Neurotoxic Zanthoxylum chalybeum root constituents invoke mosquito larval growth retardation through ecdysteroidogenic CYP450s transcriptional perturbations

Intracellular effects exerted by phytochemicals eliciting insect growth-retarding responses during vector control intervention remain largely underexplored. We studied the effects of Zanthoxylum chalybeum Engl. (Rutaceae) (ZCE) root derivatives against malaria (Anopheles gambiae) and arbovirus vector (Aedes aegypti) larvae to decipher possible molecular targets. We report dose-dependent biphasic effects on larval response, with transient exposure to ZCE and its bioactive fraction (ZCFr.5) inhibiting acetylcholinesterase (AChE) activity, inducing larval lethality and growth retardation at sublethal doses. Half-maximal lethal concentrations (LC50) for ZCE against An. gambiae and Ae. aegypti larvae after 24-h exposure were 9.00 ppm and 12.26 ppm, respectively. The active fraction ZCFr.5 exerted LC50 of 1.58 ppm and 3.21 ppm for An. gambiae and Ae. aegypti larvae, respectively. Inhibition of AChE was potentially linked to larval toxicity afforded by 2-tridecanone, palmitic acid (hexadecanoic acid), linoleic acid ((Z,Z)-9,12-octadecadienoic acid), sesamin, β-caryophyllene among other compounds identified in the bioactive fraction. In addition, the phenotypic larval retardation induced by ZCE root constituents was exerted through transcriptional modulation of ecdysteroidogenic CYP450 genes. Collectively, these findings provide an explorative avenue for developing potential mosquito control agents from Z. chalybeum root constituents.


