The potential use of Azolla pinnata as an alternative bio-insecticide

Four different tests showed the effectiveness of Azolla pinnata plant extracts against Aedes aegypti and Aedes albopictus mosquitoes. In the adulticidal test, there was a significant increase in mortality as test concentration increases and A. pinnata extracts showed LC50 and LC95 values of 2572.45 and 6100.74 ppm, respectively, against Ae. aegypti and LC50 and LC95 values of 2329.34 and 5315.86 ppm, respectively, against Ae. albopictus. The ovicidal test showed 100% eggs mortality for both species tested for all the concentrations tested at 1500 ppm, 1000 ppm, 500 ppm, 250 ppm and 125 ppm. Both tested samples of Ae. aegypti and Ae. albopictus did not lay any eggs in the plastic cups filled with the A. pinnata extract but instead opted to lay eggs in the plastic cups filled with water during the oviposition deterrence test. Similarly, the non-choice test of Ae. aegypti mosquitoes laid eggs on the sucrose solution meant for the nutrient source of the mosquitoes instead of in the plastic cup that was designed to facilitate oviposition filled with the extract. This clearly indicates the presence of bioactive compounds which are responsible in adulticidal and ovicidal activity in Aedes mosquitoes and at the same time inducing repellence towards the mosquitoes. The LC–MS results showed mainly three important chemical compounds from A. pinnata extracts such as 1-(O-alpha-D-glucopyranosyl)-(1,3R,25R)-hexacosanetriol, Pyridate and Nicotinamide N-oxide. All these chemicals have been used for various applications such as both emulsion and non-emulsion type of cosmetics, against mosquito vector such as Culex pipens and Anopheles spp. Finally, the overall view of these chemical components from A. pinnata extracts has shown the potential for developing natural product against dengue vectors.

Dengue is transmitted by viral infected female mosquitoes known as Aedes albopictus and Aedes aegypti, primary vectors for vector-borne diseases which affect humans 1 . Dengue is known as one of the most common infections for humans, which is transmitted through bites of infected Aedes mosquitoes. It is considered to be a major health problem in tropical and subtropical countries 2 . Currently, Malaysia has recorded 80,000 dengue cases, with 113 deaths between January and August 2019 3 .
Physical control method is always a daunting task as it provides temporary solutions and is currently used as equipment's such as bed nets, clothes and mosquito rackets 4 . Meanwhile, the use of chemical method such as temephos and pyrethroids is more prominent but it has resistance challenges. Problems of resistance in mosquitoes towards the application of permethrin and temephos has been reported in two major cities in Malaysia, namely Kuala Lumpur and Penang 5,6 . The insecticide resistances in mosquitoes are caused by factors of alterations in its targeted sites and the increase in insecticide metabolisms rate 7 .
Current application of the synthetic chemical controls and its constant repetitive applications have resulted in resistant mosquitoes and environmental pollutions 8 . Thus, the limited success of biocontrol programs on Aedes has encouraged the necessity for new insecticide search. Plant products have produced positive outcomes as an alternative to chemicals in dengue vector control programs. With this concept in mind, the use of Azolla pinnata has been recently reported against Aedes vectors 4,6,[8][9][10] . Most of A. pinnata studies were only conducted for Aedes larvicidal effects and for its chemical compositions analyzed with emitted compounds by gas chromatographic  4 . Thus, it is vital to investigate the applications of direct contact from A. pinnata plant extracts against adult Aedes mosquitoes and to its entire life cycle satges. Moreover, it is necessary to investigate the chemical constituent of A. pinnata in its liquid compositions, which may be responsible against Aedes mosquitoes' interactions. Meanwhile, unlike conventional insecticides, the advantages of plant-derived insecticides composed by botanical blends of multiple chemical compounds is that it may act concertedly on both physiological and behavioural processes 11 . The alternative uses of bio-insecticides will provide a more suitable and sustainable solution against Ae. aegypti and Ae. albopictus. In line with United Nations (UN) global sustainable goals and following a safer and greener alternative conception from A. pinnata plant, the purpose of this study is to evaluate the effectiveness of A. pinnata liquid extracts on Aedes adulticidal, ovicidal and ovipositional deterring activity and its liquid chemical compounds.

