Comparative Efficacy of Selected Biorational Insecticides against Larvae of Southern House Mosquito Culex quinquefasciatus Say (Diptera: Culicidae)

Mujahid Tanvir1, Muhammad Asam Riaz1*, Muhammad Zeeshan Majeed1, Mazhar Iqbal Zafar2, Muhammad Tariq3 and Muhammad Bilal Tayyab1 1Department of Entomology, College of Agriculture, University of Sargodha, Sargodha 40100, Pakistan 2Department of Environmental Sciences, Faculty of Biological Sciences, Quaid-I-Azam University, Islamabad 45320, Pakistan 3Department of Entomology, Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi 46000, Pakistan Article Information R ceived 05 July 2021 Revised 26 August 2021 Accepted 02 September 2021 Available online 12 November 2021


INTRODUCTION
M any arthropod species vector direct and indirect transmission of different bacterial, viral, and protozoan diseases in humans. The most common vector borne diseases which affect humans are typhus transmitted by human louse, plague caused by fleas, enteric diseases caused by houseflies, sleeping sickness caused by tsetse fly, chagas disease vectored by triatomine bugs (Manguin and Boëte, 2011;Dacey and Chain, 2020). Similarly, several mosquito species belonging to Aedes, Anopheles and Culex genera are medically important and vector many viral diseases such as chikungunya, malaria and O n l i n e

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2002; Paily et al., 2007;De Wispelaere et al., 2017). In early 19 th century, transmission of malaria and avian pox virus in Hawaiian bird populations was caused by Culex quinquefasciatus mosquitoes and resulted in suppression of the population of native Hawaiian honeycreepers (Atkinson and LaPointe, 2009). Mosquitos are primarily controlled by extensive applications of persistent synthetic insecticides such as DDT, malathion, chlorpyrifos, deltamethrin etc. and many field populations of mosquitoes including C. quinquefasciatus have attained high resistance against these synthetic insecticides (Tikar et al., 2008;Senthil-Nathan, 2020). Therefore, there is a dire need of searching for biorational mosquito control methods such as botanical, microbial and reduced-risk synthetic insecticides (Rose, 2001;Benelli, 2015). Plant based pesticides usually have low mammalian toxicity and have been emerging as promising alternatives to synthetic insecticides for the control of mosquitoes (Sukumar et al., 1991;Isman, 2008;Zhu et al., 2008;Senthil-Nathan, 2020). Similarly, microbial pesticides are usually based on entomopathogenic fungal, bacterial or viral strains and have been demonstrated as safe and effective against a wide range of insect pest species including mosquitos (Federici, 1995;Regis et al., 2000;Bukhari et al., 2013;Dacey and Chain, 2020). This research work was hence aimed to determine the effectiveness of selected microbial and synthetic insecticides and indigenous botanical extracts against the larvae of C. quinquefasciatus.

Collection and preparation of plant samples
Indigenous flora consisted of stems, leaves, flowers and fruits of local plant species (including herbs, shrubs, bushes and trees) were collected from six different sites of Soon Valley located in North-West of district Khushab (Punjab, Pakistan) (Table I). Collected samples were labeled and were brought to the Laboratory of Entomology at College of Agriculture, University of Sargodha, Pakistan. These samples were cleaned manually to remove all foreign material followed by washing with distilled water and were shade-dried at room temperature (27°C). Dried samples were weighed and ground to fine powder with an electric blender. Powdered samples were stored in hermetic plastic zipped-locked bags to avoid any contamination.

Botanical extraction
As the ordinary method of extraction was not efficient to yield good amount of phyto-constituents, Soxhlet extractor (DAIHAN Scientific North America Inc., USA) was employed for the extraction of prepared plant samples using n-hexane as extraction solvent following a previously described protocol (Majeed et al., 2020). In brief, extractor thimble was filled with a known amount (50 g) of ground plant material of each sample and was plugged with a piece of cotton to stop the entry of crude extract into the siphoning tube. A known volume (500 ml) of n-hexane (purity ≥ 99.0%) was filled into the flask (1 L) installed on the mantle of heating device. The temperature of heating mantle was maintained at 68±5°C. The extraction process took 5 to 6 h for each sample. The crude botanical extract obtained from Soxhlet apparatus was concentrated by evaporating excess of solvent using rotary evaporator (DAIHAN Scientific North America Inc., USA). Final concentrated extracts were preserved in hermetic dark glass vials in a refrigerator at 4°C until their downstream use in toxicity bioassays. Collection of mosquitoes Mosquito (C. quinquefasciatus) larvae were collected from the water pound near the College of Agriculture (32°06' N to 72°39' E) with the help of an aquatic net. It was ensured that collection site was never exposed to any insecticide application. These larvae were brought to the laboratory for identification and were reared up to F 3 to get a homogeneous population.