Background
Renewed interests in search of environmentally friendly alternative insecticides have lately led to the gradual substitution of chemical-based insecticides in global markets.However, as much as the public demand for biocides over their synthetic counterparts continues to increase and considerable appreciation of plant-derived bioactive compounds in pest control [1][2][3], the mechanistic effects mediated by these insecticidal agents at a molecular level as well as their target proteins remain largely elusive.Only studies focusing on phytophagous insects highlight structurally-based concerted cellular interference of vital physiological processes by growth-reducing plant compounds [4].Conversely, detailed molecular toxicity studies to demystify the mechanisms of action of various mosquitocidal agents are few.In a recent study, tea proanthocyanidins interfered with mosquito larval growth and reproduction tness by physiologically disrupting the juvenile hormone biosynthetic pathway [5].Using a yeast two-hybrid reporter system, Lee et al. showed that juvenile hormone mimics derived from Lindera erythrocarpa (Lauraceae) and Solidago serotina (Asteraceae) effectively antagonized mosquito juvenile hormone receptor, methoprene-tolerant (Met); killing mosquito larvae and retarding follicle development in the ovaries [6].Larvicidal activity of annonaceous acetogenin (squamocin) was associated with multitarget midgut gene effects in Aedes aegypti larvae [7].In other studies, terpenes and terpenoids, polyphenols, alkaloids, and prenylpropanoid compounds were found to target the larval neurotransmission process, inducing sudden neuronal toxicity and death [8].
The post-embryonic insect molting, developmental timing, and morphological remodelling events controlled by periodical ecdysteroid pulses are under neural regulatory and nutritional inputs [9,10].These metamorphosis behavioral changes are orchestrated by biosynthesis and release of growth hormones upon decoding sensory environmental cues from insulin/insulin-like signaling and target of rapamycin (IIS/TOR) pathway.Ecdysteroids synthesized through sequential enzymatic oxidation of dietary cholesterol in the prothoracic glands are released into the hemolymph where they are activated into 20hydroxyecdysone (20E) [11].Major advancements have been made to understand ecdysone functions in insect physiology.For instance, through comprehensive molecular analyses in model insects (e.g.Drosophila melanogaster) and other related species, it is evident that ecdysteroidogenic pathway is transcriptionally regulated by Halloween genes that encode CYP450 enzymes; Neverland, non-molting glossy (nmg), CYP307A1/spook, CYP307A2/spookier, CYP306A1/phantom, CYP302A1/disembodied, CYP315A1/shadow and CYP314A1/shade, and a number of nuclear transcription factors [9,12,13].
Following ecdysone activation to 20E within the peripheral tissues by shade, it binds to the ecdysone receptor (EcR) forming a heterodimeric complex with Ultraspiracle (USP).The resultant trimeric complex 20E/EcR/USP binds ecdysone response elements (EcREs) activating transcriptional expression of 20Einducible genes; E74, E75A, HR3, Broad, βFtz-F1 and other downstream proteins involved in metamorphosis and morphogenesis [14].Biosynthesis and release of ecdysteroids from neurosecretory cells in metamorphosing juvenile insects could apparently be halted under modulatory chemical environment, underscoring the anti-ecdysteroid-inducing effects of certain xenobiotic stressors.
In multicellular organisms, neuronal coordination network of nerve circuits is regulated by a serine hydrolase acetylcholinesterase (AChE; E.C 3.1.1.7),which rapidly terminates synaptic signals by hydrolyzing the neurotransmitter, acetylcholine (ACh), into inactive derivatives [15].Besides this primary function, the implication of AChE in atypical noncholinergic roles [16] including the regulation of insect growth and development is characterized.Exempli ed by this important functional role in cellular development and survival, AChE continues to be explored in biotechnological and chemical-based control of crop pests and insect vectors [17][18][19].As a biochemical target of organophosphates and carbamates, through phosphorylation or carbamoylation of a conserved serine residue (Ser200), AChE delineates a classical pest control chokepoint, despite its vulnerability to point mutations associated with decreased insecticide sensitivity.Various RNA interference (RNAi) studies targeting Ace of diverse insects including Helioverpa armigera (Noctuidae), Chilo suppresalis (Crambidae), Plutella xylostella (Plutellidae), Tribolium castenum (Tenebrionidae), Bemisia tabaci (Aleyrodidae), fall armyworm Spodoptera frugiperda (Noctuidae), and Bombyx mori (Bombycidae) have reported adverse effects on larval growth and survival, delayed pupation and adult emergence, reduced motor control and female reproductive viability [20][21][22][23][24][25][26], implicating a signi cant role of AChE in insect growth physiology.Most likely, the mentioned ndings could postulate a direct involvement of AChE in the regulation of insect hormonal activities.In fact, previous studies in metamorphosing brain of Tenebrio molitor, and cell lines of Chironomus tentans, Ae. aegypti, S. frugiperda, and D. melanogaster have demonstrated relationship between AChE activity and its regulation by ecdysteroid hormone levels and vice versa [27][28][29][30].Studies using D. melanogaster and Manduca sexta as models have further provided signi cant insights into how insect larval-pupal transition behavior through ecdysis and tissue differentiation is driven by motoneuron networks controlled by ecdysteroid hormone and EcR activity [31][32][33].Based on these interactions and interdependent co-regulatory mechanisms during growth, transcriptional dysregulation of either AChE or ecdysteroid genes by chemical intervention, RNAi and/or genetic ablation could adversely affect hormone-regulated insect growth by inducing toxicity and retardation phenotypes of inter-instar and larval-pupal transitions.In spite of this knowledge, the underlying molecular mechanisms responsible for the growth retardation effects exerted by inhibitory in uences on insect AChE remain unclear.
Underscoring the public health importance of mosquitoes in the transmission of life-threatening diseases, particularly malaria and arboviral infections, vector control aiming at area-wide suppression of adult populations are reconsidering targeting the juvenile larval stages [34].This is largely due to the evergrowing concerns of insecticide resistance and other consequential behavioral effects associated with intensi ed adult vector interventions.Due to cost and environmental concerns associated with synthetic mosquito larvicides, community-based vector control interventions have shown great interest in the application of naturally occurring botanicals for mosquito control around human dwellings [35][36][37][38].In that regard, intensi ed laboratory screening of plant derivatives have reported a number of effective larvicides, among them derived from Zanthoxylum plant species [39][40][41][42][43].While several plant-derived AChE antagonists are potent insecticides, the knowledge of how they functionally affect insect larval development is underexplored.No existing studies of Z. chalybeum (knobwood) bioactivity against mosquitoes and therefore the current study reports the effects of its root chemical constituents on developing An.gambiae and Ae.aegypti juveniles.We demonstrate that dysregulation of mosquito larval nervous coordination upon exposure to ZCE root extract and its bioactive fraction (ZCFr.5)retards larvalpupal transitions through transcriptional perturbation of ecdysteroidogenic CYP450 regulatory genes and effector transcription factors.

