IGR Activity of Three Indigenous Plants belonging to Asteraceae Family and its Potential Role on Control of Filarial Vector, Culex quinquefasciatus Say (Diptera: Culicidae)

SR Ahalya, E Pushpalatha 1,2Biopesticides & Toxicology Division, Department of Zoology, University of Calicut, Malappuram, Kerala, India. DOI: https://doi.org/10.24321/0019.5138.202014 Background: Insect Growth Regulatory and larvicidal role of three indigenous plants belonging to the family Asteraceae is tested against the filarial vector, Culex quinquefasciatus Say. Methods: IGR and larvicidal bioassays were conducted using methanol and water extracts of the selected plants such as Blumea mollis, Vernonia cinerea and Centratherum punctatum against I instar larvae of Cx. quinquefasciatus. Result: Lethal concentrations (LC50 and LC90) were estimated for methanol and aqueous extracts of the selected plants. Treatment with methanolic extracts exhibited prolonged larval and pupal duration, when compared to control. Total developmental period was also significantly extended. The larvae treated with aqueous extracts showed shortening of larval and pupal durations. Conclusions: The present study highlights the importance of plantbased materials and their efficacy to control the mosquito vectors with special to Culex quinquefasciatus. Treatment with methanol extracts of the selected plants showed extension of larval duration where as aqueous extracts show precocenes like activity and the total developmental period shortened with the emergence of almost 90 % of the treated larvae.


Introduction
Since mosquitoes are most abundant group of insect disease agents, control of them has become a major challenge to eradicate major epidemic diseases. Most of the developing countries have been suffering from frequent disease outbreaks. Several ways are in practice to control mosquitos like, physical, chemical, biological, and genetic ways. From 1920 onwards, chemical control J. Commun. Dis. 2020; 52 (2) ISSN: 0019-5138 DOI: https://doi.org/10.24321/0019.5138.202014 became a predominant way of insect control. Due to the adverse effects of extensive use of chemical insecticides, the idea of biopesticides became more relevant. Since the effects of biopesticides are not persistent throughout years, it will not contribute to bio magnification and does not cause much development of resistance in organisms. The less toxic effects to non-target organisms is also an important feature of biopesticides. Since the sources of bio pesticides are plants and phytochemicals, they are easily biodegradable and eco-friendly. As the Integrated Vector Management (IVM) is the current strategy which includes a combination of all management techniques to control insect pests and vectors.
From the biopesticides point of view, phytochemicals do possess anti-insect properties which are either purely insecticidal or act as feeding deterrents, growth inhibitors, growth regulators, repellents or oviposition inhibitors against a variety of insect species. Biopesticides/ biolarvicides generally have several advantages compared to conventional pesticides 1,2 as chemical pesticides are withdrawn owing to resistance problem, or because they are no longer commercially viable and, thus, immense opportunities are emerging for bio rational solutions.
IGRs are classified as (i) Juvenile hormone analogues (JHAs also called as Juvenoids) (ii) Ecdysteroid agonists and (iii) Chitin synthesis inhibitors 3 (CSIs or moult-inhibitors). IGRs belong to a group of compounds which are not directly toxic, but selectively affect the growth and development, metamorphosis and reproduction of insects through the disruption of hormonally regulated physiological processes. 4 Compounds with anti JH activities can be used as an insect control agent. The present study was carried out to assess insect growth regulatory and larvicidal properties of the three indigenous plants belonging to Asteraceae family and its potential role on control of filarial vector, Culex quinquefasciatus Say.

Collection and Maintenance of Culex quinquefasciatus
Larvae of Culex quinquefasciatus were collected from botanical garden of Calicut University and Kalluthan Kadavu colony, Kozhikode and brought to laboratory and reared. The adult mosquitoes were maintained at 29±2°C and 75±5% relative humidity at 12-hour day and night cycle mimicking the natural condition in a 15 inch square cage covered with mesh. The adults were fed on 10 % sucrose solution soaked in cotton and placed in a glass jar. Adequate blood meals were provided to 2 to 3 day old adult mosquitoes from an immobilized quail in twilight hours and a bowl containing water provided in the cage to facilitate oviposition. Egg rafts laid were transferred to a beaker and allowed to hatch. The freshly hatched larvae were fed by fine powder of yeast and dog biscuits in the ratio 3:1 and maintained in the laboratory.

Preparation of Methanol Extracts
The aerial parts of selected weed plants, Blumea mollis, Vernonia cinerea and Centratherum punctatum (Plate 1-3) were collected from the botanical garden of the University of Calicut, brought to the laboratory, washed thoroughly with water and shade dried at room temperature. The dried parts were powdered using a grinder and extracted in analytical grade methanol using a soxhlet apparatus. 15 to 20 cycles were fixed for each extract and collected separately and evaporated to dryness using a flash evaporator. The net weight of the extract was estimated and yield was calculated and 10% stock solution were prepared and stored in refrigerator for bioassay tests.

Preparation of Aqueous Extracts
20 g plant powder mixed with 250 ml distilled water in a conical flask was stirred for 24 hours in magnetic stirrer. The extract was then filtered and collected separately and evaporated to dryness. The yield of the plant material was calculated from the dried extract and 10% stock solution was prepared and stored in refrigerator.

Estimation of Lethal Concentration LC 50 and LC 90
Bioassay for the estimation of larvicidal activity using WHO protocol (1996) with slight modifications was adopted for the study. Methanol and water extracts of selected plants is tested against early first instar larvae of Culex quinquefasciatus. The appropriate volume of 1% stock is diluted to 100 ml 0.08% saline water in a disposable glass to obtain the desired test medium. 10 larvae were released to each tumbler containing the test medium as 100 ml test medium and also in the control set. Triplicates were maintained for each set. Observations on mortality of the larvae after 24 hr was taken and percentage mortality was calculated. Using the percentage mortality, LC 50 and LC 90 was calculated using SPSS statistical programme.

