Bioefficacy of Epaltes Divaricata (L.) N-Hexane Extracts and Their Major

molecules

Article Bioefﬁcacy of Epaltes divaricata (L.) n-Hexane Extracts and Their Major Metabolites against the Lepidopteran Pests Spodoptera litura (fab.) and Dengue Mosquito Aedes aegypti (Linn.)

Kesavan Amala 1,†, Sengodan Karthi 2,†, Raja Ganesan 3 , Narayanaswamy Radhakrishnan 4 , Kumaraswamy Srinivasan 4, Abd El-Zaher M. A. Mostafa 5 , Abdullah Ahmed Al-Ghamdi 5, Jawaher Alkahtani 5 , Mohamed Soliman Elshikh 5, Sengottayan Senthil-Nathan 2,* , Prabhakaran Vasantha-Srinivasan 1,* and Patcharin Krutmuang 6,7,*

1 Department of Biotechnology, St. Peter’s Institute of Higher Education and Research, Avadi-600 054 Chennai, Tamil Nadu, India; [email protected] 2 Division of Bio-Pesticides and Environmental Toxicology, Sri Paramakalyani Centre for Excellence in Environmental Sciences, Manonmaniam Sundaranar University, Alwarkurichi, 627412 Tirunelveli, Tamil Nadu, India; [email protected] 3 Department of Internal Medicine, Hallym University College of Medicine, Chuncheon 200 704, Korea; [email protected] 4 Department of Biochemistry, St. Peter’s Institute of Higher Education and Research, Citation: Amala, K.; Karthi, S.; Avadi-600 054 Chennai, Tamil Nadu, India; [email protected] (N.R.); [email protected] (K.S.) Ganesan, R.; Radhakrishnan, N.; 5 Department of Botany and Microbiology, College of Sciences, King Saud University, P.O. Box 22452, Srinivasan, K.; Mostafa, A.E.-Z.M.A.; Riyadh 11495, Saudi Arabia; [email protected] (A.E.-Z.M.A.M.); [email protected] (A.A.A.-G.); Al-Ghamdi, A.A.; Alkahtani, J.; [email protected] (J.A.); [email protected] (M.S.E.) Elshikh, M.S.; Senthil-Nathan, S.; et al. 6 Department of Entomology and Plant Pathology, Faculty of Agriculture, Chiang Mai, University, Bioefﬁcacy of Epaltes divaricata (L.) Muang, Chiang Mai 50200, Thailand n-Hexane Extracts and Their Major 7 Innovative Agriculture Research Center, Faculty of Agriculture, Chiang Mai University, Metabolites against the Lepidopteran Chiang Mai 50200, Thailand Pests Spodoptera litura (fab.) and * Correspondence: [email protected] (S.S.-N.); [email protected] (P.V.-S.); [email protected] (P.K.) Dengue Mosquito Aedes aegypti † These authors contributed equally to this work. (Linn.). Molecules 2021, 26, 3695. https://doi.org/10.3390/ Abstract: The present research investigated the chemical characterization and insecticidal activity of n- molecules26123695 Hexane extracts of Epaltes divaricata (NH-EDx) along with their chief derivatives n-Hexadecanoic acid

Academic Editor: Natalizia Miceli (n-HDa) and n-Octadecanoic acid (n-ODa) against the dengue vector Aedes aegypti and lepidopteran pest Spodoptera litura. Chemical screening of NH-EDx through GC–MS analysis delivered nine major

Received: 12 May 2021 derivatives, and the maximum peak area percentage was observed in n-Hexadecanoic acid (14.63%) Accepted: 10 June 2021 followed by n-Octadecadienoic acid (6.73%). The larvicidal activity of NH-EDx (1000 ppm), n-HDa Published: 17 June 2021 (5 ppm), and n-ODa (5 ppm) against the A. aegypti and S. litura larvae showed significant mortality rate in a dose-dependent way across all the instars. The larvicidal activity was profound in the A. Publisher’s Note: MDPI stays neutral aegypti as compared to the S. litura across all the larval instars. The sublethal dosages of NH-EDx with regard to jurisdictional claims in (500 ppm), n-HDa (2.5 ppm), and n-ODa (2.5 ppm) also showed alterations in the larval/pupal published maps and institutional affil- durations and adult longevity in both the insect pests. The enzyme activity revealed that the α- iations. and β-carboxylesterase levels were decreased significantly in both the insect pests, whereas the levels of GST and CYP450 uplifted in a dose-dependent manner of NH-EDx, n-HDa, and n-ODa. Correspondingly, midgut tissues such as the epithelial layer (EL), gut lumen (GL), peritrophic matrix (Pm), and brush border membrane (BBM) were significantly altered in their morphology across Copyright: © 2021 by the authors. both A. aegypti and S. litura against the NH-EDx and their bioactive metabolites. NH-EDx and their Licensee MDPI, Basel, Switzerland. bioactive metabolites n-HDa and n-ODa showed significant larvicidal, growth retardant, enzyme This article is an open access article inhibition, and midgut toxicity effects against two crucial agriculturally and medically challenging distributed under the terms and insect pest of ecological importance. conditions of the Creative Commons Attribution (CC BY) license (https:// Keywords: Epaltes; n-HDa; n-ODa; lepidopteran; dengue vector; insecticide creativecommons.org/licenses/by/ 4.0/).

Molecules 2021, 26, 3695. https://doi.org/10.3390/molecules26123695 https://www.mdpi.com/journal/molecules Molecules 2021, 26, 3695 2 of 20

1. Introduction Phytochemicals are generally secondary metabolites that aid as a solid defensive action in plants against the constant selection pressure of herbivore predators, microbial pathogens, and other ecological risks [1]. Plant extracts hold surplus phytocompounds that are not utilized as nutrients for their growth [2]. Presently, research on insect toxicology has chiefly focused on green extracts and their major derivative warehouse to manage harmful pests of both agricultural and medical importance. Generally, plant extracts/essential oils/bioactive compounds have performed significant roles as mosquitocides and insecticides specially targeting lepidopteran insects [3,4]. The class of “plant extracts” generally covers a huge blend of phytochemicals, many of which delivered a higher impact in control- ling insect pests [5]. Global researchers have proved that plant extracts and their bioactive compounds predominantly control harmful agriculture pests and disease-spreading arthropods with similar toxicity as displayed by chemical pesticides [6–8]. Moreover, plant extracts have been widely utilized by human beings across the world and merged into integrated pest management (IPM) for effective control measures [9]. Previous natural product research has evidenced that plant-derived phytocompounds have multipotent toxicity against insect pests, with diverse functions such as insect growth inhibitors, larvicides, repellents, and adulticides, as well as having ovipositional attractants [10–13]. Insect pests play a crucial role in damaging agricultural crops apart from other natural problems. In particular, Lepidoptera, the largest family, embraces most caustic pests that can damage crops both directly and indirectly [14]. Among the lepidopteran pests Spodoptera litura Fab. (Lepidoptera: Noctuidae) is most significant and harmful pest, reported to target >150 plant species in south Asian countries [15]. S. litura is a firm flier and splits a wide-ranging distance annually all over the summer season. It is recognized as the most harmful agriculture pest of tropical nations such as China, Japan, and India, as it causes significant financial damage to diversified vegetables and grain crops [15,16]. Correspondingly, dengue is the most rampant human arbovirus, infecting half of the global population across 120 countries [17]. The chief culprit for spreading this disease is Aedes aegypti (Diptera: Culicidae) Linn., which is considered to be one of the crucial mosquito vectors capable of spreading the dengue virus and causing a significant mortality rate by spreading dengue hemorrhagic fever (DHF) [7]. It is estimated that >2.5 billion people are at high risk of dengue in more than 100 nations [18,19]. In India, it is estimated that 34% of global cases occur, as the disease is endemic, and that it mainly spreads four different serotypes: DENV-1, -2, -3, and -4 [20]. Synthetic chemical application is the major tool in managing both pests (S. litura and A. aegypti). However, the selection pressure levied by commercial insecticides is uplifting the resistance ratio in both of these pests and more importantly causing adverse effects to nontargets [21–23]. Green-based products such as crude extracts, essential oils, and chief derivatives have been consider as a potential insect pest management agent. Chemical pesticides are recognized as chief agents to manage insect pests due to their general acceptance [24]. Resistance to major commercial insecticides has been associ- ated with an upsurge in the level of metabolism in insects, such as the up-regulation of detoxifying enzymes or enzyme structural changes that increase the capacity of metabolic rate [25–29]. Previous research on diverse plant extracts and chief metabolites against mosquito vectors or agriculture pests has delivered that many botanical sources proved as potential alternatives, or as rotational chemistries, to heavily-used synthetic chemical insecticides [30]. Plant derivatives may deliver alternative sources of commercial pesticides, since they are enriched with bioactive insecticidal compounds that are easily degradable [2,31,32]. The genus Epaltes is widely used by traditional healers in South Asian countries such as Sri Lanka, India, Java, and China for treating diverse medical complications including jaundice, acute dyspepsia, and urethral discharges [33]. Traditionally, the roots of Epaltes species are utilized as healing tonics for treating insect or reptile bites [34]. Among these species, Epaltes divaricata (L.), which belongs to the family Compositae, is a traditional Molecules 2021, 26, 3695 3 of 20

medicinal plant used in Ayurveda medicine for healing diverse medical problems [35,36]. Previous research on ethyl acetate extracts of E. divaricata displayed profound inhibition against Staphylococcus aureus multiple drug resistant strains (MRSA) [37]. However, the chemical characterization of these extracts in screening their phytochemicals and their insecticidal activity remains scanty. Thus, the present research aimed to investigate the following objectives: (a) chemical characterization and larvicidal activity of n-Hexane extracts of E. divaricata (NH-EDx), along with their chief derivatives n-Hexadecanoic acid (n-HDa) and n-Octadecanoic acid (n-ODa), against A. aegypti and S. litura; (b) detection of developmental changes in the larval and pupal duration in both insect pests; (c) detection of enzyme inhibition activity of NH-EDx, n-HDa, and n-ODa in both lepidopteran pest and dengue vector and (d) evaluation of gut-histological activity in both of the pests after treatment with the extracts and major derivatives of E. divaricata.

2. Methodology 2.1. Insect Culture A. aegypti rearing was maintained unexposed to any chemicals at the Insect Toxicol- ogy Laboratory (ITL) at St. Peter’s Institute of Higher Education and Research, Chennai, India, and the complete rearing procedure followed the adapted methodology of Thani- gaivel et al. [19]. The cultures were conserved, and all the experiments were carried out in our laboratory at 27 ± 2 ◦C and 75–85% RH under a 14:10 L/D photoperiod without exposure to any chemical insecticides. Brewer‘s yeast, dog biscuits (Choo Stix Biskies), and algae collected from pools in a ratio of 3:2 were fed as a diet to the larvae. Pupae that emerged from the larvae were shifted to a plastic cup (round, 250 mL capacity) containing tap water and located in breeding cages (60 × 60 × 60 cm dimensions) for adult emergence. Wet raisins (dried grapes) and 10% sucrose solution soaked in cotton were fed to the adults. The adult females were destitute of sucrose from 6h and then provided a mouse placed in a breeding cage overnight for blood feeding. Adult mosquitoes were maintained under sterile conditions as similar to the larvae. The ﬁrst-generation larvae were used for conducting the experiment. Correspondingly, the S. litura culture was maintained since 2019 without exposure to any pesticides or chemicals under in vitro conditions. The eggs were sterilized in surface using 10% formaldehyde for 10 min and washed with double distilled water for 5 min, then ﬁnally air-dried and allowed to hatch. The adults that emerged were shifted to a hygienic plastic container having castor leaves for oviposition and were fed with 8% honey solution on cotton wool located in a small glass cap. Generally, eggs laid in batches on paper were kept for hatching in sterilized plastic boxes. The environment was set as 25–27 ◦C and 60–70% relative humidity in an ecologically controlled chamber. The day/night period was adjusted to 13:11 h. Second-generation larval instars were used for the bioassay.

2.2. Plant Harvesting and Crude Extract Preparation Fresh leaves of the plant material Epaltes divaricata (L.) Cass. were harvested during the early morning hours (6.00 AM) from the Southern Ghats region, Tirunelveli, Tamil Nadu, India. Harvesting was authenticated by a botanist from Plant Biology and Plant Biotechnology, Presidency College, Chennai, India (Voucher Specimen No.00629), and leaves were deposited in the Department of Botany, Captain Srinivasa Murti Drug Research Institute for Ayurveda, Chennai, India for reference. The external morphology of E. divaricata is displayed in Figure1. Molecules 2021, 26, x FOR PEER REVIEW 4 of 22

Molecules 2021, 26, 3695 leaves were deposited in the Department of Botany, Captain Srinivasa Murti Drug4 of Re- 20 search Institute for Ayurveda, Chennai, India for reference. The external morphology of E. divaricata is displayed in Figure 1.

FigureFigure 1.1. ExternalExternal morphologymorphology ofofEpaltes Epaltes divaricata divaricata(L.) (L.) Cass. Cass. collected collected from from the the southwestern southwestern Ghats regionGhats ofregion Tirunelveli, of Tirunelveli, Tamil Nadu, Tamil India.Nadu, India.

