Background
The operational scale up of indoor residual spraying (IRS), long-lasting insecticide treated nets (LLINs) and artemisinin-based combined therapy (ACT) over the last decade progressively declined transmission rates of malaria to vulnerable children and pregnant women [
1]. An estimate of 69% fewer malaria cases has been reported in sub-Saharan Africa between 2001–2015 following the widespread deployment of the three key interventions [
1]. However, evolution of resistance to the active ingredients of these tools and little consideration of developing new complementary compounds targeting the ever changing behavioral traits of Afrotropical malaria vectors have greatly challenged efforts geared to bring malaria under control [
2‐
4]. Nevertheless, vector control forms the integral platform of integrated malaria management aimed to reduce malaria reproduction rate to less than 1 [
5,
6].
Larviciding, a less-practiced component of integrated vector management (IVM) and larval source management (LSM), appears a promising approach of suppressing both indoor and outdoor feeding mosquito populations [
7‐
9]. Impressive stories from Brazil and Egypt following its impactful malaria eradication motivates its revival [
10] with current operation in Kenya [
11], The Gambia [
8], Burkina Faso, Benin [
12] and Tanzania [
13]. Low immobility, confinement to shallow water bodies, susceptibility to chemical attacks and less chances of developing resistance favor this vector control approach [
14,
15]. Additionally, manipulation and/or modification of larval habitat bio-physicochemical parameters negatively influence vector competence of resultant mosquitoes suggesting a feasible target of mosquito control [
16,
17]. For millennia, mosquito control has considerably relied on chemicals that inevitably reduced environmental quality and facilitated emergence of resistant mosquito strains a phenomenon that has limited their continued reliance, prompting for alternative chemistries [
18].
One feasible way of averting the aforementioned drawbacks is prospecting for novel compounds with less environmental impacts and selectively toxic to target arthropods [
19,
20]. In addition to being a rich source of bioactive pharmacophores, plants produce allelochemicals with great potential of controlling crop pests and disease-transmitting vectors [
21,
22]. Among these are essential oils documented to repel nuisance human biting mosquitoes in addition to inducing toxicity to developing juveniles [
23‐
25]. Non-volatiles, for instance, Azadirachtin and its derivatives from neem tree and plant-based ecdysteroidal analogs potentially inhibit larval development and adult emergence terminating insect metamorphosis immaturely [
25,
26]. Additionally, these compounds induce growth disruption effects resulting into mortalities and non-viable females incapable of lineage progression [
27‐
29]. Taken together, plant-derived compounds are promising sources of effective insecticides with meager chances of resistance development afforded by multimodal targets [
30,
31].
Agerantum conyzoides L. is an
Asteraceae herbaceous weed that grows in many countries worldwide. Ethnopharmacological surveys of this polyherbal plant have documented biological activities such as analgesic, anti-inflammatory, purgative, febrifuge, anti-asthmatic, antibacterial, antifungal, antispasmodic, anti-diarrhoeic, headache relief, antihelmintic and nematicidal [
32,
33]. Phytochemically, the plant contain various bioactive compounds including alkaloids, coumarins, flavonoids, tannins and essential oils [
34,
35]. Of considerable interest, the plant extracts have shown detrimental effects on survival, development and adult emergence of mosquitoes such as
Aedes albopictus [
36],
Culex quinquefasciatus [
37],
Aedes aegypti and
Anopheles stephensi [
38] which has been attributed to possibility of compounds with anti-juvenile hormone activity. However, effectiveness of the plant extracts to control the principal Afrotropical malaria vectors
An. gambiae sensu stricto and
An. arabiensis remain obscure. Therefore, we sought to evaluate the larvicidal and developmental disruption effects of
A. conyzoides against
An. gambiae s.s and
An. arabiensis. Our findings demonstrate for the first time to the best of our knowledge that, the methanolic leaf extract of
A. conyzoides had considerable larvicidal and development inhibition activities in a dose-dependent manner against Afrotropical malaria vectors. In addition, we identified alkaloids, aglycone flavonoids, triterpenoids, tannins and coumarins as phytochemicals that were associated with the observed bioactivities.
Discussion
In search for better insecticides to replace or complement the synthetic insecticides and alleviate resistance pressure on malaria vectors, scientists have turned interests into nature for alternative controls. Many plants have been reported around the globe to have bioactivity against mosquitoes and their multiple targets of actions against mosquitoes assure effectiveness as alternative bio-insecticides. The toxic efficacy of these botanicals against various mosquito vectors vary depending on different factors such as the part of the plant used, method of extraction adopted, solvent used, geographical locality the plant was obtained, the concentration of the extract used and photosensitivity of some plant compounds [
43].
In the current study, we challenged late third (L3) instar larvae of
An. gambiae s.s and
An. arabiensis with crude extract of
A. conyzoides to evaluate their responses. Our data demonstrate that the extract had detrimental effects on both survival and development of
An. gambiae s.s and
An. arabiensis in a dose-dependent response manner. High dosages of 250 ppm and 500 ppm evoked acute toxicity to the developing larvae while the sublethal doses of 50 and 100 ppm induced developmental disruptions as shown by Fig.
