Comparative Toxicant Efficacy of Some Novel Insecticides against Metamorphosis of Papilio demoleus (L.) Larvae

The lemon butterfly, Papilio demoleus (L.) is a key pest of citrus orchards found throughout the year. Their infestation causes defoliation, retards growth, and yields reduction. The five insecticides namely; Emamectin Benzoate 1.9 EC, Belt 480g/L SC (Flubendiamide), Helmet 40 EC (Chlorpyrifos), Coragen (Chlorantraniliprole 18.5% SC), and Regent 5% SC (Fipronil) were tested on C. limon orchard leaves against the larvae population of the lemon butterfly under laboratory conditions during, 2020. The maximum cumulative effect of all treatments was observed in three replications. The mortality % of larvae and efficacy of insecticides were recorded after 24, 48, 72, 120, and 168 hours of post-sprays. The result revealed that Belt pesticide proved with maximum larval mortality % (66.64±8.16) followed by Regent (60.26±0.76), Coragen (59.66±7.72), Helmet (57.14±7.65), Emamectin and (56.18±7.50), respectively. It is concluded that the Belt insecticide provided a better mortality % against the larval population. The one-way ANOVA showed a significant difference among all tested insecticides at (P< 0.05) level. After insecticide application larvae reduced movement, skin became wrinkled, thin, and yellow-brown pigmentation visibly appeared with loose muscle concentration by releasing hyper-salivation and semi-liquid frass. Thus, it is recommended to be applied at a time interval basis to suppress the population density of lemon butterflies.


Introduction
Citrus, the thorny aromatic orchards that originated first in the Indian Sub-continent has obscure and long domestication history (Rao et al., 2021).Citrus is the largest evergreen prime genus and contains leathery leaves, and stems with sharp spines, the most significant crop in the world with antioxidant values and high content of vitamin C (Gorinstein et al., 2021).China, India, USA, Pakistan, Japan, Korea, and Spain produce massive citrus fruit, but Brazil is the topmost citrus fruitproducing country in the world (Mahato et al., 2019).These fruits are thirst-quenching, and widely popular due to their refreshing fragrance and their nutritional and phytochemical content depending upon maturity, fruit storage, growing conditions, processing, and varieties (Singh et al., 2019).The lemon orchards rapidly grow in large proportion in the summer season, they may be seedy or seedless, spread upright, grow in open habitats, oblong to obovate, and due to their delightful taste, therapeutic and potential healthy citrus fruits are widely preferred (Czech et al., 2021).The Lepidoptera are highly recognized and widely distributed scaly-winged insects with differences in distribution and lifestyles, more in number than Diptera, Hymenoptera, and Coleoptera (Powell, 2009).They are amazing surrogate herbivore species with marvellous body colors, their life cycle plays a main role in the food supply for spiders, reptiles, predatory insects, and birds and due to the availability of food sources, their habitat is muscularly interlinked (Haroon et al., 2013).Papilio demoleus often called "lime swallowtails" belongs to the family Papilionidae, commonly associated with evergreen forests, dry and humid areas, and human settlements (Smith and Vane-Wright, 2008).This species of pest insect is regularly seen in natural vegetation, gardens, wideopen, and rapid visual power, frequently found in Africa, Madagascar, Cape Verde Islands, Mauritius, India, Bangladesh, Burma, Ceylon, Formosa, Arabia, and all over Pakistan (Rafi et al., 1989).The citrus varieties are mostly harmed by the P. demoleus larvae and their biological development depends on environmental conditions (Narayanamma et al., 2001).From nurseries, up to mature stages of the citrus lemon are vigorously harmed mostly 4 th and 5 th instar stages of LBF (Mangrio et al., 2020).The Crambidae, Curculionidae, Eriophyidae, Drosophilidae, Pseudococcidae, and Cossidae families are major pest insect species to citrus (Mal et al., 2014).The female of P. demoleus exclusively lays eggs on twigs continues two to three days, their larvae pass five distinct stages that harm new growing twigs, and leaflets cause complete defoliation to citrus orchards (Mangrio and Sahito, 2022).They are widely dispersed insects with rapid growth rates and for their growth and development feed on soft green parts of the leaves, and behave as potential insect pest species to citrus (Islam et al., 2017).The lead arsenate hydrogen and sodium fluorosilicate were sprayed to control the larvae population of LBF and observed the best larvae mortality (Bindra, 1957).In Maharashtra (Sontakay, 1943), used parathion 0.025%, endrin 0.025%, malathion 0.05%, mevinphos 001%, dichlorodiphenyltrichloroethane, folithion, and diazinon against LBF larvae and found best LBF larvae mortality.Sprayed parathion insecticide and recorded 100% mortality in larvae of LBF (Saini and Sharma, 1970).The deltamethrin insecticide was applied against the P. malayanus and P. demoleus species and reported more effective against the LBF larvae population (Riaz et al., 2020).Generally, the wide application of insecticides utmost enhances the resistance power of insect pests, causing loss of natural pollinators, pest predators, bird communities, pollution, and several public health problems (Pimentel, 2005).Therefore, the present work is the first attempt to find out the control against the larvae population of the LBF under laboratory conditions in Sindh-Pakistan.

