytochemicals which act as larvicidal agent (tannine, tarpeans, isoflavinoides) and these are generally target specific and eco-friendly as they are biodegradable [6][7][8]. Therefore, application of phytochemicals [9] for mosquito control is widely recognized. Present study evaluates the larvicidal efficacy of locally available botanicals like Duranta repens against the Culex larvae under controlled laboratory condition. It is also known to us that aqueous extract of Duranta repens acts as an alternative to control the tea red spider mite [10] and ethyl acetate extract of the same also shows larvicidal activity against cotton bollworm [11]. It is known to us that 17 large trees are required to produce 1 ton of paper from virgin pulp, which leads to deforestation. There is a growing concern around the globe for preserving our forest reserves. Thus, the use of non-wood raw material is the demand of the century as it saves trees. Revival in homemade papermaking crafts paves the way to transform recycled waste into amazing prospects. Several scientists found algae as a potential cellulose fiber source, substitute to wood pulp owing to its low lignin content [12]. There is an invention that describes algal pulps, pre-pulps, and paper products made there from (WO1994004745A1 D21C). Chaudhari et al. [13] described a method of production of fast cards using 400 GSM handmade papers with ayurvedic herbs like nimbi, tulsi, haridr, and karpoor. Mosquito repellant gel and spray was prepared from various herbal formulations including that of neem by Ranasinghe et al. [14], but as per our knowledge, this is the first recorded description of production of fast cards against adult Culex that has been made from algal pulp containing added mosquitocidal properties of Duranta sp. As attention was given on the application of alternative strategies in the regulation of the population load of mosquito and emphasis was given on the system of IMM, IMM utilizes a collection of tools and strategies. The focus of IMM is to protect the health of human from diseases caused by vectors like mosquitoes, maintain healthy condition of environment through suitable utilization and disposal of insecticides, and improve the total condition of human life through rational and effective mosquito control strategies. Commonly, the active toxic ingredients of plant extracts are secondary metabolites which are developed to defend the plant themselves from primary consumers or herbivores. These toxic phytochemicals have a number of physiological and biochemical targets in the insect body. These targets are proteins, nucleic acids, and other cellular elements. This, in turn, hampers normal physiological activities of insect body in different ways; the principal of which is the development of abnormality in the nervous system [15]. Therefore, in the present study, the active ingredient of Duranta leaf extract has also been identified for its further use as Culex controlling agent.

Materials and Methods
The present experimental study was performed as follows:

Preparation of Plant Extract by Percolation Method
i. The leaves of Duranta repens were collected from Serampore College campus. Leaves were air dried in shaded condition at room temperature for 7 days and after that powdered with a blender. The powder was preserved in glass jars for successive solution preparation. A total of 15 gm of powder was added in 250 ml of ethanol (C 2 H 5 OH a polar solvent) in a brown flask bottle and kept at room temperature for 3 days. ii. After 3 days, the mixture was filtered through Whatman no. 1 filter paper. Then, the yield % and the concentration of the stock solution were measured. iii. The remaining stock solution was refrigerated at 4 °C until the subsequent larvicidal bioassay.
To determine the yield % of the stock solution weight of the remaining dried material of sample dust in filter paper after percolation, was measured in a watch glass. Firstly, weight of a blank clean watch glass was recorded and secondly, weight of the watch glass with 1 ml of stock solution precipitated in it (by incubating at 30 °C for few minutes) was recorded.
In present study, the calculation of yield % is as follows: Yield % = total amount of plant material before percolation − total amount of plant material after percolation × 100 / total amount of plant material before percolation.
For Duranta repens leaves = (15 gm − 12 gm) / 15 × 100 = 20% The calculation of concentration of stock solution is as follows: Precipitation of stock solution = weight of the watch glass containing precipitate − weight of the blank watch glass (mg/ml)

Collection and Rearing of Larvae
Larvae of Culex mosquito were collected from surrounding areas of Serampore College (22.75,°/ N, 88,35°/ E) West Bengal, in early morning with small glass jars and transferred to the laboratory of Department of Zoology, Serampore College. In the laboratory, the larvae were transferred to enamel tray for the purpose of emergence of adult mosquito. Water in rearing tray was refreshed each day by removing a minute part of water from the rearing tray and putting back fresh water. Afterwards, the adult mosquitoes were identified at the species level. In our laboratory, mosquitoes were reared at 26-28 °C and at 80-85% relative humidity. A total of 10% sucrose solution was used as the food of adult mosquito [16].
In order to maintain a mosquito colony, the adult female mosquitoes were blood fed by the use of chick. For the purpose of egg laying, petri dishes with tap water lined by filter paper were settled inside the cages. Then the eggs were transferred to enamel trays for rearing of larvae. Larval food (dog biscuits and yeast powder in 3:1 ratio) was used in our laboratory [17].