Perturbed acetylcholinesterase (AChE) activity linked to larval lethality
Transient exposure of mosquito larvae to ZCE and its bioactive fraction for <5 h exhibited no obvious morphological aberrations but drastically reduced larval swimming behavior, induced muscle paralysis and immobilization leading to death.These observations are closely linked to neuromuscular toxicity effects reported by Tomé et al. [45].To ascertain whether the observed acute toxicity on mosquito larvae was as a result of suppressive effects of ZCE and its bioactive fraction on AChE, we performed the biochemical colorimetric Ellman's enzymatic assay and RT-qPCR gene expression analysis.Due to the low amount of ZCFr.5, we only assayed ZCE for larval AChE activity inhibition.ZCE exhibited dosedependent AChE inhibitory effect in mosquito larvae (Fig. 3B), attaining IC 50 136.0µg/mL (95% CI 111.3-547.3)and 277.5 µg/mL (95% CI 47.4-293.4)for Ae.aegypti and An.gambiae, respectively.The AChE inhibition achieved by ZCE was preferably 1.4-2.73fold higher in Ae. aegypti (44.19-81%) than An.gambiae (16.11-59.59%).Irrespective of ZCE dosage, the toxicity susceptibility of AChE signi cantly varied between Ae. aegypti and An.gambiae (Welch two-sample t-test, p < 0.001), highlighting a speciesspeci c response.Relative to propoxur, a speci c carbamate-based irreversible AChE inhibitor, the mean ZCE activity varied signi cantly between the species (An.gambiae; t-test, t= -8.4174, df = 4, p = 0.001055; Ae. aegypti; t=-2.9963,df = 4, p = 0.03648).Agreeably, the larval treatment with ZCFr.5 was associated with 0.0764 and 0.017-fold Ace transcriptional changes in Ae. aegypti and An.gambiae, respectively (Fig. 3A), but these expressions were not signi cantly different from each other (t-test = 2.4406, df = 4, p = 0.066).Meaningful and signi cant (p > 0.05) negative correlation coe cients between the mean LC 50 and AChE inhibition values of ZCE were obtained, but not varied between the two mosquito species (Supplementary Table S3).

ZCFr.5 modulates mosquito larval ecdysteroid biosynthetic and transcriptional regulatory genes
Lignan-based prenylpropanoid and 2-tridecanone compounds are believed to inhibit ecdysteroids and their biosynthetic-dependent cytochrome P450 activities [44] , [46], interfering with insect growth.As expected, the sesamin-rich ZCE root fraction altered the transcriptional expression of ecdysteroid biosynthetic Halloween and regulatory genes (Fig. 4).Notably, the larvicide treatment signi cantly downregulated the expression of larval 'black box' cytochrome P450 spook (Cyp307a1), βFtz-F1, disembodied (Cyp302a1), and ecdysteroid receptor (EcR) genes.We further noted that, with an exception of Cyp302a1 in Ae. aegypti, the gene expressions of Cyp314a1, Cyp306a1, and dHAT were not signi cant from each other (t-test, p > 0.05; Fig. 4A-B), but remarkably modulated relative to the controls.Intriguingly, while juvenile hormone (JH) expression levels decrease during the last larval instar to allow 20E-induced transformation into pupal stages, we established that the expression of JH biosynthetic rate-limiting enzyme, JH acid O-methyltransferase (JHAMT), in ZCFr.5-treated larvae remained relatively higher compared to that of controls (Fig. 4C-D).This nding could suggest low levels of circulating hemolymph 20E to suppress JH and further underscoring the observed larval growth retardations and precocious pupations.These ndings con rmed that indeed the larval treatment that targeted AChE activity also perturbed ecdysteroidogenic pathway associated genes delaying larval-pupal transitions and inducing retardant phenotypes.