Sample collection site:
The new 4 th and 5 th instar stages of larvae of LBF were collected from each talukas of district Naushahro Feroze, during, 2020.After the collection of the larvae population from the infested C. limon leaves were kept in plastic jars and for aeration, plastic jars were covered with a muslin cloth.The five different insecticide solutions were prepared and tested and also compared with the control group against the larvae of LBF under laboratory examination.The techniques and procedures were adopted to testify to the insecticidal efficacy against the larvae of LBF at 28.5±°C and 70±5% RH in the laboratory.Experimental design: The five treatments were tested against the larvae of LBF and all treatments were replicated three times.The (n=05) petri plates were arranged and (n=40) new green emerging leaves and (n=20) larvae of the LBF (n=200) green lemon were placed individually.For each insecticide examination, a total of (n=100) 4 th and 5 th instar stage LBF larvae (n=200) insecticide containing green lemon leaves and (n=100) LBF larvae (n=200) without insecticide lush green lemon leaves were used for control.The experiment was done in the Entomology laboratory, new emerged green leaves were dipped in T1= Belt (0.62ml), T2= Helmet (2.5ml), T3= Coragen (0.62ml), T4= Regent (6.25ml) and T5= Emamectin Benzoate (6.25ml) per litter.For the recommended dosage of each insecticide, the leaves were dipped for two minutes and then kept on blotting paper for dehydration (Table -1).After that, insecticides containing leaves were placed (6x6) in diameter containing petri plates and placed Whatsman ® filter paper for moisture absorption.The insecticide efficacy and larvae mortality % were recorded after the 2 nd , 3 rd , 4 th , 6 th , and 8 th days of insecticide application.For the determination of each insecticide application against LBF larvae (Muhammad et al., 2019) described methodology was used.Statistical analysis: The analysis of variance and means were significant difference was counted through student package software (8.1) and LBF larvae mortality % was taken through the application of (Abbott, 1925), introduced formula.
Insect population in treated plot/lab after treatment Corrected (%)=1-_________________________________________________________________________________ ) x 100 Insect population control plot/lab after treatment   spray, after the application of Emamectin insecticides under laboratory conditions.Emamectin insecticide against the larvae of LBF was found to best control during earlier days as compared to the later days of the post-spray.ANOVA was found with a non-significant difference in treatments at (DF= 2; F= 0.9; P= 0.44) and a significant difference (DF= 4; F= 49.66; P= 0.02), in post-spray days.T-test was found at (P<0.05), further validation is given in (Fig- 5).Overall efficacy of five different insecticides against larvae of LBF on different days under laboratory conditions: All five insecticides proved with the most efficacy power against the 4 th and 5 th larvae stages of LBF under laboratory conditions.Belt insecticides proved to have a maximum mortality % as compared to Emamectin, Regent, Helmet, and Coragen against the pest population.
The overall maximum to minimum mortality % against the larvae after the application of all five insecticides recorded (78.65±8.86)(2 nd day), (73.41±8.65)(3 rd day), (67.37±8.20)(4 th day), (50.28±7.09)(6 th day), (30.18±5.49)(8 th day), respectively.Above all tested insecticides were found with maximum mortality % (2 nd day) compared with other post-spray days.The ANOVA was found with a significant difference at (DF= 4; F= 56.18; P= 0.02), among the post-treatment.The LSD during the post-spray days was found through (SWX) software (8.1),USA at (P<0.05).The Origin 2017 64Bit graphing and analysis software was used for the minimum to maximum mortality % of larvae on different days after post-treatment as given in (Fig- 7).