TheThe collectedcollected plantplant material material was was shade-dried shade-dried under under laboratory laboratory conditions conditions (27 (27± ±2 2 ◦°C).C). TheThe drieddried leaves were were powdered powdered finely finely using using a ablender, blender, and and the the dried dried leaf leafpowder powder was wasextracted extracted by a bya Soxhlet a a Soxhlet apparatus apparatus using usingn-hexanen-hexane solvent solvent as adapted as adapted from Ponsankar from Pon- et sankaral. [11]; et Further, al. [11]; 500 Further, g air-dried 500 gplant air-dried powder plant was powder dissolved was with dissolved 700 mL withof n-hexane 700 mL (60– of n80-hexane °C) and (60–80 then ◦extractedC) and then with extracted 750 mL with distilled 750 mLn-hexane distilled inn a-hexane Soxhlet in apparatus a Soxhlet for appara- 16 h. tusThe for extract 16 h. Thewas extractfiltered was and filtered concentrated and concentrated in a rotary in flash a rotary evaporator flash evaporator at 60 °C; atthe 60 con-◦C; thecentrated concentrated n-hexanen-hexane extract extractwas poured was poured into the into same the volume same volume of methanol of methanol while stirring while stirringand then and filtered. then filtered. The solvent The solvent was further was further removed removed by vacuum by vacuum evaporation evaporation in a rotary in a rotaryevaporator, evaporator, and the and deposits the deposits were werestored stored at 4 °C at 4in◦ Can in airtight an airtight glass glass bottle bottle for forfurther further ex- experiments.periments. The The yield yield of of n-hexanen-hexane extract extract was was 2.03 2.03 g. g.

2.3.2.3. CharacterizationCharacterization ofof PlantPlant VolatileVolatile throughThrough GC–MS GC–MS TwoTwo microliters microliters of of NH-EDx NH-EDx were were dissolved dissolved in inmethanol methanol(HPLC-grade) (HPLC-grade) and andprojected projected toto GCGC and and MS MS JEOL JEOL GC mateGC mate prepared prepared with secondary with secondary electron multiplierelectron multiplier (JEOL GCMATE (JEOL IIGCMATE GC-MS (Agilent II GC-MS Technologies (Agilent Technologies 6890N Network 6890N GC Network system)). GC The system)). HP5 column The HP5 was column linked with silica 55 m × 0.25 mm I.D. Analysis conditions were (i) HPLC-grade—20 min at 100 ◦C; was linked with silica 55 m × 0.25 mm◦ I.D. Analysis conditions were i) HPLC-grade—20◦ (ii)min column at 100 temperature—3°C; ii) column temperature—3 min at 235 C; and min (iii) at injector235 °C; temperatureand iii) injector 240 temperatureC. Helium was 240 the°C. carrierHelium gas, was and the thecarrier split gas, ratio and was the 5:4. split Further ratio was experimental 5:4. Further procedure experimental was procedure adapted fromwas adapted the previous from methodologythe previous [methodology37]. The molecular [37]. The structure, molecular molecular structure, weight, molecular and formulaweight, ofand the formula individual of the compounds individual of compounds test materials of weretest materials ascertained were by ascertained interpretation by of the mass spectrum from GC–MS using the database of the National Institute of Standards interpretation of the mass spectrum from GC–MS using the database of the National In- and Technology (NIST). stitute of Standards and Technology (NIST). 2.4. Chemicals 2.4. Chemicals GC–MS analysis results of NH-EDx delivered peak areas in n-Hexadecanoic acid (n-HDa)GC–MS followed analysis by resultsn-Octadecadienoic of NH-EDx delivered acid (n-ODa). peak areas Thus, in to n-Hexadecanoic detect the efﬁcacy acid of(n- theseHDa) selectivefollowed metabolitesby n-Octadecadienoic of NH-EDx acid against (n-ODa). insect Thus, pests, to detect the plant the chemicalsefficacy of werethese commerciallyselective metabolites purchased of NH-EDx from Sigma-Aldrich against insect (≥ 99%pests, (capillary the plant GC)). chemicals were commercially purchased from Sigma-Aldrich (≥99% (capillary GC)). 2.5. Larvicidal Bioassay 2.5.1.2.5. LarvicidalA. aegypti BioassayLarvicidal Assay 2.5.1.The A. aegypti second, Larvicidal third, and Assay fourth instar larvae were placed into 200 mL sterile disposable plasticThe cups second, each third, covering and fourth 20 mL instar of different larvae were dosages placed of NH-EDxinto 200 mL (200, sterile 400, disposable 600, 800, andplastic 1000 cups ppm) each and covering the bioactive 20 mL of derivatives different dosagesn-HDa andof NH-EDxn-ODa (1,(200, 2, 3,400, 4, 600, and 800, 5 ppm) and and incubated at 27 ◦C. A solution containing n-Hexane (0.1%) was employed as the negative control. During the treatment period, larval food was added to each test cup, particularly if high mortality was noted in the control. Larvae were considered dead when they were unable to reach the surface of the solution when the cups were disturbed. The Molecules 2021, 26, 3695 5 of 20

larvae were considered moribund if, at the end of 24 h, they showed no sign of swimming movements even after gentle touching with a glass rod. The dead and moribund larvae were recorded after 24 h of treatment. Larvicidal assay was adapted from the World Health Organization [38].

2.5.2. S. litura Larvicidal Assay Larvicidal assays of NH-EDx and their derivatives n-HDa and n-ODa were executed against the II, III, and IV instars of S. litura larvae with discriminating dosages utilized in the A. aegypti larvicidal assay (Section 2.5.1). The treatment dosages were gushed in sterile castor leaves and allowed to air dry. Hexane (0.1%) was used alone as a control. Second to fourth instars larvae were starved for 4 h were exposed to the treated leaves and kept under ﬁxed conditions (28 ± 2 ◦C and 75% relative humidity). The rate of mortality was observed every 24 h, compared with the control, and recorded. The calculated mortality data percentage was examined using Probit analysis to determine the lethal concentrations (LC50 and LC90). The treatments were replicated ﬁve times, and each replicated set contained one control. Analyses of the follow-up assay were carried out according to the Probit method. Percentage mortality (1) was calculated by using Abbott’s formula [39] (2).

Number of dead larvae Percentage of mortality = × 100 (1) Number of larvae introduced n in T after treatment Corrected percentage of mortality = 1 − × 100 (2) n in C after treatment

2.6. Larval and Pupal Duration Assay

The effect of sublethal dosages (LC50—dosage responsible to cause 50% insect mortality in a single exposure) of plant extracts (NH-EDx) along with their major phytochemicals (n-HDa and n-ODa) against the larval and pupal stages of both A. aegypti and S. litura, with discriminating sublethal dosages (NH-EDx—100, 200, 300, 400, and 500 ppm; n-HDa and n-ODa—0.5, 1.0, 1.5, and 2.0 ppm) were prepared. For analyzing A. aegypti larval and pupal durations, the sublethal dosages were subjected in an enamel tray of 30 × 25 × 5 cm dimensions. Fifty eggs were released into enamel tray in treated water and allowed to hatch. For analyzing S. litura larval and pupal duration, the sublethal dosages were dipped in sterile castor leaves, the eggs were released, and their duration to become larvae and pupae in days were calculated and compared with the control, which was treated with hexane alone. The total larval duration (days) was calculated from hatching to pupation. Pupae were placed in a small container closed with a transparent mesh. The pupal duration (days) was calculated from the pupal molt to adult emergence.

2.7. Enzyme Assays 2.7.1. α- and β-Carboxylesterase Activity The enzyme activity of α- and β-carboxylesterase in the A. aegypti and S. litura was determined using the adapted methodology of Agra-Neto et al. [40], The enzyme activity was investigated by the expression of one unit of active enzyme, which was determined as the amount of enzyme essential to generate 1 µmol of α- or β-naphthol expressed per minute.

2.7.2. GST and CYP450 Activity Glutathione-S transferase activity (GST) was expressed as µmol/mg protein/min substrate conjugated, and cytochrome P450 activity (CYP450) was expressed as l mol 7-OH/mg larvae/min. The complete protocol was followed based on the GST assay kit (Sigma-Aldrich (Catalog 0410, Bangalore)) that was used to evaluate the conjugation of the thiol group of glutathione to the 1-chloro-2, 4- dinotrobenzene (CDNB) substrate. CYP450 activity was determined based on the measurement of ethoxycoumarin-O-de-ethylase level in the body walls as adapted from Thanigaivel et al. [41]. Molecules 2021, 26, x FOR PEER REVIEW 6 of 22

Molecules 2021, 26, x FOR PEER REVIEW Glutathione-S transferase activity (GST) was expressed as µmol/mg protein/min sub-6 of 22

strate conjugated, and cytochrome P450 activity (CYP450) was expressed as l mol 7- OH/mg larvae/min. The complete protocol was followed based on the GST assay kit (Sigma-AldrichGlutathione-S (Catalog transferase 0410, Bangalore) activity (GST) that was usedexpressed to evaluate as µmol/mg the conjugation protein/min of thesub- thiolstrate group conjugated, of glutathione and cytochrome to the 1-chloro-2, P450 4-activity dinotrobenzene (CYP450) (CDNwas expressedB) substrate. as CYP450l mol 7- activityOH/mg was larvae/min. determined The based complete on the protocol measurement was followed of ethoxycoumarin- based on theO GST-de-ethylase assay kit level(Sigma-Aldrich in the body walls(Catalog as adapted 0410, Bangalore) from Thanigaivel that was usedet al. to[41]. evaluate the conjugation of the thiol group of glutathione to the 1-chloro-2, 4- dinotrobenzene (CDNB) substrate. CYP450 2.8.activity Gut-Histological was determined Assay based on the measurement of ethoxycoumarin-O-de-ethylase Molecules 2021, 26, 3695 6 of 20 levelThe in NH-EDx-,the body walls n-HDa-, as adapted n-ODa-, from treated Thanigaivel and control- et al. fourth [41]. instars of A. aegypti and S. litura were fixed in Bouin’s solution overnight, flowingly dehydrated, and mounted in wax2.8. blocksGut-Histological using paraffin. Assay Larval blocks of tissue were segmented using the microtome 2.8. Gut-Histological Assay instrumentThe NH-EDx-, (Leica, Germany), n-HDa-, andn-ODa-, the active treated se ctionsand control- were mounted fourth instars on sterile of A. microscopic aegypti and The NH-EDx-, n-HDa-, n-ODa-, treatedglassS. litura andslides, were control- stained fixed fourth inwith Bouin’s instars eosin solution ofandA. aegyptihemato overnight,andxylin, flowingly and observed dehydrated, for histopathological and mounted in S. litura were fixed in Bouin’s solution overnight,changeswax blocks and flowingly photographedusing paraffin. dehydrated, using Larv andal light blocks mounted a micros of tissue incope were (Optika segmented vision lite using 2.0 ML)the microtome pre-con- wax blocks using paraffin. Larval blocksnectedinstrument of tissue with were a (Leica, laptop. segmented Germany), Midgut using cells and theof the A. microtome active aegypti se ctionsand S. were litura mounted were photographed, on sterile microscopic and the instrument (Leica, Germany), and the activechangesglass sections slides, in the were midgutstained mounted cellswith of oneosin treated sterile and microscopiclarvae hemato werexylin, observed and observedand matched for withhistopathological control [6]. glass slides, stained with eosin and hematoxylin,changes and and photographed observed for using histopathological light a microscope (Optika vision lite 2.0 ML) pre-con- changes and photographed using light2.9.nected a Data microscope withAnalysis a laptop. (Optika Midgut vision cells lite of 2.0 A. ML) aegypti pre- and S. litura were photographed, and the connected with a laptop. Midgut cellschanges of A.The aegypti larvicidal in theand midgut experimentalS. litura cells wereof treated data photographed, were larvae exposed were observed to analysis and of matched variance with(ANOVA control and [6]. and the changes in the midgut cells ofsquare treated root larvae transformed were observed percentages), and matched and the with obtained statistical results were considered control [6]. as2.9. a five Data replicate Analysis means. Statistical significance within each larvicidal group was analyzed using Tukey’sThe larvicidal multiple experimental range test data(significance were exposed at p < to0.05), analysis and the of variancelethal concentrations (ANOVA and 2.9. Data Analysis ofsquare larvae root in 24 transformed h were calculated percentages), by Probit and analysis the obtained with statisticala dependability results intervalwere considered of 95% The larvicidal experimental data wereusingas exposeda fivethe replicateMinitab to analysis® means. 17 program. of Statistical variance For (ANOVA significancedetermining and within enzyme each inhibition larvicidal level, group Microcal was analyzed Soft- square root transformed percentages), andwareusing the (Sigma obtainedTukey’s plot statisticalmultiple 11) was rangeused results to test wereplot (significance the considered graphs. at p < 0.05), and the lethal concentrations as a five replicate means. Statistical significanceof larvae within in 24 each h were larvicidal calculated group by was Probit analyzed analysis with a dependability interval of 95% using Tukey’s multiple range test (significance3.using Results at thep < Minitab 0.05), and® 17 the program. lethal concentrations For determining of enzyme inhibition level, Microcal Soft- larvae in 24 h were calculated by Probit analysis3.1.ware Chemical (Sigma with aCharac dependabilityplot 11)terization was used intervalof NH-EDx to plot of 95% the graphs. using the Minitab® 17 program. For determining enzyme inhibition level, Microcal Software Chemical screening of NH-EDx through GC–MS analysis showed nine major deriv- (Sigma plot 11) was used to plot the graphs. atives,3. Results and the maximum peak area percentage was observed in n-Hexadecanoic acid 3. Results (14.63%)3.1. Chemical followed Charac byterization n-Octadecadienoic of NH-EDx acid (6.73%). Correspondingly, the NH-EDx was 3.1. Chemical Characterization of NH-EDxalso composedChemical of screening Hexacosane of NH-EDx (6.13%), through n-Hexatriacontane GC–MS analysis (5.13%), showed Ethyl nineHexadecanoate major deriv- (1.32%), Tetratriacontane (1.3%), n-Eicosane (1.13%), Neophytadiene (0.83%), and finally Chemical screening of NH-EDx throughatives, GC–MS and the analysis maximum showed peak nine area major percentage deriva- was observed in n-Hexadecanoic acid Hexadecane (0.75%) (Table 1). All of these derivatives might play a significant role in caus- tives, and the maximum peak area percentage(14.63%) wasfollowed observed by n-Octadecadienoic in n-Hexadecanoic acid acid (6.73%). Correspondingly, the NH-EDx was ing mortality rate in both of the pests. Since these metabolites are the major active deriva- (14.63%) followed by n-Octadecadienoic acidalso (6.73%).composed Correspondingly, of Hexacosane the(6.13%), NH-EDx n-Hexatriacontane was (5.13%), Ethyl Hexadecanoate tives of the NH-EDx, they all synergistically act together, which results in displaying pro- also composed of Hexacosane (6.13%), n-Hexatriacontane(1.32%), Tetratriacontane (5.13%), (1.3%), Ethyl Hexadecanoate n-Eicosane (1.13%), Neophytadiene (0.83%), and finally (1.32%), Tetratriacontane (1.3%), n-EicosanefoundHexadecane (1.13%), activity Neophytadiene (0.75%)against (Tableboth the 1). (0.83%), pests. All of Despitethese and finallyderivatives this, the major might peak play areaa significant derivatives role n in-HDa caus- Hexadecane (0.75%) (Table1). All of thesefolloweding derivativesmortality by n-ODa rate might inmight both play signify of a the significant pests.a profound Since role levelthese in of metabolites contribution are against the major the activeinsect deriva-pests. causing mortality rate in both of the pests.Thus,tives Since theseof the these two NH-EDx, compounds metabolites they all were are synergistically the purchased major active coactmmercially, together, which and theirresults bioefficacy in displaying against pro- derivatives of the NH-EDx, they all synergisticallybothfound the activity pests act together,(A. against aegypti which both and the resultsS. litura pests. in) wasDespite displaying examined this, the parallel major topeak that area of the derivatives NH-EDx. n -HDa profound activity against both the pests.followed Despite by this, n-ODa the majormight peaksignify area a profound derivatives level of contribution against the insect pests. Table 1. Chemical characterization of NH-EDx (n-Hexane extracts of E. divaricate). R/T: retention n-HDa followed by n-ODa might signify aThus, profound these level two ofcompounds contribution were against purchased the insect commercially, and their bioefficacy against time; PA: peak area. pests. Thus,Molecules these two 2021 compounds, 26, x FOR PEER were REVIEWboth purchased the pests commercially, (A. aegypti and and S. theirlitura bioefficacy) was examined parallel to that of the NH-EDx. 7 of 22 A. aegypti S. litura against both the pests ( and ) was examined parallel to that of the NH-EDx. Table 1. Chemical characterization of NH-EDx (n-Hexane extracts of E. divaricate). R/T: retention Molecules 2021, 26, x FOR PEER REVIEW 7 of 22 Table 1. Chemical characterization of NH-EDx (n-Hexane extracts of E.time; divaricate PA:). peak R/T: area. retention time; PA: peak area.