Larvicidal bioassays with botanical extracts
In initial screening bioassays, only one concentration (0.5%) of each plant extract was used. Twenty five late 3 rd or early 4 th fourth instar larvae of C. quinquefasciatus were released in 30 ml of 0.5% aqueous solution of each plant extract in disposable glasses (100 ml). The experimental layout was CRD with five replications for each treatment and was performed under controlled condition (25±2ºC and 60±5% RH) with 16:8 light and dark hours, respectively. The mortality of mosquito larvae was recorded at 24 and 48 h post-exposure. Ten plants exhibiting significant larvicidal activities in screening bioassays were further O n l i n e F i r s t A r t i c l e bioassayed to determine their detailed toxicity. A volume of 30 ml of following concentrations (2.0, 1.0 and 0.5%) were prepared form stock solution of plant extracts in disposable plastic glasses (100 ml). Late 3 rd or early 4 th instar larvae (n = 25) of C. quinquefasciatus were released in these plastic cups with the help of a dropper. The mortality of larvae was observed at 12, 24 and 48 h postexposure. Each treatment was replicated four times.

Larvicidal activities of synthetic and microbial insecticides
Larvicidal activities of synthetic and microbial insecticides were determined by performing bioassays according to WHO protocol with insecticidal formulations detailed in Table II. One drop of Tween 80 was used to solubilize the microbial insecticides in water. Three concentrations (800, 400 and 200 ppm) of microbial insecticides were used and water with Tween 80 was used as control. However, four concentrations (5.0, 2.5, 1.25 and 0.62 ppm) of synthetic insecticides, causing mortality from 10 to 90%, were employed and only water was used as control. Late 3 rd or early 4 th instar larvae (n = 25) of C. quinquefasciatus were tested in disposable glasses. The mortality of mosquito larvae was recorded at 24 and 48 h post-exposure for synthetic and microbial insecticides, respectively. The experiment was repeated four times and was performed under controlled condition (25±2°C and 60±5% RH) with 16:8 light and dark hours, respectively.

Statistical analysis
Prior to statistical analysis, data regarding the mosquito larval mortality were corrected using Abbott's formula (Abbott, 1925). Lethal concentration (LC 50 and LC 90 ) values were calculated by Probit analysis using POLO ® Plus version 2.0 (LeOra Software). Mortality data was subjected to one-way ANOVA and the treatment means were compared by Tukey's HSD at 95% level of significance.

Identification of plants
Botanical extracts are world widely used for insect control. They are effective against insects without considerable deleterious effects on the environment. This study focuses on the identification of plants from salt range to assess their toxicity potential against mosquito larvae. The plants were collected from different locations of Soon Valley and its surrounding salt range (Punjab, Pakistan). These plants were identified up to species level with the help of botanists from the Department of Botany, University of Sargodha, Sargodha. The vernacular names provided by the native inhabitants, botanical names and literature-based phyto-constituents of collected plants are given in Supplementary Table SI. Interestingly, all plants collected from salt range constitute of alkaloids, flavonoids, terpenoids, tannins and saponins in common, showing their anti-insect potential. This plant collection and characterization would serve as baseline data about the indigenous flora of study area.
Toxicity bioassay with the most effective plant extracts against C. quinquefasciatus larvae Based on the results of initial screening bioassays, ten plants, exhibiting significant mortality (more than 70%), were further evaluated against C. quinquefasciatus larvae. Results of this toxicity bioassay (  (Table  IV).

Larvicidal activities of microbial and synthetic insecticides against C. quinquefasciatus larvae
The results of larvicidal bioassay conducted with microbial insecticides ( Fig. 2A) showed that all insecticidal formulations caused significant larval mortality (p ≤ 0.05) as compared to control. M. anisopliae was the most effective larvicidal treatment exhibiting significantly highest mortality (83%), followed by B. thuringiensis (60%), B. bassiana (58%), while the lowest larval mortality was recorded for I. fumosorosea (50%) at 800 ppm at 48 h post-exposure. Similarly, entomopathogenic fungi M. anisopliae had the lowest LC 50 value i.e. 325 ppm and was the most toxic larvicide as compared to other three microbial insecticides (95 % CI did not overlap). B. thuringiensis, B. bassiana and I. fumosorosea showed similar toxicity against C. quinquefasciatus larvae ( Fig.  2A). Larvicidal evaluation of synthetic insecticides against C. quinquefasciatus showed that permethrin exhibited 70% mortality at 0.62 ppm. Indoxacarb showed 86% mortality at 5 ppm. Lambda-cyhalothrin displayed 73% at 2.5 ppm and pyriproxyfen showed 86% mortality at 200 ppm at 24 h post-exposure. Indoxacarb had the lowest LC 50 value i.e. 0.14 ppm, and was the most toxic synthetic insecticide as compared to other three tested insecticides (95 % CI did not overlap). Permethrin and lambda-cyhalothrin were moderately toxic larvicide as compared to pyriproxyfen which was proved to be the least toxic synthetic chemical (Fig. 2B).