Larvicidal Efficacy Test of Plant Extract on Culex Larvae
Third, instar larvae were placed in a tray containing water. Then larvicidal activity, i.e., % of larval mortality, was calculated according to guidelines of WHO (2005) [18]. A total of 25 larvae were released with the help of a dropper into each paper glass. Forty milliliters of working solution was prepared from stock solution by the following formula: VlSl = V2S2 Volume (x) is to be taken from sock solution X. Stock solution concentration = volume to be prepared for working solution × concentration of working solution.
Following the above formula, 40 ml of working solution was distributed into each of the 2 paper glass in the following way: 1) 40 ml of working solution of 1000 ppm strength = 2 ml of stock solution + 38 ml of water 2) 40 ml of working solution of 500 ppm strength = 1 ml of stock solution + 39 ml of water Paper glass as control contained 38 ml of water + 2 ml ethanol. Larvae were considered dead if they exhibited no sign of movement even after being touched with a glass rod. The % of mortality was recorded after 24 h of larvicidal exposure.

Preparation of Algae Mixture
i. Algae were collected directly from field from a pond in the crop research and seed multiplication farm of The University of Burdwan, West Bengal.
ii. At first, collected algae were cleaned in water and filtered. A total of 180 gm of the algae was taken in a mixer grinder with added water and beaten to make a bolus. iii. Then, algae bolus was taken in a pan and boiled for 30 min on an oven. During this time, period water was added for maintaining the liquid phase. iv. A total of 10 gm of rice husk was added in pan which provided cementing property to the mixture. v. A total of 10 gm of saw dust was added with the boiling mixture to provide thickness property to the mixture.
In this mixture formation, the ratio that was taken is 90:5:5, i.e., in a 50-gm algae mixture, there is 40 gm algae, 5 gm rice husk, and 5 gm saw dust.

Development of an Anti-Mosquito Fast Card
i. Algae mixture was cooled; after cooling, ethanol extract of Duranta leaf was added in the mixture. The mixture was then stirred for proper mixing and kept for 30 min.
In the present study, 50:50 ratio was taken, i.e., 50 gm of algae mixture was added with 50 ml of ethanolic plant extract. ii. After that, the mixture was taken in Deckle and mold tray and excess water was drained. Then, the algal mold was pressed in a hydraulic pressing machine with temperature controller to provide the proper shape. iii. After that, the final prepared fast card was subjected to subsequent bioassays.

Test of Efficacy of Prepared Fast Card
Bioassay was carried out in the laboratory on adult Culex pipiens. Twenty mosquitoes were released in two big bowls like jars by an aspirator. A 1 × 1 cm 2 diameter Duranta ethanolic algae cards were burnt and suspended inside the first jar and 1 × 1 cm 2 diameter commercial fast card was burnt in the second jar and a thermocol piece was placed on each of the opening mouth of the jar. Cards were burnt slowly and smoke was released inside the jar. The process was allowed for 20 min, and then cards were taken out. Cotton mass soaked with sugar solution was placed inside the jar and the opening was covered by a small net and allowed for next 24 h. After 2 h, the cotton mass was removed and behavior of mosquitoes was recorded in a note book.

Evaluation of Emitted Gases by Toxic Indicator
Emitted gases from Duranta fast card and commercially available fast card were identified and evaluated by toxic indicator of Model PPMTM gaZguard Tx (Fig. 1).
For investigating anti-mosquito repellent fast card activity, the prepared card was checked for its flammability, burning efficiency with respect to burning time, and eventually, its effective repellent activity.
From the produced smoke, emission of CO 2 , CO, and O 3 were observed and recorded.

GC-MS Study
For GC-MS study, the viscous dark mass of yielded plant product was dissolved in little quantity of chloroform and reduced to the volume of 2 ml. The dissolved 5-µl sample diluted to 50 µl and has been filtered with the help of High-Performance Liquid Chromatography technique and the component analysis has been done by GC-MS (GC) using TR-WAX MS column (30 mm × 0.25 mm ID × 0.25 μM df, composed of PEG binding material, Polar column). Helium (99.99%) was used as carrier gas. An injection volume of 1 μl was used (split ratio of 10:1). The oven temperature was set from 60 °C (isothermal for 2 min), with a rise of 10 °C/min, to 110 °C, then 5 °C/min to 260 °C, ending with a 10-min isothermal condition at 260 °C. Mass spectra have to be taken at 70 eV. Forty-nine minutes is the total running time for the study. Identification of the components was performed with the help of the datasheet of the National Institute of Standards and Technology.