Weight R/T PA Chemical Name Formula Molecules 2021, 26, x FOR PEER REVIEW Chemical Structure 7 of 22 (g/mol) (min) (%)

Hexadecane C16H34 226.4 9.033 0.75

n-Eicosane C20H42 282.5 13.23 1.13

Tetratriacontane C34H70 478.9 19.76 1.45

Ethyl-Hexadecanoate C H O 284.5 21.46 1.32 18 36 2

Neophytadiene C20H38 278.5 21.97 0.83

Weight R/T PA Chemical Name Formula Chemical structure (g/mol) (min) (%) Hexadecane C16H34 Weight226.4 9.033R/T PA0.75 Chemical Name Formula Chemical structure n-Eicosane C20H42 (g/mol)282.5 (min)13.23 (%)1.13 TetratriacontaneHexadecane CC1634HH3470 226.4478.9 9.03319.76 0.751.45 Weight R/T PA ChemicalEthyl-Hexadec-n-Eicosane Name FormulaC20H 42 282.5 13.23 1.13 Chemical structure C18H36O(g/mol)2 284.5 (min) 21.46 (%) 1.32 Tetratriacontaneanoate C34H70 478.9 19.76 1.45 Hexadecane C16H34 226.4 9.033 0.75 Ethyl-Hexadec-Neophytadiene C20H38 278.5 21.97 0.83 n-Eicosane C20CH18H42 36O2 282.5284.5 13.2321.46 1.13 1.32 n-Hexatri-anoate Tetratriacontane C34HC7036 H74 478.9507 19.7622.46 1.45 5.13 Neophytadieneacontane C20H38 278.5 21.97 0.83 Ethyl-Hexadec- Hexacosanen-Hexatri- C18HC36O26H2 54 284.5366.7 21.46 24.67 1.32 6.13 anoate C36H74 507 22.46 5.13 n-Octadecadi-acontane Neophytadiene C20CH1838H 32O2 278.5280.4 21.97 31.78 0.83 14.63 Hexacosaneenoic acid C26H54 366.7 24.67 6.13 n-Hexatri- nn-Hexadecanoic-Octadecadi- C 36H74 507 22.46 5.13 acontane CC1816HH3232OO22 280.4256.4 31.7833.45 14.636.73 enoicacid acid Hexacosane C26H54 366.7 24.67 6.13 n-Hexadecanoic n-Octadecadi- C16H32O2 256.4 33.45 6.73 3.2. Larvicidalacid ActivityC18H32O 2 280.4 31.78 14.63 enoic acid The larvicidal activity of NH-EDx against A. aegypti larvae showed a significant mor- n-Hexadecanoic 3.2.tality Larvicidal rate in a Activity dose-dependentC16H32 O2 256.4 way across33.45 all6.73 the instars. The mortality rate was profound acid The larvicidal activity of NH-EDx against A. aegypti larvae showed a significant mortality rate in a dose-dependent way across all the instars. The mortality rate was profound 3.2. Larvicidal Activity The larvicidal activity of NH-EDx against A. aegypti larvae showed a significant mortality rate in a dose-dependent way across all the instars. The mortality rate was profound Molecules 2021, 26, x FOR PEER REVIEW 7 of 22

Molecules 2021, 26, x FOR PEER REVIEW 7 of 22

Molecules 2021, 26, 3695 7 of 20

Table 1. Cont.

Weight R/T PA Chemical Name Formula Chemical Structure (g/mol) (min) (%)

n-Hexatriacontane C H 507 22.46 5.13 36 74

Hexacosane C26H54 366.7 24.67 6.13

n-Octadecadienoic acid C18H32O2 280.4 31.78 14.63

n-Hexadecanoic acid C16H32O2 256.4 33.45 6.73

Weight R/T PA Chemical Name Formula Chemical structure 3.2. Larvicidal Activity (g/mol) (min) (%) Hexadecane C16H34 Weight226.4 R/T9.033 PA 0.75 The larvicidal activity of NH-EDx Chemical against NameA. aegypti Formulalarvae showed a significant Chemical structure mortality rate in a dose-dependent way acrossn-Eicosane all the instars.C20H42 The (g/mol) mortality282.5 (min)13.23 rate (%) was 1.13 Tetratriacontane 16C3434H70 478.9 19.76 1.45 profound at the maximum dosage (NH-EDx—1000Hexadecane ppm), C andH it was significantly226.4Weight 9.033 differentR/T 0.75 PA from other dosages in II instars (97.65%—FnEthyl-Hexadec-Chemical-Eicosane4,20 = 28.68, NameCp 20≤ FormulaH0.001),42 282.5 III instars 13.23 (94.11%— 1.13 Chemical structure C18H36O2 (g/mol)284.5 21.46(min) 1.32(%) F4,20 = 20.32, p ≤ 0.001), and IV instarsTetratriacontane (92.94%—Fanoate4,20 =C 18.88,34H70 p ≤478.90.001). Despite19.76 1.45 this, Hexadecane C16H34 226.4 9.033 0.75 there was no significant difference between NH-EDx 800 and 600 ppm across all the treated Molecules 2021, 26, x FOR PEER REVIEW Ethyl-Hexadec-Neophytadiene C20H38 Weight278.5 R/T21.97 PA9 of0.83 22 Chemicaln-Eicosane Name FormulaC18HC36O20H2 42 284.5282.5 21.46 13.23 1.32 1.13 Chemical structure larval instars. Even so, all of the treatment dosagesanoaten-Hexatri- were significantly(g/mol) different (min) from control(%) (Figure2A). Tetratriacontane CC3634HH7470 478.9507 22.4619.76 5.131.45 NeophytadieneHexadecaneacontane CC1620HH3438 226.4278.5 9.03321.97 0.750.83 Ethyl-Hexadec- nn-Eicosane-Hexatri-Hexacosane C20CHC182642HH 3654O 2282.5 366.7284.5 13.23 24.6721.46 1.13 6.131.32 140 Control 100 anoate C36H74 507 22.46 5.13 NH-EDx 200 ppm Tetratriacontaneacontanen-Octadecadi-a C34H70 a 478.9 19.76 1.45 NH-EDx 400 ppm Control Neophytadiene C18CH2032HO382 280.4278.5 a 31.7821.97 14.630.83 120 NH-EDx 600 ppm ABn-HDa 1 ppm b 80 Ethyl-Hexadec-Hexacosaneenoic acid C26H54 366.7 24.67 6.13 NH-EDx 800 ppm n-HDa 2 ppm n-Hexatri- C18H36Ob 2 284.5 b21.46 1.32 a a 36 74 100 NH-EDx 1000 ppm a n-HDan 3-Octadecadi-n ppm-Hexadecanoicanoate c cC H 507c 22.46 5.13 n-HDa 4 ppm acontane 18C1632H322 O2 256.4 33.45 6.73 b b C H O 280.4 31.78 14.63 b b 60 n-HDaNeophytadiene 5 enoicppm acid C20H38 278.5 21.97 0.83 80 b b Hexacosaned C26H54 366.7 24.67 6.13 d d c (%) mortlaity c n-Hexadecanoicn-Hexatri- n-Octadecadi- 36 74 60 c 40 CC16HH32O 2 256.4507 22.4633.45 5.136.73 mortality (%) (%) mortality 3.2.e Larvicidal ActivityC18 H32O2 280.4 31.78 14.63 acontaneacid e enoic acid e 40 d d d HexacosaneThe larvicidal C26 activityH54 of366.7 NH-EDx 24.67 against 6.13 A. aegypti larvae showed a significant mor- 20 n-Hexadecanoic A. aegypti 3.2. Larvicidal Activity C16H32O2 256.4 33.45 6.73

A. aegypti tality rate in a dose-dependent way across all the instars. The mortality rate was profound 20 n-Octadecadi-f f f acid C18H32O2 280.4 31.78 14.63 e e e 0 enoicThe larvicidalacid activity of NH-EDx against A. aegypti larvae showed a significant mor- 0 II instar III instar IV instar talityn-Hexadecanoic rateII instar in a dose-dependent III instar way IV acrossinstar all the instars. The mortality rate was profound 3.2. Larvicidal ActivityC16H32O 2 256.4 33.45 6.73 NH-EDx treatment dosage (ppm) acidn-HDa treatment dosage (ppm) 100 The larvicidal activity of NH-EDx against A. aegypti larvae showed a significant mor- Control n-ODa 1 ppm tality rate in a dose-dependent way across all the instars. The mortality rate was profound 80 n-ODa 2 ppm a 3.2. LarvicidalC Activity n-ODa 3 ppm a a a n-ODa 4 ppm a a 60 n-ODa 5 ppm b The larvicidal activity of NH-EDx against A. aegypti larvae showed a significant mor- b b c tality rate in a dose-dependent way across all the instars. The mortality rate was profound mortality (%) b b 40 c d c 20 A. aegypti e d d 0 II instar III instar IV instar n-ODa treatment dosage (ppm)

Figure 2. Percentage mortality of second, third, and fourth instar larvae of A. aegypti after treatment with NH-EDx (A), nFigure-HDa 2. (B Percentage), and n-ODa mortality (C). Means of second, (±SEM) third, followed and fourth by the instar same larvae letters of A. above aegypti bars after indicate treatment nosigniﬁcant with NH-EDx difference (A), n- (HDap ≤ 0.05) (B), and according n-ODa to (C a). Tukey’s Means test.(±SEM) followed by the same letters above bars indicate no significant difference (P ≤ 0.05) according to a Tukey’s test.

Control 100 Control NH-EDx 200 ppm n-HDa 1 ppm a a NH-EDx 400 ppm 80 a a a n-HDa 2 ppm NH-EDx 600 ppm a b 80 NH-EDx 800 ppm a b ABn-HDa 3 ppm b NH-EDx 1000 ppm b b n-HDa 4 ppm b b b 60 c n-HDa 5 ppm c c 60 c c d d 40 d mortality (%) 40 c d (%) mortlaity e d d e 20 e

S. litura 20 S. litura f f f e e e 0 0 II instar III instar IV instar II instar III instar IV instar NH-EDx treatment dosage (ppm) n-HDa treatment dosage (ppm) 80

Control a a 60 n-ODa 1 ppm a C n-ODa 2 ppm n-ODa 3 ppm b n-ODa 4 ppm b n-ODa 5 ppm b 40 c

mortlaity (%) mortlaity c c d 20 d d e

S. litura e e e e e

0 II instar III instar IV instar n-ODa treatment dosage (ppm) Figure 3. Percentage mortality of second, third, and fourth instar larvae of S. litura after treatment with NH-EDx (A), n- HDa (B), and n-ODa (C). Means (±SEM) followed by the same letters above bars indicate no significant difference (P ≤ 0.05) according to a Tukey’s test. Molecules 2021, 26, x FOR PEER REVIEW 9 of 22

140 Control 100 NH-EDx 200 ppm a a NH-EDx 400 ppm Control a 120 NH-EDx 600 ppm ABn-HDa 1 ppm b 80 NH-EDx 800 ppm n-HDa 2 ppm b b a a Molecules100 2021NH-EDx, 26, 1000 3695 ppm a n-HDa 3 ppm c c c 8 of 20 n-HDa 4 ppm b b b b 60 n-HDa 5 ppm 80 b b d d d c (%) mortlaity c 60 c 40 mortality (%) (%) mortality e e Similar results were obtained from phytochemical treatment (n-HDae and n-ODa). 40 d d d 20 The mortality rate was foundA. aegypti to be concentration-dependent. There was a signiﬁcant