Discussion
The impact of plant-derived insecticidal compounds on insect tissues elicits pleiotropic growth effects for which a number of insecticides speci cally targeting neuromuscular activity have overlooked at the molecular level.Herein we investigated the bioactivity of Z. chalybeum root constituents against An.gambiae and Ae.aegypti larvae with a keen focus on elucidating the possible downstream toxicity effects.Our ndings demonstrate that the fast-acting ZCE constituents elicit larvicidal activities in a dosedependent fashion by targeting the neuromuscular actions regulated by AChE and further invoking larval retardation at sublethal doses through disrupted ecdysteroidogenesis.While similar observations have previously been reported using RNAi assays in other insects [20][21][22][23][24][25][26], we report for the rst time the effect of ZCE root constituents to dysregulate neural-linked ecdysteroidogenesis in mosquito larvae, inducing retardants that fail or exhibit delayed pupation.
Zanthoxylum plant species have been reported to possess insecticidal, repellent and larvicidal activities exerted by compounds mostly concentrated within the hexane-soluble portions [40][41][42][47][48][49][50][51][52][53][54].Similarly, Z. chalybeum larvicidal activity reported in this study was more pronounced in hexane-soluble fraction suggesting that the lipophilicity parameter was key in mediating the observed acute toxicity effects.Compared to their reported bioactivities exerted at relatively higher LC 50 doses, ZCE and its active fraction (ZCFr.5)provoked larval toxicities at low LC 50 doses (1.58-12.26ppm) against An.gambiae and Ae.aegypti, possibly due to species-speci c distribution of the secondary metabolites.While different compounds from the stem barks, seeds, and leaves were reported to differentially interfere with mosquito survival, we detected for the rst time the presence and abundance of sesamin, 2-tridecanone, (Z,Z)-9,12octadecadienoic acid (linoleic acid), hexadecanoic acid (palmitic acid), and β-caryophyllene in Z. chalybeum root extract that have previously been reported to exhibit growth reducing effects in insects.For instance, the phenylpropanoid lignan, sesamin, has been reported in Zanthoxylum stem barks to elicit moderate larvicidal activity (LC 50 >150 μg/mL) [41] but relatively with similar bioactivity to ZCE against Ae.aegypti at 14.28 mg/L [42].Further, an aliphatic methyl ketone (2-tridecanone) exhibited repellent effects against both Amblyomma americanum (Ixodidae) and Dermacentor variabilis (Ixodidae) ticks, and An.gambiae [55,56].Strikingly, this carbonyl compound (2-tridecanone) retards insect growth by down-regulating Halloween CYP450 genes [46], a feature corroborated in the current study.In addition, the ubiquitous caryophyllene and fatty acids, such as linoleic acid and palmitic acid, have previously exhibited larval toxicity mediated through AChE inhibition [57].Ingestion and/or cuticular penetration of this blend of compounds presumably interacted with larval tissues, thus incapacitating mosquito larvae during 24-h of treatment.Considering the larvicidal activity of ZCE compounds appeared to be associated with muscle paralysis manifesting in uncoordinated motility, inhibition against AChE appeared to be as a result of the lipophilic nature of these compounds that transverse cell membranes to their target site, possibly increasing ACh levels and overstimulating neuromuscular action akin organophosphates.Our ndings agree with those of Calderón et al. [58], that reported acute toxicity of Mexican Gutierrezia microcephala (Asteraceae) on S. frugiperda larvae through AChE inhibition, in addition to reduced growth and delayed pupation.
Residual growth effects are often associated with insecticides but have largely been underexplored for Zanthoxylum extracts and other insecticidal compounds with AChE inhibition properties.Lack of any induced morphological aberrations associated with insect growth regulators at sublethal concentrations motivated our hypothesis that the ZCE/ZCFr.5treatment could have profound effects on the ecdysteroid pathway inducing larval retardation.The post-embryonic insect metamorphosis, associated with various morphological and behavioral changes particularly during the late third instar of larval-pupal transitions, is accompanied by ~100-fold neurogenesis and neural remodeling events for the maturation of adultspeci c neuronal lineages [25,59,60].It has been demonstrated using various insect models that this phenomenon is coincidentally linked to ecdysteroid hormone activity regulated by stage-speci c expression of biosynthetic Halloween CYP450 genes [61,62].Since decreased mRNA levels of neuronal AChE gene could positively correlate with the observed changes in 20E biosynthetic enzymes and the fact that ecdysteroid receptor isoforms (EcR-A and EcR-B1) have been previously found in insect neuronal cells [63,64], toxicity perturbation of these neurosecretory cells by ZCE constituents could have resulted in reduced biosynthesis, release and signaling, insu cient to in uence ecdysone-mediated larval growth and pupation.These ndings, akin to nicotinic acetylcholine receptor (nAChR) neonicotinoid effects, depicted that reduced expression of AChE in uenced larval-pupal transition by perturbing ecdysteroidogenesis pathway.It is possible to postulate that tissue injuries on the developing larvae imparted by the larvicide treatment could have severely constrained ecdysteroid biosynthesis, hemolymph titres and signaling, arresting growth to allow regeneration as previously observed in Drosophila [65].Furthermore, it could be possible that these cholinergic injuries had negative effects on larval prothoracic gland (PG) innervations among them the 5-hydroxytryptamine (5HT)-producing serotonergic neurons [66] modulating inositol 1,4,5-triphosphate/calcium (InsP 3 /Ca 2+ ) signaling pathway and reducing downstream ecdysone production that deprived pupal transformation.Nevertheless, the perturbed expression of steroidogenic CYP450s and especially spook (Cyp307a1) in the hypothetical ratelimiting "black box" clearly suggested either a partial or an absolute inhibition of 7-dehydrocholesterol (7dC) conversion to 5b-ketodiol resulting into diminished activation of EcR and effector signaling transcription factors E75A and βFtz-F1.In fact the genetic loss and/or RNAi-mediated gene silencing of spook results in slowed development and molting interference that impairs growth [67,68] due to low ecdysteroid titres as the prothoracicotropic hormone (PTTH) target SPOOK translation and phosphorylation in the "black box" for ecdysteroidogenesis.Additionally, the low hemolymph 20E titers in treated larvae were accompanied by slightly high JH levels (Fig. 4C-D) that agreeably sustained the phenotypic expression of larval retardants.Disruption of enzymes and genes associated with ecdysteroid biosynthetic and signaling pathway by plant-derived compounds has been investigated extensively [69].Therefore, treatment with lignans and neolignans disrupts insect development by destabilizing steroid hormones [46].Sesamin inhibits CYP3A4 by antagonizing steroid nuclear receptor [70], and its presence and abundance in ZCE (Fig. 2B) could have had signi cant inhibitory effects on ecdysteroid CYP450s and 20E response effectors due to its methylene dioxy phenyl (piperonyl) structure.Moreover, the previously observed retardant effects caused by plant-derived 2-tridecanone on H. armigera as a result of down-regulation of 20E biosynthetic genes, particularly Cyp307a1 could also be involved in this study [46].Previous studies further demonstrated inhibitory regulation of CYP450s by saturated and unsaturated fatty acids [71,72], and the exogenous presence of linoleic acid and palmitic acid alongside other compounds in the hexane-soluble fraction could interfere with hormonal metabolism in developing mosquito larvae.
Our ndings demonstrate that Z. chalybeum root constituents are acutely toxic to mosquito larvae.This toxicity being mediated through disruption of neuromuscular coordination and further dysregulating ecdysteroidogenic-associated CYP450s at sublethal doses deserves further toxicological investigations aimed at designing potential mosquito control agents.Additionally, with the promising larvicidal effects herein demonstrated, we propose a comprehensive environmental safety assessment of ZCE constituents on non-target organisms prior to the commercial formulation, recommendation, and application in disease vector control programmes.