HPLC Analysis
The leaf extract of Duranta was further analyzed by a "Hitachi Chromaster quarternary gradient reverse phase HPLC system" with 5160 pump, 5420 UV visible detector for confirmation of the presence of propionic acid. The extracts were filtered with syringe filter of 0.22 micron (High Media). The mobile phase for this experiment was prepared by mixing of 50% HPLC grade Acetonitrile (Merck) and 50% HPLC grade water (Merck) followed by filtering the mixture with solvent filtration system (Borosil

Observation and Result
The present study on the plant extract has revealed the efficacy of Duranta repens as a potential mosquito larvicide. Table 1 depicts the impact of ethanolic extract of this plant on the mortality of third and fourth instar larvae of Culex sp. after 24 h of larvicidal exposure. Ethanolic plant leaf extract at a concentration of 1000 ppm and 500 ppm shows 100% mortality of the studied mosquito larvae (Table1). The present investigation also unfolds that the exposure of mosquitoes in Duranta extract algae mixture card shows 100% knock down in the first 20 min in comparison of 95% knock down by commercially available fast card. After 1 h of exposure, the mosquitoes are treated with 10% of sugar solution on cotton wool and kept for 24 h at 27 ± 2 °C and 80 ± 10% relative humidity. Seventy percent of mosquitoes died and 30% showed knockdown. Contrarily, commercially available fast card was responsible for 25% mortality and 65% knockdown ( Table 2 and Fig. 2) and the rest 10% of mosquitoes were active or in flying condition after this treatment with commercially available fast card.
During the burning of prepared card, the amount of some toxic gaseous products was considered. Result revealed that commercially available fast card discharged 2.20 ppm CO 2 , 1.72 ppm CO, and 1.73 ppm O 3 . On the flip side, released amount of gases from the prepared organic fast card were 0.79 ppm, 1 ppm, and 0.48 ppm, respectively (Fig. 2).
From the GC-MS analysis of the leaf extract, the presence of 21 phytochemical constituents was revealed which are responsible for the larvicidal quality of the Duranta. The Propionic acid was the major bio active component of Duranta plant extract as it showed the highest area peak (49.70%). HPLC analysis of Duranta was performed. The crude extract and fractions from Duranta plants was investigated for the presence of propionic acid (Fig. 3). Figure 3 also represents that the component which appeared in the peak number five and investigated at 3.260 min showed high similarity with the standard compound at 3.487 min that confirmed the presence of propionic acid in the Duranta leaf extract. Further study of the larvicidal bioassay of the pure Propionic acid in ethanol solvent showed that this compound is highly effective as they caused 100% mortality of the fourth instar larvae in comparison to 13% mortality caused by the solvent alone.

Discussion
One of the most effective unorthodox approaches under the biological control program is the exploration of the plant biodiversity to develop safer insecticides of botanical origin as a manageable and tenable method of mosquito control. In the current investigation, the ethanolic extract of Duranta repens shows 100% mortality of Culex larvae. Therefore, Duranta extract might be employed as a potent larvicide. Duranta repens are locally available also, hence provides means to protect the human health from vector mosquitoes. The ethanolic crude extract of Duranta leaves leads to 100% mortality of all instar (Culex pipiens) larvae at both 1000 ppm and 500 ppm concentration. So, it can be presumed from the present study that the crude extract of Duranta leaves have larvicidal efficacy that can be used to reduce the population load of Culex vector. Literature survey reveals that the ethanolic leaf extract of Duranta offered some protections against diseases related to the muscles and they also have confirmed the non-toxicity of the plant extract to the kidney of rat [20]. In our study, the dominant compound found in the ethanolic extract of Duranta sp. was propionoic acid, which was further confirmed by the GC-MS study, and the US Environmental Protection Agency considers it quite safe and therefore, has no limitation on its use [21]. Generally, the active toxic ingredients of plant extracts are secondary metabolites that are evolved to protect them from herbivores. Hence, these are generally non-toxic for aquatic flora. As the propionic acid, in the environment, acts as a carbon source for various microbes and is metabolized to carbon dioxide and water, it might assist in photosynthesis in the aquatic plants to some extent. To increase the shelf-life of this botanic larvicide, our future plan of work is to develop silver nanoparticles (AgNPs) using plant extracts as stabilizing and capping agents. This formulation will combine the antimicrobial functions of silver, the larvicidal property of the sorted plant with high efficacy because of the increased surface-volume ratio [22] due to the tiny size of the particles (1-100 nm). The present experiment also reveals that prepared card from Duranta ethanol extract with algae mixture possesses potentiality to be utilized as a mosquito repellent. On the basis of existing result, it can be inferred that such kind of organic fast card exhibits much better result in mosquito controlling methods and in addition, it causes insignificant hazard on human health than commercially available mosquito repellent fast card (Fig. 2). It is observed that in the recent few years, various government sectors as well as non-governmental organizations are coming forward to boost products and technologies that lead to the conservation of nature. Therefore, the fast card prepared in this research carries great promise in the coming days as an eco-friendly alternative to commercially available fast cards. This algal-Duranta fast card might also create a way of alternate livelihood generation specifically for the rural people. It can also be confirmed that the major active compound is propionic acid, a naturally occurring carboxylic acid in Duranta which is responsible for both the larvicidal and adulticidal properties against Culex mosquito. Occurrence of propionic acid in Duranta was also confirmed by the HPLC analysis. From this study, it can be concluded that leaves of Duranta may serve as new potential source of mosquitocide due to