A. aegypti 20 difference in the mortality rate at the maximumf dosagef of 5 ppm in IIf instars (98.86%— e e e 0 0 F4,20 = 14.22, p ≤ 0.001), III instars (95.21%—F4,20 = 14.55, p ≤ 0.001), and IV instars II instar III instar IV instar II instar III instar(93.89%—F IV instar4,20 = 19.18, p ≤ 0.001), in the n-HDa treatment (Figure2B). Likewise, a higher NH-EDx treatment dosage (ppm) n-HDa treatment dosage (ppm) 100mortality rate was recorded in the 5 ppm dosage of n-ODa treatment in II instars (98.93%— F Control= 13.33, p ≤ 0.001), III instars (96.87%—F = 17.89, p ≤ 0.001) and IV instars (94.75%— 4,20 n-ODa 1 ppm 4,20 80 F n-ODa= 16.90, 2 ppm p ≤a 0.001) (Figure2C). However,C there was no signiﬁcant difference between 4,20 n-ODa 3 ppm a a a n-HDan-ODa or 4 ppmn -ODa treatment.a a 60 n-ODa 5 ppm b b b The larvicidalc activity of NH-EDx against the lepidopteran pest S. litura showed a mortality (%) b b 40prominent mortality rate at the maximum lethal dosage of 1000 ppm across the II instars c d c (91.35%—F4,20 = 43.84, p ≤ 0.001), III instars (89.88%—F4,20 = 18.45, p ≤ 0.001), and IV 20 A. aegypti instars (86.18%—F4,20 = 34.67, p ≤ 0.001), and it was signiﬁcantly different from other e d d 0treatment dosages and from control (Figure3A). Like the NH-EDx treatment, the bioactive metabolite IIn instar-HDa showed III instar a higher IV mortalityinstar rate at the maximum dosage of 5 ppm in II instars (89.45%—Fn-ODa4,20 treatment= 17.34, dosagep (ppm)≤ 0.001), III instars (86.66%—F4,20 = 42.15, p ≤ 0.001) and IV instars (85.17%—F4,20 = 19.45, p ≤ 0.001) (Figure3B). The maximum dosage of n-ODa Figure 2. Percentage mortalityalso of showedsecond, third, higher and larval fourth mortality instar larvae at theof A. maximum aegypti after dosage treatment of with 5 ppm NH-EDx across (A II), instarsn- HDa (B), and n-ODa (C). Means(75.45%—F (±SEM)4,20 followed= 18.58, by pthe≤ same0.001), letters III instarsabove bars (67.66%—F indicate 4,20no significant= 32.90, p difference≤ 0.001), (P and ≤ IV 0.05) according to a Tukey’sinstars test. (63.15%—F4,20 = 12.45, p ≤ 0.001) (Figure3C).

S. litura 20 S. litura f f f e e e 0 0 II instar III instar IV instar II instar III instar IV instar NH-EDx treatment dosage (ppm) n-HDa treatment dosage (ppm) 80

Control a a 60 n-ODa 1 ppm a C n-ODa 2 ppm n-ODa 3 ppm b n-ODa 4 ppm b n-ODa 5 ppm b 40 c

mortlaity (%) mortlaity c c d 20 d d e

S. litura e e e e e

0 II instar III instar IV instar n-ODa treatment dosage (ppm) Figure 3. PercentagePercentage mortality mortality of of second, second, third, third, and and fourth fourth instar instar larvae larvae of ofS. S.litura litura afterafter treatment treatment with with NH-EDx NH-EDx (A), (A n),- nHDa-HDa (B (),B ),and and n-ODan-ODa (C ().C Means). Means (±SEM) (±SEM) followed followed by the by thesame same letters letters above above bars bars indicate indicate no significant no signiﬁcant difference difference (P ≤ (0.05)p ≤ 0.05) according according to a Tukey’s to a Tukey’s test. test. Molecules 2021, 26, x FOR PEER REVIEW 10 of 22

Molecules 2021, 26, 3695 9 of 20

3.3. Developmental Changes ParallelOverall, to the larval larval mortality, mortality the rate sublethal was found dosages to be of higher NH-EDx in the (500A. aegyptippm), nthan-HDa in (2.5 the ppm),S. litura andacross n-ODa all the(2.5 dosages; ppm) also the significantly maximum significance decreased the was growth observed rates in theof larvae 1000 ppmand pupaedosage of (F both4,20 = A. 15.33, aegyptip ≤ and0.001). S. litura Also,. The the larval mortality and pupal rate of duration phytocompounds of S. litura seems after treat- to be mentprofound with againstNH-EDx the (500A. aegyptippm) increasedlarvae as significantly compared to as the compared NH-EDx. to In other contrast, treatment the larval con- centrationsmortality was and significantcontrol (larvae: in the F4,20 crude = 11.33, extract P ≤ 0.001; (NH-EDx) pupae: as F compared4,20 = 17.90, toP ≤ the 0.001) phytocom- (Figure 4A,D).pounds Correspondingly, (n-HDa and n-ODa) the againstn-HDa sublethalS. litura larvae. dosage The also LC showed50 (lethal significant concentration upsurge to kill in the50% larval of larvae) and pupal was observedduration of at S. 500 litura ppm at inthe the maximum NH-EDx dosage and 2.5 of ppm 2.5 ppm in the in nboth-HDa larvae and (Fn-ODa4,20 = 9.13, across P ≤ both 0.001) pests. and pupae (F4,20 = 10.63, P ≤ 0.001) (Figure 4B,E). Similar results were observed in the n-ODa treatment, as the larval and pupal duration of S. litura extended prominently3.3. Developmental when Changesthe treatment dosage was increased. However, there was no significant differenceParallel in the to larvallarval and mortality, pupal thegrowth sublethal rates between dosages the of control NH-EDx and (500 the ppm), 0.5 ppmn-HDa dosage(2.5 (F ppm),4,20 = 8.13, and Pn -ODa≤ 0.001) (2.5 (Figure ppm) 4 also C,F). significantly decreased the growth rates of larvae and pupaeThe larval of both andA. pupal aegypti durationand S. lituraof A.. aegypti The larval significantly and pupal increased duration when of S.litura the treat-after menttreatment dosage with of NH-EDxNH-EDx, (500 n-HDa ppm) and increased n-ODa significantlywas increased. as comparedThe duration to other of both treatment larvae andconcentrations pupae was andprofound control in (larvae: the maximum F4,20 = 11.33, sublethalp ≤ 0.001;dosage pupae: of 500 F ppm4,20 = of 17.90, NH-EDxp ≤ 0.001) (larvae:(Figure F4,204 =A,D). 28.94, Correspondingly, P ≤ 0.001; pupae—F the4,20 n=-HDa 14.88, sublethalP ≤ 0.001), dosageand it was also significant showed significantcompared withupsurge other in treatment the larval dosages and pupal and duration the control of S.(Figure litura 5A,D).at the maximumSimilar trends dosage were of observed 2.5 ppm inin the both n-HDa larvae treatment, (F4,20 = 9.13, as pthe≤ 0.001)maximum and pupaesublethal (F4,20 dosage= 10.63, (2.5p ppm≤ 0.001) and (Figure2.0 ppm)4B,E). in- creasedSimilar resultsthe larval were and observed pupal days in the ofn -ODaA. aegypti treatment, steadily as as the compared larval and to pupal other duration treatment of dosagesS. litura extended(Figure 5 prominentlyB,E). However, when there the was treatment no significant dosage was difference increased. in the However, duration there of larvaewas no and significant pupae in difference A. aegypti in from the larval 0.5 to and1.5 ppm pupal dosages growth of rates n-ODa between (larvae—F the control4,20 = 8.54, and Pthe ≤ 0.001; 0.5 ppm pupae—F dosage4,20 (F 4,20= 7.32,= 8.13, P ≤ p0.001)≤ 0.001) (Figure (Figure 5C,F).4C,F).

30 a 40 A a D b NH-EDx S. litura b b NH-EDx S. litura 30 bc 20 c c d d d 20 e 10

Larval duration Larval(days) duration 10 Pupal duration (days)

0 0 Control 100 ppm 200 ppm 300 ppm 400 ppm 500 ppm Control 100 ppm 200 ppm 300 ppm 400 ppm 500 ppm 40 a a B 20 E b 30 n-HDa S. litura b b b n-HDa S. litura b c c c d 20 d 10

10 Pupal Pupal duration (days) Larval duration (days) 0 Control 0.5 ppm 1 ppm 1.5 ppm 2 ppm 2.5 ppm 0 Control 0.5 ppm 1 ppm 1.5 ppm 2 ppm 2.5 ppm

a 30 a b F C n-ODa S. litura b 20 b b n-ODa S. litura c c b 20 c d d 10 10 Pupal duration (days) Larval duration (days)

0 0 Control 0.5 ppm 1 ppm 1.5 ppm 2 ppm 2.5 ppm Control 0.5 ppm 1 ppm 1.5 ppm 2 ppm 2.5 ppm

Figure 4. DevelopmentalDevelopmental time in in days days for for S.S. litura litura larvaelarvae after after treatment treatment with with NH-EDx NH-EDx ( A), n-HDa-HDa ( B), and and n-ODa-ODa ( (C), and and forfor S. litura pupae after treatment with NH-EDX (D), n-HDa ((E),), andand n-ODa-ODa ((F),), respectively.respectively. MeansMeans ((±SEM)±SEM) followed followed by thethe same same letters above bars indicate no significant signiﬁcant difference ((pP ≤≤ 0.05)0.05) according according to to a Tukey’s test. Molecules 2021, 26, 3695 10 of 20

The larval and pupal duration of A. aegypti significantly increased when the treatment dosage of NH-EDx, n-HDa and n-ODa was increased. The duration of both larvae and pupae was profound in the maximum sublethal dosage of 500 ppm of NH-EDx (larvae: F4,20 = 28.94, p ≤ 0.001; pupae—F4,20 = 14.88, p ≤ 0.001), and it was significant compared with other treatment dosages and the control (Figure5A,D). Similar trends were observed in the n-HDa treatment, as the maximum sublethal dosage (2.5 ppm and 2.0 ppm) increased the larval and pupal days of A. aegypti steadily as compared to other treatment dosages Molecules 2021, 26, x FOR PEER REVIEW 11 of 22 (Figure5 B,E). However, there was no significant difference in the duration of larvae and pupae in A. aegypti from 0.5 to 1.5 ppm dosages of n-ODa (larvae—F4,20 = 8.54, p ≤ 0.001; pupae—F4,20 = 7.32, p ≤ 0.001) (Figure5C,F).

a a NH-EDx A. aegypti 20 a b a 20 b NH-EDx A. aegypti b b c c bc d 10 10 D Larval duration (days) A Pupal duration (days) duration Pupal 0 0 Control 100 ppm 200 ppm 300 ppm 400 ppm 500 ppm Control 100 ppm 200 ppm 300 ppm 400 ppm 500 ppm

a a 20 n-HDa A.aegypti a 20 b n-HDa A.aegypti a b b b b c c c 10 10 E

B Pupal duration (days) 0

Larval duration (days) 0 Control 0.5 ppm 1 ppm 1.5 ppm 2 ppm 2.5 ppm Control 0.5 ppm 1 ppm 1.5 ppm 2 ppm 2.5 ppm 20 a a 20 a n-ODa A.aegypti b b b n-ODa A.aegypti b b b c c c 10

C Pupal duration (days) F

Larval duration (days) 0 0 Control 0.5 ppm 1 ppm 1.5 ppm 2 ppm 2.5 ppm Control 0.5 ppm 1 ppm 1.5 ppm 2 ppm 2.5 ppm

FigureFigure 5.5. DevelopmentalDevelopmental time time in in days days for forA. A. aegypti aegyptilarvae larvae after after treatment treatment with with NH-EDx NH-EDx (A), (nA-HDa), n-HDa (B) and(B) nand-ODa n-ODa (C) and (C) forandA. for aegypti A. aegyptipupae pupae after after treatment treatment with with NH-EDx NH-EDx (D), (nD-HDa), n-HDa (E), and(E), andn-ODa n-ODa (F), respectively.(F), respectively. Means Means (±SEM) (±SEM) followed followed by theby the same same letters letters above above bars bars indicate indicate no signiﬁcantno significant difference difference (p ≤ (P0.05) ≤ 0.05) according according to a to Tukey’s a Tukey’s test. test.