Mosquito larvae
Experimental bioassays were performed with third instar mosquito larvae (L3) of insecticide-susceptible An. gambiae (Mbita strain) and Ae.aegypti obtained from a mosquito culture maintained at the International Centre of Insect Physiology and Ecology (icipe), Nairobi.The insects were maintained as previously described [5] at a density of 200 larvae per 1 L of dechlorinated water under controlled insectarium conditions: temperature 30 ± 2˚C, 60-80% relative humidity (RH), and 12-h light/dark cycles.
Throughout the experimental period, the larvae were fed on Tetramin sh meal (Tetra ® , Melle, Germany).

Mosquito bioassays
Larvicidal bioassays were set up as outlined in WHO guidelines [73] under controlled insectarium rearing conditions.Preliminary assay with ZCE at 25-500 ppm revealed 100% mosquito larval mortality within < 3 h, prompting lower dosages.Late third instar (L3) mosquito larvae (n = 25) were separately introduced into ve beakers containing 100 mL of test solutions at different concentrations (5-25 ppm and 1-10 ppm of ZCE and its solvent fractions, respectively).These test solutions were separately formulated in 0.01% (v/v) ethanol and dispensed into respective beakers for the assays.A parallel experimental setup containing essential oil of neem rich in triterpenoid azadirachtin which is a potent larvicide was used as positive control while the negative control setup constituted mosquito larvae in water with 0.01% (v/v) ethanol.Larval mortalities in each experimental setup were recorded in each experimental replicate for 24-h post-treatment.To assess the effects of ZCE and its active fraction (ZCFr.5) on larval development, 20 newly molted (synchronous) early L3 instars (4-5 days post egg hatching) added into three beakers were treated with sublethal dosages (ZCE: 8 ppm, 10 ppm; ZCFr.5: 1.0 ppm, 3 ppm for An.gambiae, and Ae.aegypti, respectively).Time taken for the mosquito larvae to reach pupal stages was recorded, and percentage pupation for each treatment replicate calculated (n = 3).Parallel setups constituting distilled water and 0.01% ethanol served as references.

