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

Highlights

• Zanthoxylum chalybeum root extract and fraction (ZCFr.5) are active larvicidals.

• Appreciable larvicidal potency peaked within 24-h of exposure.

• Larvicidal effects were associated with neuromuscular toxicity.

• Larval growth retardations associated with perturbed ecdysteriodogenic regulations.

• Z. chalybeum is a potential candidate source for biolarvicide development.

Abstract

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.

Introduction

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 (George et al., 2014; Lengai et al., 2019; Muema et al., 2017a), 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 (Mithöfer and Boland, 2012). Conversely, detailed molecular toxicity studies to demystify the mechanisms of action of various mosquitocidal agents are few. In our recent study, tea proanthocyanidins interfered with mosquito larval growth and reproduction fitness by physiologically disrupting the juvenile hormone biosynthetic pathway (Muema et al., 2017b). 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 (Lee et al., 2015). Larvicidal activity of annonaceous acetogenin (squamocin) was associated with multitarget midgut gene effects in Aedes aegypti larvae (da Silva Costa et al., 2016). In another study, terpenes and terpenoids, polyphenols, alkaloids, and phenylpropanoid compounds were found to target the larval neurotransmission process, inducing sudden neuronal toxicity and death (Hematpoor et al., 2016).

The post-embryonic insect molting, developmental timing, and morphological remodeling events controlled by periodical ecdysteroid pulses are under neural regulatory and nutritional inputs (Koyama and Mirth, 2018; Niwa and Niwa, 2014). 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 20-hydroxyecdysone (20E) (Ou et al., 2016). 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 (Niwa and Niwa, 2016, Niwa and Niwa, 2014; Pankotai et al., 2010). 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 20E-inducible genes; E74, E75A, HR3, Broad, βFtz-F1 and other downstream proteins involved in metamorphosis and morphogenesis (Fletcher and Thummel, 1995). Biosynthesis and release of ecdysteroids from neurosecretory cells in metamorphosing juvenile insects could apparently be halted under a modulatory chemical environment, underscoring the anti-ecdysteroid-inducing effects of certain xenobiotic stressors.

In multicellular organisms, the 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 (Toutant, 1989). Besides this primary function, the implication of AChE in atypical noncholinergic roles (Soreq and Seidman, 2001) including the regulation of insect growth and development is characterized. Exemplified 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 (Dou et al., 2013; Mutunga et al., 2019; Pang et al., 2012). 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 Helicoverpa 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 (He et al., 2012; Hui et al., 2011; Kumar et al., 2009; Lu et al., 2012; Malik et al., 2016; Saini et al., 2018; Ye et al., 2017), implicating a significant role of AChE in insect growth physiology. Most likely, the mentioned findings 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 (Cohen, 1981; Jean-Jacques et al., 2005; Jenson et al., 2012; Spindler-Barth et al., 1988). Studies using D. melanogaster and Manduca sexta as models have further provided significant 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 (Novicki and Weeks, 1993; Truman, 1996; Veverytsa and Allan, 2013). 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 influences 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 (Tusting et al., 2013). This is largely due to the ever-growing concerns of insecticide resistance and other consequential behavioral effects associated with intensified 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 (Demissew et al., 2016; Gianotti et al., 2008; Imbahale and Mukabana, 2015; Trudel and Bomblies, 2011). In that regard, intensified laboratory screening of plant derivatives have reported a number of effective larvicides, among them derived from Zanthoxylum plant species (Kim and Ahn, 2017; Moussavi et al., 2015; Overgaard et al., 2014; Pavela and Govindarajan, 2017; Zhang et al., 2009). While several plant-derived AChE antagonists are potent insecticides, the knowledge of how they functionally affect insect larval development is underexplored. There are 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 larval-pupal transitions through transcriptional perturbation of ecdysteroidogenic CYP450 regulatory genes and effector transcription factors.