Correspondingly,Correspondingly, thethe effecteffect onon adultadult longevitylongevity (male(male andand female)female) waswas alsoalso foundfound toto be profound in S. litura larvae treated with 500 ppm NH-EDx (F = 18.32, p ≤ 0.001) be profound in S. litura larvae treated with 500 ppm NH-EDx (F4,20 4,20= 18.32, P ≤ 0.001) (Fig- (Figure6A), 2.5 ppm n-HDa (F = 13.45, p ≤ 0.001) (Figure6B) and 2.5 ppm n-ODa ure 6A), 2.5 ppm n-HDa (F4,20 =4,20 13.45, P ≤ 0.001) (Figure 6B) and 2.5 ppm n-ODa (F4,20 = ≤ (F15.77,4,20 = P 15.77, ≤ 0.001)p (Figure0.001) (Figure6C). Likewise,6C). Likewise, the longevity the longevity of adults of in adults A. aegypti in A. displayed aegypti dis- a playedsignificant a signiﬁcant decline in decline a dose-dependent in a dose-dependent manner. manner. Both male Both and male female and longevity female longevity signifi- signiﬁcantly decreased in 500 ppm NH-EDx (F4,20 = 14.88, p ≤ 0.001) (Figure6D), 2.5 ppm cantly decreased in 500 ppm NH-EDx (F4,20 = 14.88, P ≤ 0.001) (Figure 6D), 2.5 ppm n-HDa n-HDa (F4,20 = 13.56, p ≤ 0.001) (Figure6E), and 2.5 ppm n-ODa (F4,20 = 17.11, p ≤ 0.001) (F4,20 = 13.56, P ≤ 0.001) (Figure 6E), and 2.5 ppm n-ODa (F4,20 = 17.11, P ≤ 0.001) (Figure 6F). (Figure6F). MoleculesMolecules2021 2021, ,26 26,, 3695 x FOR PEER REVIEW 1112 of of 20 22

A. aegypti female longevity 8 10 A. aegypti male longevity

S. litura female longevity 6 S. litura male longevity

5 4 Adult longevity (days) Adult longevity (days) A D 2 Control 100 ppm 200 ppm 300 ppm 400 ppm 500 ppm Control 100 ppm 200 ppm 300 ppm 400 ppm 500 ppm NH-EDx treatment concentration (ppm) NH-EDx treatment dosage (ppm) 14

10 12

S. litura female longevity A. aegypti female longevity 8 10 S. litura male longevity A. aegypti male Longevity 8 6 6

4 Adult longevity (days) 4

Adult longevity (days) B E 2 2 Control 0.5 ppm 1 ppm 1.5 ppm 2 ppm 2.5 ppm Control 0.5 ppm 1 ppm 1.5 ppm 2 ppm 2.5 ppm n-HDa treatment dosage (ppm) n-HDa treatment dosage (ppm) 14

10 A. aegypti female longevity 12 A. aegypti male longevity S. litura female longevity 8 S. litura male longevity 10

6 8

4 6 Adult longevity (days)

Adult Adult longevity (days) C F 2 4 Control 0.5 ppm 1 ppm 1.5 ppm 2 ppm 2.5 ppm Control 0.5 ppm 1 ppm 1.5 ppm 2 ppm 2.5 ppm n-ODa treatment dosage (ppm) n-ODa treatment dosage (ppm)

Figure 6. Developmental time in days for S. litura adults after treatment with (A) NH-EDx, (B) n-HDa, and (C) n-ODa, and forFigureA. aegypti 6. Developmentaladults after treatmenttime in days with for (D S.) litura NH-EDx, adults (E )aftern-HDa, treatment and (F with) n-ODa, (A) NH-EDx, respectively. (B) n-HDa, and (C) n-ODa, and for A. aegypti adults after treatment with (D) NH-EDx, (E) n-HDa, and (F) n-ODa, respectively. 3.4. Enzyme Activity 3.4. Enzyme Activity 3.4.1. α-Carboxylesterase 3.4.1. α- Carboxylesterase The sublethal dosage of NH-EDx significantly controlled the level of α-carboxylesterase activityThe in sublethalS. litura fourth dosage instar of NH-EDx larvae in significantly a concentration-dependent controlled the level manner. of α The-carboxyles- level of terase activity in S. litura fourth instar larvae in a concentration-dependent manner. The α-carboxylesterase reduced at the maximum dosage of 500 ppm (F4,20 = 13.55, p ≤ 0.001), whichlevel of result α-carboxylesterase was significant reduced against at other the maximum treatment dosage dosages of and500 ppm control (F4,20 (Figure = 13.55,7A). P ≤ Likewise,0.001), which the maximum result was dosages significant of n -HDaagainst and othern-ODa treatment (3.0 ppm) dosages also exhibited and control strong (Figure inhi- bition7A). Likewise, in the level the of maximumα-carboxylesterase dosages of as n compared-HDa and ton-ODa other (3.0 treatment ppm) also dosages exhibited and control strong (Finhibition4,20 = 19.77, in pthe≤ level0.001; of and α-carboxylesterase F4,20 = 23.11, p ≤ as0.001, compared respectively) to other (Figure treatment7 B&C). dosages The max- and imumcontrol sublethal (F4,20 = 19.77, dosage P ≤ of 0.001; NH-EDx and F (5004,20 = ppm) 23.11, also P ≤ 0.001, reduced respectively) the level of (Figureα-carboxylesterase 7 B&C). The inmaximumA. aegypti sublethalfourth instar dosage larvae of NH-EDx significantly (500 ppm) as compared also reduced to other the level treatment of α-carboxyles- concentra- tions.terase However, in A. aegypti there fourth was noinstar significant larvae significantly difference between as compared control to and other 300 treatment ppm NH-EDx con- (Fcentrations.4,20 = 18.22, However,p ≤ 0.001) there (Figure was7D). no The signif prominenticant difference sublethal between dosage control of n-HDa and (3.0 300 ppm) ppm significantlyNH-EDx (F4,20 decreased = 18.22, P the ≤ 0.001) enzyme (Figure rate as7D). compared The prominent to other sublethal treatment dosage concentrations of n-HDa and(3.0 controlppm) significantly (F4,20 = 19.44, decreasedp ≤ 0.001) the (Figure enzyme7E). rate Similar as compared trends to were other observed treatment in concen-n-ODa treatmenttrations and (3.0 control ppm), which(F4,20 = reduced 19.44, P the ≤ 0.001)α-carboxylesterase (Figure 7E). Similar in the fourth trends instars were ofobservedA. aegypti in n-ODa treatment (3.0 ppm), which reduced the α-carboxylesterase in the fourth instars of Molecules 2021, 26, x FOR PEER REVIEW 13 of 22 Molecules 2021, 26, 3695 12 of 20

A. aegypti larvae. However, there was no significant difference between the control and larvae. However, there was no signiﬁcant difference between the control and the 1.0 ppm the 1.0 ppm dosage (F4,20 = 23.22, P ≤ 0.001) (Figure 7F). dosage (F4,20 = 23.22, p ≤ 0.001) (Figure7F).

1.4 1.0 1.2 A D

0.8 a 1.0 a

NH-EDx A. aegypti 0.8 b a a NH-EDx S. litura 0.6 c b c 0.6 b b c d 0.4 0.4 250 300 350 400 450 500

-Carboxylase mM/min/mg/protein) 250 300 350 400 450 500 α -Carboxylase mM/min/mg/protein) NH-EDx treatment dosage (ppm) NH-EDx treatment dosage (ppm) α 1.0 1.0

0.8 B E a 0.8 a a a n-HDa S. litura n-HDa A. aegypti b b 0.6 b b b 0.6 c c 0.4 d 0.4 -Carboxylase mM/min/mg/protein) -Carboxylase mM/min/mg/protein) 0.51.01.52.02.53.0 0.5 1.0 1.5 2.0 2.5 3.0 α α n-HDa treatment dosage (ppm) n-HDa treatment dosage (ppm) 1.0 1.0

0.8 C F a 0.8 a a a n-ODa S. litura n-ODa A. aegypti b b 0.6 b 0.6 b b c c 0.4 d 0.4

0.51.01.52.02.53.0 0.51.01.52.02.53.0 -Carboxylase mM/min/mg/protein) -Carboxylase mM/min/mg/protein)

α n-ODa treatment dosage (ppm) α n-ODa treatment dosage (ppm) FigureFigure 7.7. α-Carboxylesterase-carboxylesterase enzyme enzyme activity activity of of the the fourth fourth instars instars of of S. litura after treatment with ( A) NH-EDx, (B) n-HDa, andand ((CC)) n-ODa,-ODa, andand of fourth instars of A. aegypti after treatment treatment with ( (D)) NH-EDx, NH-EDx, ( (E)) nn-HDa,-HDa, and and ( (FF)) nn-ODa,-ODa, respectively. respectively. TheThe datadata werewere ﬁttedfitted onon aa polynomialpolynomial (regression)(regression) model.model. VerticalVertical bars bars followed followed by by letters letters indicate indicate standard standard error error ( ±(±SEM).SEM).

3.4.2. β- Carboxylesterase 3.4.2. β-Carboxylesterase The level of β-carboxylesterase in the fourth instars of S. litura was significantly re- The level of β-carboxylesterase in the fourth instars of S. litura was signiﬁcantly duced by treatment with sublethal dosages of NH-EDx, n-HDa, and n-ODa, with maxi- reduced by treatment with sublethal dosages of NH-EDx, n-HDa, and n-ODa, with maximum reductions at 500 ppm (F4,20 = 17.47, P ≤ 0.001) (Figure 8A), 3.0 ppm (F4,20 = 13.99, P ≤ mum reductions at 500 ppm (F4,20 = 17.47, p ≤ 0.001) (Figure8A), 3.0 ppm (F 4,20 = 13.99, 0.001) (Figure 8B), and 3.0 ppm (F4,20 = 15.88, P ≤ 0.001) (Figure 8C), respectively. Corre- p ≤ 0.001) (Figure8B), and 3.0 ppm (F 4,20 = 15.88, p ≤ 0.001) (Figure8C), respectively. Cor- respondingly,spondingly, the the maximum maximum sublethal sublethal dosages dosages of of NH-EDx, NH-EDx, nn-HDa,-HDa, and n-ODa decreaseddecreased the β-carboxylesterase-carboxylesterase level level in in the the fourth fourth instars instars of A. of aegyptiA. aegypti significantlysigniﬁcantly at 500 at ppm 500 ppm (F4,20 = 13.33, P ≤ 0.001) (Figure 8D), 3.0 ppm (F4,20 = 12.23, P ≤ 0.001) (Figure 8E), and 3.0 ppm (F4,20 = 13.33, p ≤ 0.001) (Figure8D), 3.0 ppm (F 4,20 = 12.23, p ≤ 0.001) (Figure8E), and (F4,20 = 16.79, P ≤ 0.001) (Figure 8F), respectively. 3.0 ppm (F4,20 = 16.79, p ≤ 0.001) (Figure8F), respectively. Molecules 2021, 26, 3695 13 of 20 Molecules 2021, 26, x FOR PEER REVIEW 14 of 22

3 A D 2 2 a NH-EDx S. litura a a a NH-EDx-A. aegypti b b b b b c c c 1 1

250 300 350 400 450 500

-Carboxylase mM/min/mg/protein) 250 300 350 400 450 500 β

-Carboxylase mM/min/mg/protein) -Carboxylase NH-EDx treatment dosage (ppm)

β NH-EDx treatment dosage (ppm)

3 B 2.5 2.0 a E a 2 a a n-HDa A. aegypti n-HDa S. litura 1.5 b b b b c c d 1.0 d 1

0.5 1.0 1.5 2.0 2.5 3.0 mM/min/mg/protein) -Carboxylase 0.5 1.0 1.5 2.0 2.5 3.0 -Carboxylase mM/min/mg/protein) β β n-HDa treatment dosage (ppm) n-HDa treatment dosage (ppm) 3

a C 2 F 2 a a a b n-ODa A. aegypti b b b n-ODa S. litura c c 1 1 d d

0.5 1.0 1.5 2.0 2.5 3.0 0.5 1.0 1.5 2.0 2.5 3.0 -Carboxylase mM/min/mg/protein) -Carboxylase -Carboxylase mM/min/mg/protein) -Carboxylase β β n-ODa treatment dosage (ppm) n-ODa-treatment dosage (ppm) FigureFigure 8. 8.β β-Carboxylesterase-carboxylesterase enzymeenzyme activityactivity ofof thethe fourthfourth instars instars of ofS. S. lituralituraafter after treatment treatment with with ( A(A)) NH-EDx, NH-EDx, ( B(B))n n-HDa,-HDa, andand (C (C) )n n-ODa-ODa and and of of fourth fourth instars instars of ofA. A. aegypti aegyptiafter after treatment treatment with with ( D(D)) NH-EDx, NH-EDx, ( E(E))n n-HDa,-HDa, and and ((FF))n n-ODa,-ODa, respectively.respectively. TheThe data data were were ﬁtted fitted on on a a polynomial polynomial (regression) (regression) model. model. Vertical Vertical bars bars followed followed by by letters letters indicate indicate standard standard error error (± (±SEM).SEM).

3.4.3.3.4.3. Glutathione Glutathione S-Transferase S-Transferase (GST) (GST) TheThe levellevel ofof GST GST increased increased in in a a dose-dependent dose-dependent mannermanner inin thethe fourthfourth instarsinstars ofofS. S. litura after treatment with 500 ppm NH-EDx (F4,20 = 18.45, P ≤ 0.001) (Figure 9A), 3.0 ppm litura after treatment with 500 ppm NH-EDx (F4,20 = 18.45, p ≤ 0.001) (Figure9A), 3.0 ppm n-HDa (F4,20 = 21.56, P ≤ 0.001) (Figure 9B) and 3.0 ppm n-ODa (F4,20 = 18.23, P ≤ 0.001) n-HDa (F4,20 = 21.56, p ≤ 0.001) (Figure9B) and 3.0 ppm n-ODa (F4,20 = 18.23, p ≤ 0.001) (Figure(Figure9 C).9C). Likewise, Likewise, thethe level of of GST GST increased increased in ina dose-dependent a dose-dependent manner manner in the in fourth the instars of A. aegypti after treatment with 500 ppm NH-EDx (F4,20 = 12.35, P ≤ 0.001) (Figure fourth instars of A. aegypti after treatment with 500 ppm NH-EDx (F4,20 = 12.35, p ≤ 0.001) 9D), 3.0 ppm n-HDa (F4,20 = 11.26, P ≤ 0.001) (Figure 9E), and 3.0 ppm n-ODa (F4,20 = 28.33, (Figure9D), 3.0 ppm n-HDa (F4,20 = 11.26, p ≤ 0.001) (Figure9E), and 3.0 ppm n-ODa P ≤ 0.001) (Figure 9F). However, there is no significant difference between 500, 400, and (F4,20 = 28.33, p ≤ 0.001) (Figure9F). However, there is no signiﬁcant difference between 500,450 400,ppm and doses 450 of ppm NH-EDx doses or of between NH-EDx 3.0, or between2.5, and 3.0,2.0 ppm 2.5, and doses 2.0 of ppm n-HDa. doses of n-HDa.