Results
Adulticidal activity. As presented in Fig. 1 Oviposition deterrence assay. Both Aedes mosquitoes preferred to lay eggs only in the untreated cups.
This can be clearly seen in Fig. 3, 4, 5, 6 and further proven through the data on Table 3 Fig. 6.          2010), with the Ervatamia coronaria and Caesalpinia pulcherrima plant extracts caused 100% egg mortality on Culex quinquefasciatus, Ae. aegypti and Anopheles stephensi at higher than 300 ppm. Meanwhile, the eggs in the control treatment had 0% mortality when immersed in regular de-chlorinated water. Additionally, the eggs mortality was 80% till 90% for A. pinnata extract concentration of 30 ppm and 75 ppm. These proved that even at the lowest concentration,the efficacy was high in causing Aedes eggs mortality.
The oviposition deterrence test proved that the eggs can only be observed in the plain water medium and not in A. pinnata plant extracts. Additionally, the non-choice test shows that without any choice or options, the Ae. aegypti and Ae. albopictus laid their eggs on the cotton soaked with sucrose solution. As discussed earlier, it is obvious that the methanolic extract of A. pinnata had repellence properties. Thus, at 500 ppm concentration of plant extract, the Aedes mosquitoes will not lay their eggs in test cups.
In this current study, all the results of adulticidal, ovicidal, oviposition deterrence were between concentrations of 125 ppm till 6100 ppm. Meanwhile, previous larvicidal results by  on A. pinnata extract reported lethal concentration of 1000 ppm till 1500 ppm. In an overall view, the concentration of LC 50 for adulticidal, 2572.45 ppm can be used for all the assays against Aedes mosquitoes. Thus, a single concentration of A. pinnata plant extracts will be effective for further field-based applications and it may serve as alternative for vector control strategies. In order to proceed with field-based applications, some contribution of knowledge is required on the chemical compositions from A. pinnata plant extracts.
The LC-MS results (Table 5) have shown the presence of various compounds in the A. pinnata extracts which can be difficult to determined. In this study, the three most likely active compounds with promising ovicidal and repellency activities, namely 1-(O-alpha-D-glucopyranosyl)-(1,3R,25R)-hexacosanetriol, pyridate and nicotinamide N-oxide were selectively identified. Other compounds were not likely to contribute to the ovicidal and repellency activities. According to Kawasaki et al., (2006), chemical compound of 1-(O-alpha-D-glucopyranosyl)-(1,3R,25R)-hexacosanetriol is used for antibacterial cosmetic composition which is easily applied to both emulsion and non-emulsion type of cosmetics. Thus,1-(O-alpha-D-glucopyranosyl)-(1,3R,25R)hexacosanetriol compound has the potential to be used as a natural ingredient for formulations in developing mosquito repellence creams in near future. Furthermore, pyridate compound were also used in several studies against mosquito vector such as Culex pipens and has been proven effective 12 . Besides that, nicotinamide N-oxide is used as insecticides in commercialized products and tested to be effective against Anopheles 13 . Our previous studies on A. Pinnata have recorded several interesting chemical compounds with insecticidal properties such as n-hexadecanoic acid, diethyl phthalate, neophytadiene, 3,7,11,15-tetramethyl-2-hexadecen-1-ol 4,8 . The chemical analysis conducted in previous studies of A. Pinnata were based on GC-MS analysis which differs compared to the current study due to the progressive experimental efforts in classifying all the volatile compounds, as LC-MS analysis is suitable for compounds of lower volatility. The overall view of these chemical components from A. pinnata extract has shown the potential for developing natural product against dengue vectors.