DISCUSSION
Mosquitoes are responsible to transmit world's most severe life-threatening diseases (Benelli and Mehlhorn, 2016). Mosquitoes in the larval stage are more susceptible targets for chemical control because they breed in water making it easy to control in this habitat. The use of conventional pesticides in the water sources is highly risky to humans and their environment. Better alternative control means are required due to the continuous increase in resistance of mosquitoes to commonly used conventional synthetic insecticides (Tikar et al., 2008). Pakistan, particularly salt range (study area), has diverse ecological zones, rich natural resources and flora with more than   (Ahmad et al., 2009;Nawaz et al., 2012). As native vegetation of a particular area may contain insecticidal properties which need to be evaluated for their potential use in pest control (Isman, 2008), the present study was conducted to evaluate the larvicidal potential of indigenous plant species of Soon valley and surrounding range of Pakistan along with some promising microbial and synthetic insecticide formulations against 3 rd and/or 4 th instar larvae of C. quinquefasciatus. Most of the plant species collected belonged to Apocynaceae, Amaranthacea, Fabaceae, Lamiaceae and Solanaceae families and are usually enriched in such phyto-constitutes as alkaloids, carbohydrates, cardiac glycosides, cyanogenic glycosides, flavonoids, phenols, resins oxalates, steroids, saponins and tannins as described in Supplementary Table  S1. Our results revealed that the extract of M. arenaria was most effective against mosquito larvae. Aqueous extract of this plant species constitutes of alkaloids, phenolics, phytosterols and saponins (Ali et al., 2008) which would be responsible for the observed significant mortality of mosquito larvae. Likewise, the extracts of N. indicum have different alkaloids and terpenoids which showed anti-feeding, ovicidal, larvicidal and repellant activities against a wide range of insect pests including mosquitoes (Hiremath et al., 1997;Srivastava et al., 2003;Saxena and Sharma, 2005;Rahuman et al., 2008;Dey et al., 2017). Acetone and methanolic extracts of N. indicum at 0.02 to 0.03% concentrations showed significant mortality (more than 50%) of C. quinquefasciatus larvae ( (Dimetry et al., 2015) and homopterous pests (Díaz et al., 2015). Similarly, many species of Oleaceae family contain toxic compounds potentially effective against different insect pests. For instance, O. europaea constitutes of higher phenolic contents and a triterpene compound (maslinic acid) exhibiting significant toxicity against aphids (Myzus persicae) and stored grain insect pests (Sitophilus granaries and Tribolium confusum) (Kisa et al., 2018).
In addition, W. coagulans and S. fruticosa extracts contain different alkaloids and phenols, and α-pinene and borneol, respectively (Koliopoulos et al., 2010;Mathur et al., 2011), and these plant extracts (10%) have shown to cause significant mortality (63%) in Callosobruchus chinensis (Gupta and Srivastava, 2008) and up to 50% mortality in larvae of Culex pipiens (Koliopoulos et al., 2010). Our results are in line with the findings of Teressa et al. (2019) showing 60% mortality in Anopheles mosquito larvae by the extract of O. europea plant. Similarly, 0.03% hexane extract of A. capillus-veneris caused 80 and 70% mortality in Plutella xylostella and Aphis craccivora, respectively (Sharma and Sood, 2012). Taken together, the screened plants could provide a baseline for their insecticidal potential. The extract of highly effective plants could be used for the development of organic mosquito repellent at commercial level and their bioactive fractions could be further developed as botanical mosquitocidal formulations.
Nevertheless, microbial pesticides also appear as

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Comparative Efficacy of Selected Biorational Insecticides 7 alternative to chemical insecticides with target specificity and ecological safety so that they are used individually or in combination with other pest management programs. Among entomopathogenic formulations tested, M. anisopliae showed significant mortality of C. quinquefasciatus larvae. The possible mode of action of this fungus could be the floating conidia come in contact with larvae. Conidia break the water tension with their peri-spiracular valves for air intake. The fungal conidia germinate and penetrate into the siphon which blocks the breathing mechanism. In warm and moist conditions, conidiophores grow on the cuticle and cover the whole insect with conidia (Daoust et al., 1982;Lacey et al., 1988). The presence of different toxic proteins increases the larvicidal activity and suppresses the development of resistance. Unfortunately, there is no ideal mosquito-pathogenic fungal strain presently known which effectively kill the mosquito larvae. Among the synthetic insecticides, indoxacarb showed highest larval mortality. Indoxacarb is a neurotoxic insecticide that blocks voltagedependent sodium channels, resulting in insect paralysis and death and is considered safe for environment (Wing et al., 2010) and has shown excellent results against pyrethroid resistant mosquitos including Anopheles and Culex species (N'Guessan et al., 2007;Shah et al., 2016).

CONCLUSIONS
Overall study results provide preliminary database regarding the insecticidal potential of indigenous plant species of Soon valley and surrounding salt range of Pakistan. These above mentioned effective plants extracts along with microbial insecticides are therefore recommended for the biorational management of mosquitoes and to minimize the contemporary issues of environmental contamination and health hazards associated with the use of persistent synthetic insecticides. Further biochemical characterization of effective plant extracts and field evaluation of these selected botanical, microbial and synthetic insecticides against mosquito larvae and their non-target effects on the environment constitute the future perspectives of this study. Sustainable, safe, and environment-friendly control methods should be established that can target different mosquito species.

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Comparative Efficacy of Selected Biorational Insecticides