3.4.4. Cytochrome P-450 (CYP450) The level of CYP450 was directly proportional to the dosage of the treatment, similarly to GST, in the fourth instars of S. litura after treatment with 500 ppm NH-EDx (F4,20 = 15.54, p ≤ 0.001) (Figure 10A), 3.0 ppm n-HDa (F4,20 = 18.65, p ≤ 0.001) (Figure 10B), and 3.0 ppm n-ODa (F4,20 = 12.22, p ≤ 0.001) (Figure 10C). Likewise, the level of CYP450 increased in a dose-dependent manner in the fourth instars of A. aegypti after treatment with 500 ppm NH-EDx (F4,20 = 18.45, p ≤ 0.001) (Figure 10D), 3.0 ppm n-HDa (F4,20 = 21.56, p ≤ 0.001) (Figure 10E), and 3.0 ppm n-ODa (F4,20 = 18.23, p ≤ 0.001) (Figure 10F). However, there was no signiﬁcance between the NH-EDx doses of 350, 400, 450, and 500 ppm in either S. litura (F4,20 = 15.23, p ≤ 0.001) or A. aegypti (F4,20 = 11.88, p ≤ 0.001). Molecules 2021, 26, 3695 14 of 20

3.5. Gut-Histological Activity The midgut cells were severely infected in the fourth instar larvae of A. aegypti after treatment with sublethal dosages of NH-EDx (500 ppm) (Figure 11B), n-HDa (Figure 11C), and n-ODa (Figure 11D) as compared to the control (Figure 11A). The control midgut cells appeared normal, with a clearly visible epithelial layer (EL), well distinguished gut lumen (GL), and normal peritrophic matrix (Pm). Midgut cells treated with NH-EDx and their major phytochemicals showed severe morphological alterations in the GL, EL, and Pm. The damage rate was particularly severe for NH-EDx as compared to its metabolites n-HDa and n-ODa. Correspondingly, the fourth instar larval midgut cells of S. litura were signiﬁcantly affected after treatment with sublethal dosages of NH-EDx (Figure 11F), n-HDa (Figure 11G), and n-ODa (Figure 11H) as compared to the control (Figure 11E). Moreover, Molecules 2021, 26, x FOR PEER REVIEW 15 of 22 the brush border membrane (BBM), EL, and GL displayed the most signiﬁcant cellular damage in NH-EDx, followed by n-HDa and n-ODa.

0.8 a a NH-EDx A. aegypti a a 1.0 0.6 NH-EDx S. litura b b a a a a AD

mol/mg/min) ab 0.4 mol/mg/min) μ

b μ 0.5 0.2

0.0

GST ( activity 0.0 250 300 350 400 450 500 250 300 350 400 450 500 GST ( activity NH-EDx treatment dosage (ppm) NH-EDx treatment dosage (ppm) 1.0 0.8 a a n-HDa S.litura a a B 0.8 E b a 0.6 n-HDa A. aegypti a a 0.6 a mol/mg/min) mol/mg/min) c ab μ μ b 0.4 0.4

0.2 0.2 GST ( activity GST activity ( activity GST 0.0 0.0 0.51.01.52.02.53.0 0.5 1.0 1.5 2.0 2.5 3.0 n-HDa treatment dosage (ppm) n-HDa treatment dosage (ppm) 0.8 0.8 a a n-ODa A.aegypti b n-ODa S.litura a a a a C 0.6 b F c 0.6 b c c 0.4 mol/mg/min) mol/mg/min) μ 0.4 μ 0.2 0.2 0.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 GST ( activity GST activity ( GST activity 0.5 1.0 1.5 2.0 2.5 3.0 n-ODa treatment dosage (ppm) n-ODa treatment dosage (ppm) Figure 9. GST enzymeenzyme activityactivity ofof the the fourth fourth instars instars of ofS. S. litura lituraafter after treatment treatment with with (A )( NH-EDx,A) NH-EDx, (B) n(B-HDa,) n-HDa, and and (C) n(-ODaC) n- ODaand ofand fourth of fourth instars instars of A. of aegypti A. aegyptiafter after treatment treatment with (withD) NH-EDx, (D) NH-EDx, (E) n -HDa,(E) n-HDa, and ( Fand) n-ODa, (F) n-ODa, respectively. respectively. The data The weredata wereﬁtted fitted on a polynomialon a polynomial (regression) (regression) model. model. Vertical Vertical bars followed bars followed by letters by letters indicate indicate standard standard error (error±SEM). (±SEM).

3.4.4. Cytochrome P-450 (CYP450) The level of CYP450 was directly proportional to the dosage of the treatment, similarly to GST, in the fourth instars of S. litura after treatment with 500 ppm NH-EDx (F4,20 = 15.54, P ≤ 0.001) (Figure 10A), 3.0 ppm n-HDa (F4,20 = 18.65, P ≤ 0.001) (Figure 10B), and 3.0 ppm n-ODa (F4,20 = 12.22, P ≤ 0.001) (Figure 10C). Likewise, the level of CYP450 increased in a dose-dependent manner in the fourth instars of A. aegypti after treatment with 500 ppm NH-EDx (F4,20 = 18.45, P ≤ 0.001) (Figure 10D), 3.0 ppm n-HDa (F4,20 = 21.56, P ≤ 0.001) (Figure 10E), and 3.0 ppm n-ODa (F4,20 = 18.23, P ≤ 0.001) (Figure 10F). However, there was no significance between the NH-EDx doses of 350, 400, 450, and 500 ppm in either S. litura (F4,20 = 15.23, P ≤ 0.001) or A. aegypti (F4,20 = 11.88, P ≤ 0.001). Molecules 2021, 26, 3695 15 of 20 Molecules 2021, 26, x FOR PEER REVIEW 16 of 22

12 12

10 10 A NH-EDx A. aegypti a a NH-EDx S. litura a a D a 8 a 8 a a ab ab b b 6 6

4 4 mol 7-OH/mgmol larvae/min) mol 7-OH/mgmol larvae/min) μ 2 2 μ

0 0 250 300 350 400 450 500

CYP450 ( 250 300 350 400 450 500 CYP450 ( NH-EDx treatment dosage (ppm) NH-EDx treatment dosage (ppm) 15 10 n-HDa S. litura a aB a a ab E b 10 nHDa A. aegypti a a a a 5 ab b

5 mol 7-OH/mgmol larvae/min) mol 7-OH/mgmol larvae/min) μ μ 0 0.5 1.0 1.5 2.0 2.5 3.0 0 0.5 1.0 1.5 2.0 2.5 3.0

CYP450 ( nHDa treatment dosage (ppm) CYP450 ( nHDa treatment dosage (ppm)

10 C nODa A. aegypti a n-ODa S. litura 10 a a a a a F a a ab ab b b

5 5 mol 7-OH/mgmol larvae/min) mol 7-OH/mg larvae/min) 7-OH/mg mol μ 0 μ 0 0.5 1.0 1.5 2.0 2.5 3.0 0.5 1.0 1.5 2.0 2.5 3.0 nODa treatment dosage (ppm) nODa treatment dosage (ppm) CYP450 ( CYP450 ( Figure 10. CYP450CYP450 enzyme enzyme activity activity of of the the fourth fourth instars instars of ofS. S.litura litura afterafter treatment treatment with with (A) ( ANH-EDx,) NH-EDx, (B) (nB-HDa,) n-HDa, and and (C) n-ODa and of fourth instars of A. aegypti after treatment with (D) NH-EDx, (E) n-HDa, and (F) n-ODa, respectively. The Molecules(C) 2021n-ODa, 26, and x FOR of PEER fourth REVIEW instars of A. aegypti after treatment with (D) NH-EDx, (E) n-HDa, and (F) n-ODa, respectively. The17 of 22 data were fitted on a polynomial (regression) model. Vertical bars followed by letters indicate standard error (±SEM). data were ﬁtted on a polynomial (regression) model. Vertical bars followed by letters indicate standard error (±SEM).

3.5. Gut-Histological Activity The midgut cells were severely infected in the fourth instar larvae of A. aegypti after treatment with sublethal dosages of NH-EDx (500 ppm) (Figure 11B), n-HDa (Figure 11C), and n-ODa (Figure 11D) as compared to the control (Figure 11A). The control midgut cells appeared normal, with a clearly visible epithelial layer (EL), well distinguished gut lumen (GL), and normal peritrophic matrix (Pm). Midgut cells treated with NH-EDx and their major phytochemicals showed severe morphological alterations in the GL, EL, and Pm. The damage rate was particularly severe for NH-EDx as compared to its metabolites n- HDa and n-ODa. Correspondingly, the fourth instar larval midgut cells of S. litura were significantly affected after treatment with sublethal dosages of NH-EDx (Figure 11F), n- HDa (Figure 11G), and n-ODa (Figure 11H) as compared to the control (Figure 11E). More- over, the brush border membrane (BBM), EL, and GL displayed the most significant cellular damage in NH-EDx, followed by n-HDa and n-ODa.

Figure 11. Gut-histological analysis of cross-section of the midgut of fourth instar larvae of A. aegypti after treatment with (A) control, (B) NH-EDx (500 ppm), (C) n-HDa (5 ppm), and (D) n-ODa (5 ppm) and of fourth instar larvae of S. litura after (A) control, (B) NH-EDx (500 ppm), (C) n-HDa (5 ppm), and (D) n-ODa (5 ppm) and of fourth instar larvae of S. litura after treatment with (E) control, (F) NH-EDx (500 ppm), (G) n-HDa (5 ppm), and (H) n-ODa (5 ppm). EL: epithelial layer; GL: treatment with (E) control, (F) NH-EDx (500 ppm), (G) n-HDa (5 ppm), and (H) n-ODa (5 ppm). EL: epithelial layer; GL: gut gut lumen; Pm: peritrophic matrix; BBM: brush border membrane. lumen; Pm: peritrophic matrix; BBM: brush border membrane. 4. Discussion 4. Discussion It is marked that chemical-based insecticides/pesticides are having less impact It is marked that chemical-based insecticides/pesticides are having less impact against against both mosquitoes and agriculture pests due to the upsurge in resistance of insect both mosquitoes and agriculture pests due to the upsurge in resistance of insect pests pestsof different of different classes classes against against commercial commercial chemicals chemicals [42,43]. [42,43]. Synthetic Synthetic insecticide insecticide resistance resistance has been linked to the increased metabolism level in the insects, including the uplift of detoxifying enzymes or enzyme structural changes that increase the capacity of the metabolic rate [43]. The major factors that are responsible for insecticidal resistance include biological factors: (i) life cycle of the insect, (ii) population growth rate, (iii) genetics of the insect, and behavioral factors: (iv) movement and more importantly (v) exposure rate to chemicals [44]. In general, commercial chemicals contain artificial blends of two or three compounds that are quickly becoming resisted by pests due to their known mode of action [44]. To overcome this problem, natural-based insecticides chiefly derived from plant sources are widely evaluated for their insecticidal efficacy against different species of both mosquito vectors and agriculture pests [45–49]. Plant extracts and their major derivatives play a pivotal role in managing insect pests of both agricultural and medical importance due to their significant blends of natural chemicals including phenolic compounds, alkaloids, tannins, flavonoids, steroids, terpenes, coumarins, terpenoids, and lignins [50]. Also, the active ingredients contained in them are natural substances, not synthetic substances, with smooth degradation pathways in nature after application that result in less pollution to the environment. As botanical insecticides have many insecticidal ingredients with special modes of action, it is difficult for pests to develop pesticide resistance. Also, botanical insecticides generally have features of strong selectivity; low toxicity to humans, livestock, and natural enemies; and relatively low development and use costs [50]. Deadly diseases such as dengue spread by mosquito, affect >700 million people across the nation. The major vector responsible for spreading this disease is A. aegypti, which is widespread across all tropical and subtropical nations [23]. Correspondingly, S. litura is a crucial pest of agriculture, infesting more than 70% of plant crops globally and feeds on >290 plant species [4,51,52]. Both these pests are widespread across developing countries and threaten crops and human lives. E. divaricata belongs to the family Compositae and is widely used in traditional medicine across South Asian countries. Moreover, E. divaricata contains a blend of natural compounds, including tannins, flavonoids, and steroids, with unique modes of action against bacterial and viral infections [36]. However, their insecticidal property remains uncharted. GC–MS analysis of NH-EDx showed nine major phytochemicals, with the highest peak area observed in n-HDa followed by n-ODa. Larvicidal screening of NH-EDx showed a Molecules 2021, 26, 3695 16 of 20

has been linked to the increased metabolism level in the insects, including the uplift of detoxifying enzymes or enzyme structural changes that increase the capacity of the metabolic rate [43]. The major factors that are responsible for insecticidal resistance include biological factors: (i) life cycle of the insect, (ii) population growth rate, (iii) genetics of the insect, and behavioral factors: (iv) movement and more importantly (v) exposure rate to chemicals [44]. In general, commercial chemicals contain artificial blends of two or three compounds that are quickly becoming resisted by pests due to their known mode of action [44]. To overcome this problem, natural-based insecticides chiefly derived from plant sources are widely evaluated for their insecticidal efficacy against different species of both mosquito vectors and agriculture pests [45–49]. Plant extracts and their major derivatives play a pivotal role in managing insect pests of both agricultural and medical importance due to their significant blends of natural chemicals including phenolic compounds, alkaloids, tannins, flavonoids, steroids, terpenes, coumarins, terpenoids, and lignins [50]. Also, the active ingredients contained in them are natural substances, not synthetic substances, with smooth degradation pathways in nature after application that result in less pollution to the environment. As botanical insecticides have many insecticidal ingredients with special modes of action, it is difficult for pests to develop pesticide resistance. Also, botanical insecticides generally have features of strong selectivity; low toxicity to humans, livestock, and natural enemies; and relatively low development and use costs [50]. Deadly diseases such as dengue spread by mosquito, affect >700 million people across the nation. The major vector responsible for spreading this disease is A. aegypti, which is widespread across all tropical and subtropical nations [23]. Correspondingly, S. litura is a crucial pest of agriculture, infesting more than 70% of plant crops globally and feeds on >290 plant species [4,51,52]. Both these pests are widespread across developing countries and threaten crops and human lives. E. divaricata belongs to the family Compositae and is widely used in traditional medicine across South Asian countries. Moreover, E. divaricata contains a blend of natural compounds, including tannins, flavonoids, and steroids, with unique modes of action against bacterial and viral infections [36]. However, their insecticidal property remains uncharted. GC–MS analysis of NH-EDx showed nine major phytochemicals, with the highest peak area observed in n-HDa followed by n-ODa. Larvicidal screening of NH- EDx showed a prominent mortality rate in both the pests S. litura and A. aegypti at the maximum dosage of 1000 ppm. Moreover, the peak area compounds of NH-EDx also showed profound mortality rate against both insect pests. Previously, hexadecanoic acid ester derivatives showed a 70–98% decline in the oviposition rate in the groundnut pest Caryedon serratus at the maximum dosage of 1.0 mg L−1 [53]. Moreover, previous research proclaims that monoterpenes, alkanes, sesquiterpenes, fatty acids, and phenyl propanoids could demonstrate significant insecticidal and repellent activity against serious pests [54]. The bioactive metabolites of NH-EDx also showed high larvicidal activity against both pests. Previously, Citrullus colocynthis L. and its chief metabolite cucurbitacin E showed significant larvicidal activity (93.8%) against S. litura [4]. In parallel, bioactive andrographolide derived from Andrographis paniculata L. showed a prominent mortality rate against the dengue mosquito vector A. aegypti [55]. Comparably to the above results, the maximum lethal dosages of NH-EDx (1000 ppm), n-HDa (5 ppm), and n-ODa (5 ppm) delivered significant mortality rates against A. aegypti and S. litura. Sublethal concentrations of NH-EDx (500 ppm), n-HDa (2.5 ppm), and n-ODa (2.5 ppm) showed substantial impacts in the growth and development of both A. aegypti and S. litura. The larval and pupal duration were significantly higher in the crude NH-EDx as compared to their bioactive metabolites across both the insect pests. Previously, Shaalan et al. [56] illustrated that the larval duration is blocked or altered when the larva is exposed to higher range of toxic chemicals. The above statement was well matched with our present results, as a significant extension of duration in larval and pupal stages was observed as compared to the control in both insect pests. In contrast, the adult longevity of both males and females of both the insect pests showed a significant reduction in a dose dependent manner, with Molecules 2021, 26, 3695 17 of 20