Discussion
Belt insecticide was found with maximum LBF larvae mortality % in 3 rd spray.The same findings were reported (Pawandeep and Anita, 2020); they used quinalphos 25% EC and chlorpyriphos 20% EC with the best LBF larvae mortality % on the citrus, nursery leaves.The mortality % in 1 st and 2 nd sprays with the work agreement of (Singh et al. 1983), discussed diofenolan as the best insecticide for the control of the citrus butterfly's population also observed pyrethroid and synthetic phoxim was found to be the best mortality % against the Achaea janata larvae.The maximum mortality % of the LBF larvae counted in 3 rd spray by the application of Regent is more comparable with the work of (Imen et al. 2021), who applied acrinathrin insecticide and reported (26%) mortality of Thysanoptera on the leaves of Citrus sinensis in Tunisia.The mortality % in 1 st and 2 nd sprays is similar work of (Patil and Rajashekaragouda, 1985), who mentioned the efficacy of 18 liters of water with 35 ml of endosulfan against the larvae of LBF.
Coragen pesticide was found with more efficacy in 3 rd spray against the pest population as discussed (Prakash, 2012), who applied different insecticides such as; copper sulfate, endosulfan, lauryl alcohol ethoxylate, dimethoate, fenthion, chlorpyrifos, Parathion methyl, buprofezin, cuprous oxide and lime sulfur against larvae of citrus insect pests and recorded with best mortality %.The larvae mortality % in 1 st and 2 nd sprays were found more or less comparable with the work of (Lewis et al. 2011), they recorded 100% LBF larvae mortality % when they were given treated leaves of methionine human nutrient amino acid and observed the positive effect on the larvae amino acid modulated system.Against the larvae of LBF the Helmet insecticide in proved more effective in 3 rd spray as described (Wen et al. 2021), reported 50% and 80% mortality of Diaphorina citri at LC50 and LC80 dosages of flupyradifurone insecticide.The larvae mortality % in 1 st and 2 nd sprays are comparable with the result of (Butani, 1979), sprayed with monocrotophos 0.04 % and chlorpyriphos 0.05 % and at the rate of twenty litters per tree applied 0.04% phosphamidon and observed effects.The maximum toxicant efficacy of the Emamectin was found in 2 nd spray with work similarity of (Radosevich and Cloyd, 2021), applied acetamiprid insecticide and counted (50%) mortality citrus mealybug on horticultural plants.The larvae mortality in 1 st spray as (Batra and Bindra, 1982), documented that Malathion, formothion, and oxydemotonmethyl insecticides are less effective compared with endosulfan but more effective compared with untreated control plants against the larvae of LBF.In 3 rd spray mortality % in the pest population counted which is comparable with the work of (Singh and Rao, 1978), applied seventeen insecticides were of which they proved methyl parathion 0.05%, methamidophos 0.05% and leptophos 0.06% with more effective against larvae of LBF.
The overall maximum toxicity recorded Belt and Regent (Ghosh et al. 2015), they administrated carbaryl against the 4 th instar of LBF larvae through foliar spray 0.25, 0.50, 1.00 mg/l and proposed that the above appropriate doses of the insecticide are recommended and fitting range against the biomonitoring and malformation of LBF larvae.The mortality % of LBF by the application of Coragen, Helmet, and Emamectin is with the work agreement of (Ghosh et al. 2015), they administrated quinallphas 2ml, B. thuringiensis 0.05%, fenitrothion 1ml, carbaryl, dimethoate 1.5ml contact and systematic insecticides against the mortality % of LBF larvae and also found adults with wrinkled and paralytic flight.By the application of all insecticides maximum, mortality % counted 2 nd the day of the spray (Afzal et al. 2018), documented mortality 73.57% against Drosicha mangiferae with the application of methidathion, 59.96% bifenthrin, 58.07%profenopfos, and 59.81% carbosulfan.The pest population on the 3 rd day of post-spray with the same findings (Bindra, 1957), administrated DDT 40 Ib, parathion 0.44 Ib, benzene-hexachloride 40 Ib, folithion 0.4 Ib, and diazinon 1.0 Ib, along with 100 gallons of water and observed all mentioned insecticides effective against the larvae population of LBF.On the 4 th day as described (Delano et al. 2011), they recorded 100 % fourth instar stage mortality by the application of methionine.On the 6 th day with work similarity of (Pawandeep and Anita, 2020), who counted 20.03 mean mortality in the control plot and larvae mortality on the 8 th day with the agreement of (Resham et al. 1986), they applied quinalphos 0.04% and juvenile hormone against the mortality % LBF larvae.