Phytochemical analysis
Activity-based screening revealed a highly toxic n-hexane-soluble fraction (ZCFr.5, eluted in 3:2 hexane/EtOAc gradient) against mosquito larvae (Table 1, Supplementary Table S1).To identify the chemical entities eliciting the toxicity effects, a GC/MS-TQ8040 system (Shidmazu Corp., Kyoto, Japan) equipped with a mass selective detector and tted with a silica capillary column (SH-Rxi-5HT; 30 m × 0.25 mm internal diameter × 0.25 µm lm thickness) was used for analysis.Ultra-pure grade helium at a ow rate of 1.44 mL/min was used as the carrier gas.The injector temperature was maintained at 280°C, and a sample volume of 1 μL injected.The initial oven temperature was programmed at 80°C for 5 min then ramped at rates of 15°C/min to 280°C (held for 8 min).For data acquisition, the GC-MS was operated in the full scan mode with Shimadzu's GC-MS Smart Pesticide Database (version 1) used as a foundation of analysis and identi cation of analytes.
Total RNA isolation, cDNA synthesis, and RT-qPCR analyses Total RNA was isolated from pools of ve mosquito larvae obtained from ZCFr.5-treated and non-treated control groups using TRIzol reagent (Invitrogen TM , Carlsbad, CA 92008, USA) according to manufacturer's guidelines.To assess the purity and concentrations of the isolated RNA, a Nanodrop 2000 UV Vis spectrophotometer (Thermo Scienti c, USA) was used.cDNA rst strand was synthesized from DNase I treated total RNA (500 ng) using oligo d(T) 20 primers of SuperScript ® IV First-Strand Synthesis System (Invitrogen Life Technologies, Lithuania) as per manufacturer's instructions.Gene expression analyses were performed using Agilent Stratagene Mx3005P real time qPCR system (Agilent technologies, USA) with SYBR Green ® /ROX qPCR master mix (Thermo Scienti c, USA).Primers used for RT-qPCR analyses are listed in Supplementary Table S4.Gene-speci c ampli cation reaction comprised of 6.25 µL SYBR Green/ROX, 0.5 picomoles of forward and reverse primers, 1 µL of 1:5 diluted cDNA, and topped up to 12.5 µL with nuclease-free water.The PCR cycling conditions were programmed as follows; initial step 95°C for 10 min, 40 cycles of 95°C for 30 sec, 60°C for 30 sec, and 72°C for 30 sec followed by a single cycle of dissociation melt curve set at 95°C for 30 sec, 55°C for 1 min and 95°C for 30 sec.Normalization of the gene expression levels was performed using species-speci c ribosomal protein S7.All the experiments were performed three times.The relative gene expression levels were analyzed using 2 -ΔΔCt method [74].

Biochemical acetylcholinesterase (AChE) enzymatic assay
Ellman's protocol [75] with minor modi cations was followed to measure the AChE inhibitory activities of ZCE.A pool of 10 larvae from each species was separately washed three times in phosphate buffer (1× PBS, pH 8.0) before homogenization in 1 mL PBS.The homogenates were centrifuged at 10,625 ×g for 20 min at 4°C and supernatants separately transferred into sterile clean 1.5-mL Eppendorf tubes to give the enzyme extract source for assaying.Into 150 µL of ice-cold PBS held in 96-wells plate, 10 µL of the enzyme extract was added before addition 20 µL of test extract preparations.The test extracts were dissolved in DMSO to a nal concentration of 0.1% DMSO (w/v) in PBS.The reactants were incubated at room temperature for 10 min followed by the addition of 20 µL 0.4 µM acetylthioiodide (ATChI, BDH Chemicals Ltd, England) and 0.3 µM 5,5´-DTNB (Ellman's reagent, Sigma Aldrich, USA).This was followed by a 30-min incubation at room temperature and absorbance read out using an Eppendorf BioSpectrometer ® Fluorescence at 412 nm.A set up comprising of enzyme extract, DTNB, and the buffer was used as blank while a positive reaction constituted the enzyme extract, ATChI, and DTNB.A commercial chemical Propoxur (PESTANAL ® analytical standard, Sigma Aldrich, USA) was included as the reference inhibitor.The assay was performed in four replicates and the % AChE inhibition expressed as a mean value of the replicates as given in equation ( 1

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Figure 2 Analysis
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Figure 2 Analysis
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Figure 2 Analysis
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Figure 3 Effects
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Table 1 .
Toxicity of ZCE extracts on An. gambiae and Ae.aegypti larvae 24-h post exposure.

Table 2 .
Developmental duration of mosquito larvae treated with sublethal doses of Z. chalybeum extracts