the highest reductions in the maximum dosages of NH-EDx (500 ppm), n-HDa (2.5 ppm), and n-ODa (2.5 ppm). Adult longevity is considered as a restricted aspect for viral transmis- sion in arthropods specifically, as the insect releases toxins that generally need a period of time to reach the midgut lining to begin replicate and spreads through the salivary glands to transmit virus [57]. The sublethal dosages of NH-EDx and their major metabolites showed significant alterations in the levels of midgut enzymes including α- and β-carboxylesterase and of detoxifying enzymes GST and CYP450 in both A. aegypti and S. litura. Previously, Koodalingam et al. [58] reported that the bioactive compounds derived from plant extracts contact to different insect species has displayed drift in the levels of carboxylesterase enzyme. The above statement was well supported with our present research, as the peak area compounds n-HDa and n-ODa displayed steady blockage in the esterase activity of both the pests. As dosage increased, α- and β-carboxylesterase levels decreased, and in contrast the detoxifying enzymes GST and CYP450 increased significantly. It is well known that GST and CYP450 are recognized as crucial biomarkers to detect resistance status in any insect pest [37,59]. The present results clearly display that NH-EDx decreased the enzyme ratio significantly as compared to the individual bioactive compounds n-HDa and n-ODa, and the level was slightly higher in A. aegypti as compared to S. litura. Examination of midgut toxicity of NH-EDx and their chief derivatives displayed that the sublethal dosages caused severe damage to gut tissues such as the peritrophic matrix and also exhibited major changes in the alignment of the epithelial layer and brush border membrane as compared to the control. Previously, Ray et al. [60] clearly stated that the primary target of any plant metabolite is the midgut epithelium cells. It is well evident that the bioactive extracts of NH-EDx showed higher toxicity in the midgut profile of both the insect pests. Amala et al. [13] proclaimed that the peritrophic membrane is chiefly responsible for growth stimulus in the pests. The major metabolites derived from the hexane extracts of Epaltes pygmaea blocked the peritrophic membrane in the dengue vector and in turn decreased the development and growth rate of the insect pests.

5. Conclusions The present investigation clearly displayed that NH-EDx and their bioactive metabolites n-HDa and n-ODa showed signiﬁcant larvicidal, growth retardant, enzyme inhibition, and midgut toxicity effects against the crucial agricultural and medically challenging insect pests of ecological importance. Future perspectives will be focused on detecting the resistance and susceptibility of these bioactive compounds in the targeted pests as well as detecting the nontarget impacts of n-HDa and n-ODa against beneﬁcial organisms sharing the same ecological niche of the targeted pest. Hopefully, the effective formulation of phytochemicals as commercial botanical insecticides will replace the current chemical pesticides, which are a global burden to our society, and which create chemical resistance in different insect pests.

Author Contributions: K.A., S.K. and P.V.-S. designed the research plan and drafted the manuscript. P.V.-S., S.K. and R.G. performed the experimental works and data compilation. N.R., K.S., A.E.- Z.M.A.M., A.A.A.-G., J.A. and M.S.E. coordinated the work and discussed the results. S.S.-N. and P.K. revised and formatted the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research work was partially supported by Chiang Mai University. Also, the author P.V. was ﬁnancially supported by the Early Career Research Award funded by the Science and Engineering Research Board (DST-SERB), Government of India (FILE NO.ECR/2018/000552). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data is contained within the article. Molecules 2021, 26, 3695 18 of 20

Acknowledgments: We would like to thank the Department of Biotechnology, St. Peter’s Institute of Higher Education and Research, Chennai, India and the Division of Biopesticides and Environmental Toxicology, Sri Paramakalyani Centre for Excellence in Environmental Sciences, Manonmaniam Sundaranar University, Tirunelveli, India for providing the infrastructural facility for carrying out this research work. This work was partially supported by the Post-Doctoral Program, Chiang Mai University, Thailand. All authors are grateful to the Deanship of Scientific Research at King Saud University Saudi Arabia for financial support through project No RG-1441-484. Conflicts of Interest: The authors declare that no conflict of interest exists. Sample Availability: Samples of the compounds are not available from the authors.

References 1. Senthil-Nathan, S. Physiological and biochemical effect of Neem and other Meliaceae plants secondary metabolites against Lepidopteran insects. Front. Physiol. 2013, 4, 1–17. [CrossRef] 2. Murfadunnisa, S.; Vasantha-Srinivasan, P.; Ganesan, R.; Senthil-Nathan, S.; Kim, T.J.; Ponsankar, A.; Kumar, S.D.; Chandramohan, D.; Krutmuang, P. Larvicidal and enzyme inhibition of essential oil from Spheranthus amaranthroids (Burm.) against lepidopteran pest Spodoptera litura (Fab.) and their impact on non-target earthworms. Biocatal. Agri. Biotechnol. 2019, 21, 101324. [CrossRef] 3. Karthi, S.; Uthirarajan, K.; Manohar, V.; Venkatesan, M.; Chinnaperumal, K.; Vasantha-Srinivasan, P.; Krutmuang, P. Larvicidal enzyme inhibition and repellent activity of red mangrove Rhizophora mucronata (Lam.) leaf extracts and their biomolecules against three medically challenging arthropod vectors. Molecules 2020, 25, 3844. [CrossRef] 4. Ponsakar, A.; Sahayaraj, K.; Senthil-Nathan, S.; Vasantha-Srinivasan, P.; Karthi, S.; Thanigaivel, A.; Petchidurai, G.; Madasamy, M.; Hunter, W.B. Toxicity and developmental effect of cucurbitacin E from Citrullus colocynthis L. (Cucurbitales: Cucurbitaceae) against Spodoptera litura Fab. and a non-target earthworm Eisenia fetida Savigny. Environ. Sci. Pollut. Res. 2020, 27, 23390–23401. [CrossRef] 5. Chellappandian, M.; Senthil-Nathan, S.; Vasantha-Srinivasan, P.; Karthi, S.; Thanigaivel, A.; Kalaivani, K.; Sivanesh, H.; Stanley- Raja, V.; Chanthini, K.M.; Shyam-Sundar, N. Target and non-target botanical pesticides effect of Trichodesma indicum(Linn) R. Br. and their chemical derivatives against the dengue vector, Aedes aegypti L. Environ. Sci. Pollut. Res. 2019, 26, 16303–16315. [CrossRef] 6. Senthil-Nathan, S.; Choi, M.Y.; Paik, C.H.; Seo, H.Y.; Kalivani, K.; Kim, J.D. Effect of azadirachtin on acetylcholinesterase (AChE) activity and histology of the brown plant hopper Nilaparvata lugens (Stal). Ecotoxicol. Environ. Saf. 2008, 70, 244–250. [CrossRef] 7. Thanigaivel, A.; Chandrasekaran, R.; Revathi, K.; Nisha, S.; Sathish-Narayanan, S.; Kirubakaran, S.A.; Senthil-Nathan, S. Larvicidal efficacy of Adhatoda vasica (L.) Nees against the bancroftian filariasis vector Culex quinquefasciatus Say and dengue vector Aedes aegypti L. in in vitro condition. Parasitol. Res. 2012, 110, 1993–1999. [CrossRef][PubMed] 8. Lija-Escaline, J.; Senthil-Nathan, S.; Thanigaivel, A.; Pradeepa, V.; Vasantha-Srinivasan, P.; Ponsankar, A.; Edwin, E.; Selin-Rani, S.; Abdel-Megeed, A. Physiological and biochemical effects of chemical constituents from Piper nigrum Linn (Piperaceae) against the dengue vector Aedes aegypti Liston (Diptera: Culicidae). Parasitol. Res. 2015, 114, 4239–4249. [CrossRef][PubMed] 9. Benelli, G. Plant-mediated biosynthesis of nanoparticles as an emerging tool against mosquitoes of medical and veterinary importance: A review. Parasitol. Res. 2016, 115, 23–34. [CrossRef][PubMed] 10. Senthil-Nathan, S. A review of bio pesticides and their mode of action against insect pests. In Environmental Sustainability- Role of Green Technologies; Springer: Bangalore, India, 2015; pp. 49–63. 11. Selin-Rani, S.; Senthil-Nathan, S.; Thanigaivel, A.; Vasantha-Srinivasan, P.; Edwin, E.; Ponsankar, A.; Lija-Escaline, J.; Kalaivani, K.; Abdel-Megeed, A.; Hunter, W.B.; et al. Toxicity and physiological effect of quercetin on generalist herbivore, Spodoptera litura Fab. and a non-target earthworm Eisenia fetida Savigny. Chemosphere 2016, 165, 257–267. [CrossRef][PubMed] 12. Senthil-Nathan, S. A review of resistance mechanisms of synthetic insecticides and botanicals, phytochemicals, and essential oils as alternative larvicidal agents against mosquitoes. Front. Physiol. 2020, 10, 1591. [CrossRef][PubMed] 13. Amala, K.; Ganesan, R.; Karthi, S.; Senthil-Nathan, S.; Chellappandian, M.; Krutmunag, P.; Radhakrishnan, R.; Mohammad, F.; Ponsankar, A.; Vasantha-Srinivasan, P. Larval and gut enzyme toxicity of n-hexane extract Epaltes pygmaea DC. against the arthropod vectors and its non-toxicity against aquatic predator. Toxin Rev. 2020.[CrossRef] 14. Huang, S.H.; Xian, J.D.; Kong, S.Z.; Li, Y.C.; Xie, J.H.; Lin, J.; Chen, J.N.; Wang, H.F.; Su, Z.R. Insecticidal activity of pogostone against Spodoptera litura and Spodoptera exigua (Lepidoptera: Noctuidae). Pest. Manag. Sci. 2014, 70, 510–516. [CrossRef] 15. Selin-Rani, S.; Senthil-Nathan, S.; Revathi, K.; Chandrasekaran, R.; Thanigaivel, A.; Vasantha-Srinivasan, P.; Ponsankar, A.; Edwin, E.; Pradeepa, V. Toxicity of Alangium salvifolium Wang chemical constituents against the tobacco cutworm Spodoptera litura Fab. Pest. Biochem. Physiol. 2016, 126, 92–101. [CrossRef][PubMed] 16. Wheeler, D.A.; Isman, M.B. Anti-feedant and toxic activity of Trichilia americana extract against the larvae of Spodoptera litura. Entomol. Exp. Appl. 2001, 98, 9–16. [CrossRef] 17. Koou, S.; Chong, C.; Vythilingam, I.; Lee, C.; Ng, L. Insecticide resistance and its underlying mechanisms in field populations of Aedes aegypti adults (Diptera: Culicidae) in Singapore. Parasit. Vectors 2014, 7, 471. [CrossRef] Molecules 2021, 26, 3695 19 of 20