Methodology
in larvae rearing. The eggs were hatched in de-chlorinated water for 24 h and maintained at 25-30 C, pH 6.95 to 7.03, and relative humidity of 80 ± 10% and dissolved oxygen of 5.5-6.1 mg/L in the laboratory. The larvae were then reared until they pupated. The pupae were then transferred into mosquito cages to allow the mosquitoes to emerge. Sucrose solution was provided in the mosquito cage to provide food source for the mosquitoes.
All the papers were left to dry at room temperature to evaporate off the methanol overnight 14 . The methods for the adulticidal activity were adapted from WHO, 1997. Female, Ae. aegypti and Ae. albopictus mosquitoes aging 2-5 days old of each strain were used in the separate test as they are fully developed and have reached sexual maturity at this age. A total of 25 female mosquitoes were collected with an aspirator and were placed in each holding tubes of the bioassay kits. The holding tubes were left for an hour acclimation time to check on any weak or unhealthy mosquitoes that might die in the tube. Dead mosquitoes were replaced with new ones and the test was continued. Strips of impregnated papers with different concentrations of methanolic A. pinnata extracts were rolled into the exposure tubes of the bioassay kits. The mosquitoes in the holding tubes were then blown into the exposure tubes and left in the exposure tube for an hour (Fig. 2). The numbers of the mosquitoes knocked-down were recorded at an interval of 5 min throughout the 60 min. The knockdown of mosquitoes was evaluated with the inability of the mosquitoes to stand up or fly (Jahangir et al., 2008). After 1 h, all 25 mosquitoes were then collected and placed in a paper cup with cotton bud soaked in sucrose solution containing 10% of vitamin B complex and was left for 24 h. The mortality of among the 25 post-treated mosquitoes was obtained. A negative control test was done with impregnated paper containing oil where no mortality was recorded. After the bioassay test, the remaining surviving mosquitoes after 24 h were taken out and were used for the subsequent test. A total of four replicates were done for each species and solvent used to obtain a better data set.
Ovicidal activity. The methodology for the ovicidal activity was followed by Su and Mulla (1998) with slight modification. Total of 100 eggs of Ae. aegypti and Ae. albopictus were immersed in 6 trays. 5 trays filled with methanolic A. pinnata extracts at different concentrations; 1500 ppm, 1000 ppm, 500 ppm, 250 ppm and 125 ppm. Control cup was filled with distilled water. The test was conducted in 5 replicates for each concentration. After treatment, the eggs from each treatment were counted under microscope and transferred into distilled water cup for hatching assessments. The percentage of eggs mortality was calculated based on the nonhatchability of eggs with unopened opercula. The hatch rates were assessed by 48 h post-treatment using formula by Govindarajan (2011); Egg mortality (%) = number of unhatched larvae/total number of eggs × 100% www.nature.com/scientificreports/ Oviposition deterrence activities. The oviposition deterrent activity was conducted in a laboratory using the method of Reegan et al., (2013) 15 and a dual-choice oviposition bioassay was performed on gravid females of Ae. aegypti. Fifteen, gravid females (5 days old) of Ae. aegypti were introduced into an insect cage (30 cm × 30 cm × 30 cm) under room conditions. The adults were provided with 10% glucose solution which was available at all time. Two 50 mL plastic cups were filled with dechlorinated water and two 50 mL plastic cups were filled with methanolic A. pinnata extracts for oviposition. Different concentrations of 1500 ppm, 1000 ppm and 500 ppm of a plant extract solutions were used in different cages. A support for oviposition was provided by placing a piece of filter paper (Whatman No.1) on the inner surface of each plastic cup so that the lower half of it was submerged in the treated solution or untreated solution for the whole paper to get moistened while the upper half of it was above the solution where the mosquitoes would lay their eggs on. The untreated and treated cups were placed at alternate diagonally opposite locations for each replicate to nullify any effect of their locations on oviposition. After 3 days, the numbers of eggs laid in the treated and untreated cups were counted under a stereomicroscope. A non-choice test, was also conducted by placing one 50 mL plastic cup filled with A. pinnata and placed with filter paper into the mosquito cage with 15 gravid female Aedes mosquitoes. The test was conducted with different concentrations as well. Percent effective repellency (ER percentage) for each oviposition repellent concentration was calculated using the following formula from Xue et al.,

Conclusion
In conclusion, the A. pinnata plant extracts showed promising results in adulticidal and ovicidal test against Ae. aegypti and Ae. albopictus. The oviposition deterrence test showed that there are repellent properties present from the A. pinnata extract. With the identification of chemical compounds in the A. pinnata extract, it can be developed as bio-insecticides for Aedes mosquito vector control. This study also suggests that future research work can be conducted on the field applications of A. pinnata extracts for its long-term effects on other nontarget organisms, including on human health.