Conclusion and Recommendations
It was concluded that within the comparison of five novel insecticides the Belt proved more effective followed by the Emamectin, Regent, Helmet, and Coragen against the LBF larvae hence it is recommended.It is recommended that on the residual effect of toxic insecticides more work should be carried out.Typically, for insect pest control and quality improvement of the citrus several insecticides have been administrated in many parts of the world but along with insect pest control, different insecticides are the causative agent in health hazards, soil contamination, environmental pollution, and wildlife demolition it should be wisely used.This study will be an authentic and more informative tool because previously no such type of scientific documentation has been reported from Sindh, Pakistan.

Author's Contribution
W.M. Mangrio is the main author of this research paper, who conducted the experiments in laboratory conditions and wrote the article whereas; H.A. Sahito, reviewed the research article and supervised the experiments.

Impact Statement
The lemon orchards are widely grown in the district of Naushahro Feroze with massive fruit production.It is the main economic commodity of this area but is vigorously harmed by larvae of LBF.It is necessary to bring awareness and apply certain control measures through IPM strategies to secure the citrus from potential citrus insect pests.

Declaration and Competing Interest
There is no personal relationship or financial interest regarding the authors to persuade work which is reported in this research article.
Fig-2.Overall LBF larvae mortality % at post-treatment of regent insecticide under laboratory conditions Fig-5.Overall LBF larvae mortality % at post-treatment of emamectin insecticide under laboratory conditions

Fig- 6 .
Fig-6.Overall comparative toxicant efficacy of five insecticides against LBF larvae under laboratory conditions

Fig- 7 .
Fig-7.Overall post-spray efficacy of five insecticides against larvae of LBF in different days under laboratory conditions
on C. limon leaves : To examine the efficacy of the five novel insecticides namely; Belt, Helmet, Coragen, Regent, Emamectin, and control were applied against the 4 th and 5 th instar stages of LBF larvae under laboratory conditions, during, 2020.The larvae mean population was recorded before the application of the insecticides and recorded at (20.00±1.41)and after the application of Belt insecticide the maximum to minimum mortality % was recorded during 3 rd spray (87.95±9.38)(2 nd day), followed by (87.50±9.35)(3 rd day), (72.22±8.50)(4 th day), (60.