18. Reegan, A.D.; Gandhi, M.R.; Paulraj, M.G.; Ignacimuthu, S. Ovicidal and oviposition deterrent activities of medicinal plant extracts against Aedes aegypti L. and Culex quinquefasciatus Say mosquitoes (Diptera: Culicidae). Osong Public Health Res. Perspect. 2015, 6, 64–69. [CrossRef] 19. Thanigaivel, A.; Vasantha-Srinivasan, P.; Senthil-Nathan, S.; Edwin, E.; Ponsankar, A.; Chellappandian, M.; Selin-Rani, S.; Lija-Escaline, J.; Kalaivani, K. Impact of Terminalia chebula Retz. against Aedes aegypti L. and non-target aquatic predatory insects. Ecotoxicol. Environ. Saf. 2016, 137, 210–217. [CrossRef][PubMed] 20. Sivan, A.; Shriram, A.N.; Sunish, I.P.; Vidhya, P.T. Studies on insecticide susceptibility of Aedes aegypti (Linn) and Aedes albopictus (Skuse) vectors of dengue and chikungunya in Andaman and Nicobar Islands, India. Parasitol. Res. 2015, 114, 4693–4702. [CrossRef] 21. Senthil-Nathan, S.; Kalaivani, K. Combined effects of azadirachtin and nucleopolyhedrovirus (SpltNPV) on Spodoptera litura Fabricius (Lepidoptera: Noctuidae) larvae. Biol. Control. 2006, 39, 96–104. [CrossRef] 22. Senthil-Nathan, S.; Kalaivani, K.; Sehoon, K. Effects of Dysoxylum malabaricum Bedd. (Meliaceae) extract on the malarial vector Anopheles stephensi Liston (Diptera: Culicidae). Bioresour. Technol. 2006, 97, 2077–2083. [CrossRef] 23. Yogarajalakshmi, P.; Poonguzhali, T.N.; Ganesan, R.; Karthi, S.; Senthil-Nathan, S.; Krutmuang, P.; Radhakrishnan, N.; Moham- mad, F.; Kim, T.; Vasantha-Srinivasan, S. Toxicological screening of marine red algae Champia parvula (C. Agardh) against the dengue mosquito vector Aedes aegypti (Linn.) and its non-toxicity against three beneﬁcial aquatic predators. Aquat. Toxicol. 2020, 222, 105474. [CrossRef] 24. Vasantha-Srinivasan, P.; Senthil-Nathan, S.; Ponsankar, A.; Thanigaivel, A.; Edwin, E.S.; Selin-Rani, S.; Chellappandian, M.; Pradeepa, V.; Lija-Escaline, J.; Kalaivani, K.; et al. Comparative analysis of mosquito (Diptera: Culicidae: Aedes aegypti Liston) responses to the insecticide Temephos and plant derived essential oil derived from Piper betle L. Ecotoxicol. Environ. Saf. 2017, 139, 439–446. [CrossRef][PubMed] 25. Vasantha-Srinivasan, P.; Senthil-Nathan, S.; Thanigaivel, A.; Edwin, E.S.; Ponsankar, A.; Selin-Rani, S.; Pradeepa, V.; Sakthi- Bhagavathy, M.; Kalaivani, K.; Hunter, W.B.; et al. Developmental response of Spodoptera litura Fab. to treatments of crude volatile oil from Piper betle L. and evaluation of toxicity to earthworm, Eudrilus eugeniae Kinb. Chemosphere 2016, 155, 336–347. [CrossRef][PubMed] 26. Edwin, E.; Vasantha-Srinivasan, P.; Senthil-Nathan, S.; Thanigaivel, A.; Ponsankar, A.; Selin-Rani, S.; Kalaivani, K.; Hunter, W.B.; Duraipandiyan, V.; Al-Dhabi, N.A. Effect of andrographolide on phosphatases activity and cytotoxicity against Spodoptera litura. Invert. Surv. J. 2016, 13, 153–163. 27. Annamalai, M.; Vasantha-Srinivasan, P.; Thanigaivel, A.; Chellappandian, M.; Karthi, S.; Mayabini, J.; Guru Pirasanna Pandi, G.; Totan, A.; Murugesan, A.G.; Senthil-Nathan, S. Effect of thiamethoxam on growth, biomass of rice varieties and its specialized herbivore, Scirpophaga incertulas Walker. Physiol. Mol. Plant. Pathol. 2017.[CrossRef] 28. Dinesh-Kumar, A.; Srimaan, E.; Chellappandian, M.; Vasantha-Srinivasan, P.; Karthi, S.; Annamalai, M.; Thanigaivel, A.; Ponsankar, A.; Chanthini, K.; Shyam-Sundar, N.; et al. Target and non-target response of Swietenia Mahagoni Jacq. chemical constituents against tobacco cutworm Spodoptera litura Fab. and earthworm, Eudrilus eugeniae Kinb. Chemosphere 2018, 199, 35–43. [CrossRef] 29. Shin, J.; Vasantha-Srinivasan, P.; Kim, K. The multi-faceted potential of plant-derived metabolites as antimicrobial agents against multidrug-resistant pathogens. Microbial pathog. 2018, 116, 209–214. [CrossRef][PubMed] 30. Pradeepa, V.; Senthil-Nathan, S.; Sathish-Narayanan, S.; Selin-Rani, S.; Vasantha-Srinivasan, P.; Thanigaivel, A.; Ponsankar, A.; Edwin, E.-S.; Sakthi-Bagavathy, M.; Kalaivani, K.; et al. Potential mode of action of a novel plumbagin as a mosquito repellent against the malarial vector Anopheles stephensi, (Culicidae: Diptera). Pesticide Biochem. Physiol. 2016, 134, 84–93. [CrossRef] 31. Senthil-Nathan, S.; Kalaivani, K.; Murugan, K. Effects of neem limonoids on the malaria vector Anopheles stephensi Liston (Diptera: Culicidae). Acta Trop. 2005, 96, 47–55. [CrossRef] 32. Senthil-Nathan, S.; Chung, P.G.; Murugan, K. Effect of bio-pesticides applied separately or together on Nutritional Indices of the Rice Leaffolder Cnaphalocrocis medinalis. Phytoparasitica 2005, 33, 187–195. [CrossRef] 33. Hewawasam, R.P.; Jayatilaka, K.A.P.W.; Pathirana, C.; Muddduwa, L.K.B. Hepatoprotective effect of Epaltes divaricata extract on carbon tetrachloride induced hepatotoxixity in mice. Ind. J. Med. Res. 2004, 120, 30–34. 34. Amala, K.; Saraswathy, A.; Amerjothy, S. GC-MS analysis of N-Hexane extract of Epaltes divaricata (L.) Cass. J. Pharmacog. Phytochem. 2013, 2, 33–35. 35. Chah, K.F.; Eze, C.A.; Emuelosi, C.E.; Esimone, C.O. Antibacterial and wound healing properties of methanolic extracts of some Nigerian medicinal plants. J. Ethnopharmacol. 2006, 104, 164–167. [CrossRef][PubMed] 36. Glorybai, L.; Kannan, B.K.; Arasu, M.V.; Al-Dhabi, N.A.; Agastin, P. Some biological activities of Epaltes divaricata L.—An in vitro study. Ann. Clin. Microbiol. Antimicrob. 2015, 14, 18. [CrossRef][PubMed] 37. Vasantha-Srinivasan, P.; Chellappandian, M.; Senthil-Nathan, S.; Ponsankar, A.; Thanigaivel, A.; Karthi, S.; Edwin, E.; Selin-Rani, S.; Kalaivani, K.; Maggi, F.; et al. A novel herbal product based on Piper betle and Sphaeranthus indicus essential oils: Toxicity, repellent activity and impact on detoxifying enzymes GST and CYP450 of Aedes aegypti Liston (Diptera: Culicidae). J. Asia-Pac. Entomol. 2018, 21, 1466–1472. [CrossRef] 38. World Health Organization. Instruction for Determining the Susceptibility or Resistance of Mosquito Larvae to Insecticides; WHOVBC; 1981; Volume 81, pp. 1–6. Available online: https://apps.who.int/iris/handle/10665/69615 (accessed on 10 March 2021). 39. Abbott, W.S. A method of computing the effectiveness of an insecticide. J. Econ. Entomol. 1925, 18, 265–267. [CrossRef] Molecules 2021, 26, 3695 20 of 20

40. Agra-Neto, A.C.; Napoleão, T.H.; Pontual, E.V.; Santos, N.D.L.; Luz, L.A.; Oliveira, C.M.F.; Melo-Santos, M.A.V.; Coelho, L.C.B.B.; Navarro, D.M.A.F.; Paiva, P.M.G. Effect of Moringa oleifera lectins on survival and enzyme activities of Aedes aegypti larvae susceptible and resistant to organophosphate. Parasitol. Res. 2015, 113, 175–184. [CrossRef] 41. Thanigaivel, A.; Senthil-Nathan, S.; Vasantha-Srinivasan, P.; Edwin, E.; Ponsankar, A.; Selin-Rani, S.; Pradeepa, V.; Chellappandian, M.; Kalaivani, K.; Abdel-Megeed, A.; et al. Chemicals isolated from Justicia adhatoda Linn reduce fitness of the mosquito. Arch. Insect Biochem. Physiol. 2017, 94, e21384. [CrossRef][PubMed] 42. Hemingway, J.; Ranson, H. Insecticide resistance in insect vectors of human disease. Annu. Rev. Entomol. 2000, 45, 371–391. [CrossRef] 43. Silva, W.J.; Doria, G.A.A.; Maia, R.T.; Nunes, R.S.; Carvalho, G.A.; Blank, A.F.; Alves, P.B.; Marcal, R.M.; Cavalcanti, S.C.H. Effects of essential oils on Aedes aegypti larvae: Alternatives to environmentally safe insecticides. Bioresour. Technol. 2008, 99, 3251–3255. [CrossRef] 44. Polson, K.A.; Brogdon, W.G.; Rawlins, S.C.; Chadee, D.D. Characterization of insecticide resistance in Trinidadian strains of Aedes aegypti mosquitoes. Acta Trop. 2011, 117, 31–38. [CrossRef] 45. Piesik, D.; Wenda-Piesik, A.; Weaver, D.K.; Morrill, W.L. Influence of Fusarium crown rot disease on semio-chemical production by wheat plants. J. phytopathol. 2007, 155, 488–496. [CrossRef] 46. Piesik, D.; Delaney, K.J.; Wenda-Piesik, A.; Sendel, S.; Tabaka, P.; Buszewski, B. Meligethes aeneus pollen-feeding suppresses, and oviposition induces, Brassica napus volatiles: Beetle attraction/repellence to lilac aldehydes and veratrole. Chemoecology 2013, 23, 241–250. [CrossRef] 47. Piesik, D.; Kalka, I.; Wenda-Piesik, A.; Bocianowski, J. Apion miniatum Germ. herbivory on the mossy sorrel, Rumex confertus Willd.: Induced plant volatiles and weevil orientation responses. Pol. J. Environ. Stud. 2014, 23, 2149–2156. [CrossRef] 48. Vasantha-Srinivasan, P.; Karthi, S.; Ganesan, R.; Senthil-Nathan, S.; Krutmuang, P.; Chellappandian, M.; Radhakrishnan, N.; Ponsankar, A.; Karthick, K.; Nelofer, A. The efficacy of methanolic extract of Swietenia mahagoni Jacq. (Meliaceae) and a commercial insecticide against laboratory and field strains of Aedes aegypti (Linn.) and their impact on its predator Toxorhnchites splendens. Biocatal. Agri. Biotechnol. 2021, 31, 101915. [CrossRef] 49. Thanigaivel, A.; Chanthini, M.; Karthi, S.; Vasantha-Srinivasan, P.; Ponsakar, A.; Sivanesh, H.; Stanley-Raja, V.; Shyam-Sundar, N.; Narayanan, K.R.; Senthil-Nathan, S. Toxic effect of essential oil and its compounds isolated from Sphaeranthus amaranthoides Burm. f. against dengue mosquito vector Aedes aegypti Linn. Pest. Biochem. Physiol. 2019, 160, 163–170. [CrossRef][PubMed] 50. Chellappandian, M.; Vasantha-Srinivasan, P.; Senthil-Nathan, S.; Karthi, S.; Thanigaivel, A.; Ponsankar, A.; Kalaivani, K.; Hunter, W.B. Botanical essential oils and uses as mosquitocides and repellents against dengue. Environ. Inter. 2018, 113, 214–230. [CrossRef][PubMed] 51. Benelli, G. Plant-borne compounds and nanoparticles: Challenges for medicine, parasitology and entomology. Environ. Sci. Pollut. Res. 2018, 25, 10149–10150. [CrossRef] 52. Zhao, H.H.; He, J.T.; Liu, Z.X.; Huang, J.G. Cytotoxicity of chemical constituents from Torricellia tiliifolia DC. on Spodoptera litura (SL-1) cells. Pest. Biochem. Physiol. 2018, 144, 19–26. [CrossRef][PubMed] 53. Tewari, H.; Jyothi, K.N.; Kasana, V.K.; Prasad, A.R.; Prasuna, A.L. Insect attractant and oviposition enhancing activity of hexadecanoic acid ester derivatives for monitoring and trapping Caryedon serratus. J. Stored Prod. Res. 2015, 61, 32–38. [CrossRef] 54. Harborne, J.B. Introduction to Ecological Biochemistry; Elsevier: Berkely, CA, USA, 2003; ISBN 0-12-324685-7. 55. Edwin, E.; Vasantha-Srinivasan, P.; Senthil-Nathan, S.; Thanigaivel, A.; Ponsankar, A.; Pradeepa, V.; Selin-Rani, S.; Kalaivani, K.; Hunter, W.B.; Abdel-Megeed, A.; et al. Anti-dengue efficacy of bioactive andrographolide from Andrographis paniculata (Lamiales: Acanthaceae) against the primary dengue vector Aedes aegypti (Diptera: Culicidae). Acta Trop. 2016, 163, 167–178. [CrossRef] 56. Shaalan, E.A.S.; Canyon, D.V.; Younes, M.W.F.; Abdel-Wahab, H.; Mansour, A. A review of botanical phytochemicals with mosquitocidal potential. Environ. Int. 2005, 31, 1149–1166. [CrossRef] 57. Hugo, L.E.; Jeffery, J.A.L.; Trewin, B.J.; Wockner, L.F.; Yen, N.T.; Le, N.H.; Nghia, L.T.; Hine, E.; Ryan, P.A.; Kay, B.H. Adult survivorship of the dengue mosquito Aedes aegypti varies seasonally in Central Vietnam. PLoS Negl. Trop. Dis. 2014, 8, e2669. [CrossRef][PubMed] 58. Koodalingam, A.; Mullainadhan, P.; Arumugam, M. Effects of extract of soapnut Sapindus emarginatus on esterases and phosphatases of the vector mosquito, Aedes aegypti (Diptera: Culicidae). Acta Trop. 2011, 118, 27–36. [CrossRef][PubMed] 59. Zibaee, A.; Alborzi, Z.; Hoda, H. A review on digestive TAG-lipases of insects. Arch. Phytopathol. Plant. Prot. 2014, 47, 2187–2192. [CrossRef] 60. Ray, D.; Pautou, M.P.; Meyran, J.C. Histopathological effects of tannic acid on the midgut epithelium of some aquatic diptera larvae. J. Inver. Pathol. 1999, 73, 173–181. [CrossRef][PubMed]