Insect Growth Regulatory Activity
Insect growth regulating activity according to the protocol of Mehdi et al 5 with slight modifications were conducted against freshly hatched 1 instar larvae of Cx. quinquefasciatus. Fifty numbers of I instar larvae were transferred to a beaker containing 500 ml of 0.08% saline solution. Desired sub lethal concentrations of the selected plant extracts were set to study the concentration depended IGR activity. Three replicates for experiment and controls were also maintained. Extension of total developmental duration, deformities and mortality rates were observed and recorded every day until adult emergence occurred. Larvae were provided with fine powder of dog biscuits and yeast in the ratio 3:1 respectively at fixed intervals during the observation period. Incidences of malformities were observed as larval-larval intermediates, larval-pupal intermediates and pupal-adult intermediates.

Result
Yield of methanol and aqueous extracts of selected plants, B. mollis, V. cinerea, and C. punctatum were estimated and provided in Table 1. Percentage yield of the methanol extracts of the selected plants ranged from 6.57% to 8.5% whereas that of aqueous extracts ranged from 5.79% to 9.21%. Aqueous extract of V. cinerea shows highest percentage yield.

Effect of Methanolic Extract on Developmental duration of Cx. quinquefasciatus
During developmental progress, the larval and pupal periods were observed and compared with that of control. Freshly hatched I instar larvae treated with methanolic extract, the larval duration and pupal duration showed increase resulting in extension of the total life span of the treated Culex quinquefasciatus (Figure 1) aqueous extract of V. cinereal whereas it was decreased to 7.5±0.5 when treated with 285 ppm aqueous extract of C. punctatum (Figure 2) when compared to that of control (11.5±0.5 days).

Effect of Aqueous Extracts on Developmental duration of Cx. quinquefasciatus
Effect on the total developmental duration of Cx quinquefasciatus when treatment with aqueous extracts of the three selected plants were observed, recorded, and presented ( Figure 2). When the freshly hatched first instar larvae were treated with aqueous extracts, it showed a decrease in total developmental duration when compared to that of control. The aqueous extracts induced shortening of larval-larval period, larval-pupal period, and pupal-adult period ( Figure 2). Treatment with 278 ppm of aqueous extract of B. mollis completes its developmental period within 10.0±0.0 days. Total developmental period was decreased to 8.0±0.0 days when treatment with 282 ppm

Discussion
Although several control measures for mosquito vectors have been practiced, the efforts are futile mainly due to the development of resistance against the synthetic insecticides. Insect Growth regulators (IGRs) and Chemosterilants offer considerable potential for the control of insect pests/ vectors. 6 IGRs have low mammalian toxicity and do not appear to cause spontaneous genetic mutation and these materials are relatively short lived in the environment which reduces potential of contamination. Azardirachtin is the most widely used botanical insect growth regulator. 7 Precocene, a plant product, is noted for its unique action interfering with growth by transgressing certain stages of J. development. In mosquitoes, it prevents pupal formation and adult emergence when newly hatched young larvae are exposed. 8 Despite the toxic effects and reduction in emergence rate, insect growth regulators also exhibited combined effects on the developmental period and adult emergence with extending to the developmental duration of exposed larvae. 9 The plant extracts caused larval mortality in a dose-dependent manner. All crude methanol extracts have been found to possess promising larvicidal activity with LC 50 values which is on par with several reported works.
Present study also provides the proof of IGR activity induced by the plant extracts on Cx. quinquefasciatus based on the developmental process. Treatment with methanolic extracts exhibited prolonged larval and pupal periods, when compared to control. Total developmental period was also significantly extended. On the other hand, on treatment with aqueous extracts of selected plants, larvae showed enormous growth and completed their life cycle within short period of time.
case of larval-larval intermediates, death has occurred with moulting sutures on head of the larvae, therefore it could not moult into next instars whereas, the formation of larvalpupal intermediates resulted in death of treated larvae at an early stage of pupation. The abdomen has also retracted along the larval skin and adopted the characteristic of pupal shape. The pupal abdomen has stretched like larvae with pupal head (Figure 4). Whereas, in the case of pupal-adult intermediates, death occurred after complete moulting, but some parts remained attached with pupal case ( Figure 5).
So, in larval-pupal intermediates, the moulted pupae could not attain the pupal characteristics such as coma shape or curved abdomen, but it appeared with stretched abdomen, probably due to the presence of JH in pupae. Similarly, in the case of pupal-adult intermediates, the adult which was emerged from the pupae existed with pupal exuvia, which might to be due to the presence of increasing amount of JH and decreasing amount of ecdysone on the adult mosquitoes, as only the complete absence of JH results in formation of the adults. 10

Conclusion
Methanol and aqueous extracts of the three indigenous plants, namely Blumea mollis, Vernonia cinerea and Centratherum punctatum belonging to the family Asteraceae shown its potential role of larvicidal and insect growth regulatory activities against Cx. quinquefasciatus Say. During the developmental metamorphosis, treated larvae exhibited extension of total developmental duration, such as prolonged larval and pupal duration in methanol extracts and shortened larval and pupal period in aqueous extracts. Therefore, the present study suggests that these plant materials can moderately control the population of filarial vector mosquito, Cx. quinquefasciatus Say and hence can be used as natural, ecofriendly and bio-degradable material for the control of mosquito vectors. However, isolation of the active principles of the selected plant extracts of the present study and its formulations is progressing which may lead to a better alternative to the synthetic larvicides for the control of mosquito vectors.