2(
b-
e). The toxic effect of the plant extract could be attributed to its bioactive phytochemical constituents (Table
1). The LC
50 values of the plant extract have shown significant potential of controlling
An. gambiae s.s and
An. arabiensis. It has been previously reported that the plant extract had larvicidal activity against
Ae. albopictus [
36],
C. quinquefasciatus [
37],
Ae. aegypti and
An. stephensi [
38] which is similar to our data though slight variation was noted. This could be attributed to the solvents used for extraction, susceptibility differences of mosquito vectors used and geographical differences of the plant. Methanolic extract of
A. conyzoides leaves was used in the present study and found effective against
An. gambiae s.s and
An. arabiensis larvae.
Moreover, phytochemicals extracted from many plant species have been reported to show growth inhibiting effects on the various developmental stages of different mosquito species [
27‐
29,
40,
43]. Various pre-emergent effects such as prolongation of larval instar and pupae durations, inhibition of larval and pupal molting, morphological abnormalities and mortality may occur especially during molting and melanization processes. Developmental disruption effects induced by the plant extracts can be associated with disturbed hormonal balance or interference in chitin synthesis during the molting process. Our data recorded morphological effects on
An. gambiae s.s and
An. arabiensis where the immature stages failed to transform into a normal adult leading to eventual death (Fig.
2c and
e). Similar results on morphological abnormalities were reported on
An. stephensi, Ae. aegypti and
C. quin
quefasciatus exposed to
A. conyzoides extract. The phenomenon has been reported by Okunade, [
35] as a result of perturbation of hormonal homeostasis by precocene-3,4-epoxide, a metabolite generated by cytochrome P450s in the insect body [
44]. The metabolite may either antagonize or agonize the biosynthesis and subsequent release of juvenile hormone, the regulator of insect metamorphosis.
Studies carried out by Nyamoita et al., [
41], Nathan et al., [
45] and Nathan et al., [
46] reported that in addition to their lethality, the secondary metabolites of the botanicals used resulted in protracted larval phase, disrupted growth and malformation of the exoskeleton. Although there was no elongation of gut as observed in [
29] and [
47], incomplete melanization process was observed in larvae and some pupae examined under light microscopy (Fig.
2). Our data corroborated with that obtained by Ndung’u et al., [
28] where limonoids from methanolic extracts of the root of
Turraea mombassana Hiern (
Meliaceae) resulted in larval and pupal morphological deformities in
An. gambiae s.s due to incomplete melanization. Similarly, exposure of
Anopheles stephensi to extracts of
Melia azedarch resulted in similar observations [
45]. Also, compounds from
Azadirachta indica and
Melia volkensii (
Meliceae) extracts induced growth disruption effects to mosquito larvae besides feeding deterrence and toxicity [
48]. Studies performed by Govindachari et al., [
49], Martinez and Van Emden, [
50] and Nathan et al., [
51] confirmed the above effects of Azadirachtin on insects. Elsewhere, dichloromethane extract of
Hyptis brevis (
Lamiaceae) displayed strong growth inhibition on
Spodoptera littoralis larvae by arresting metamorphosis [
52]. The same phenomenon has been reported by Cespedes et al., [
53].
Several plant species produce a myriad of bioactive chemicals as part of defenses against herbivory attacks majorly classified as volatile compounds (essential oils) and non-volatiles. The non-volatiles include the alkaloids, flavonoids, terpenoids, glucosinolates, cyanogenic glycosides, phenolic acids among others [
31]. Majority of these non-volatiles particularly phytoecdysteriods, phytojuvenoids and anti-juvenile hormones act as insect growth regulators (IGRs) reducing survival rates and development of insects upon ingestion [
54]. Previous reports indicate various insecticidal compounds isolated and identified from
A. conyzoides extracts such as steroids, flavonoids, coumarins, pyrrolizidine alkaloids, triterpenoids, and chromenes [
36,
55‐
57]. In this regard, phytochemical analysis revealed presence of main compounds such as alkaloids, terpenoids (e.g. precocene I and precocene II) [
56], flavones (e.g. ageconyflavones A, B and C) [
57], coumarins and tannins which equally agree with these reports. All these compounds may act in a concerted manner to nonspecifically induce toxicity to insects. More specifically, precocenes (terpenoids) have been reported to be anti-juvenile hormone, accelerating the development of insects and inducing dwarfness associated with low survival rates [
43]. Phytochemicals that agonize or antagonize the effects of insect development hormones have been reported to be good bio-pesticides [
53]. These compounds disrupt the normal metabolism of the insect hormones during the development of the juveniles leading to failure of adult emergence [
55].
Two important insect developmental hormones that interplay are 20-hydroxyecdysone (20-E) and juvenile hormone (JH) [
58]. It is the balance in levels of these two hormones that define the outcome of each developmental transition [
59]. Ligand-binding to the insect juvenile receptor complex disrupt insect endocrine signaling and regulation causing abnormal development and lethality [
21]. The accumulation of these plant compounds above threshold levels disrupt the insects’ developmental progression culminating into premature death or failure to emerge as a normal adult [
60]. The active compounds from
A. conyzoides extract induced toxicity and growth inhibition effects to developing mosquito larvae and could potentially be isolated for formulating effective mosquito control agents. Further, identification of molecular targets, ligand docking and simulation assays accompanied by field applications could be pursued for improved mosquito control.