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Research Article

The Use of Piper amalago var. amalago as an Eco-Friendly Insecticide Synergist in the Management of Aedes Aegypti

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DOI:

10.3791/68723

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October 24th, 2025

 ,  ,  ,  ,  ,  , 
1Caribbean Centre for Research in Biosciences, Natural Products Institute, University of the West Indies, 2Life Sciences, University of the West Indies, 3The Mosquito Control Research Unit, Department of Life Sciences, University of the West Indies, 4Cell Therapy Institute, Paracelsus Medical University, 5Vector Group, Liverpool School of Tropical Medicine

In This Article

Summary

Ethanolic extracts of Piper amalago var. amalago demonstrate synergistic effects when used with deltamethrin against Ae. aegypti. The demonstrated synergistic activity is the result of its active ingredient, piperine, which is an inhibitor of CYPs known to be upregulated in mosquitoes that show resistance to insecticides.

Abstract

Resistance to insecticides challenges the management of Ae. aegypti, the main vector of dengue, chikungunya, and Zika viruses in the Caribbean and Latin America. The development of novel vector management strategies is critical to reducing the transmission of its diseases. Piper spp., traditionally used for their culinary properties, have demonstrated insecticidal activity. The study explores extracts of Piper amalago var. amalago, a variety native to the Caribbean and Latin America, as insecticide synergists, compounds that are moderately toxic to insects but enhance toxicity when used in combination with synthetic insecticides.

The toxicity of deltamethrin on field-collected Ae. aegypti using the CDC bottle bioassay was assessed. After confirming resistance, ethanol or hexane extracts were prepared from the aerial parts of Piper amalago var. amalago. The extracts were applied to subsequent lab-reared Ae. aegypti generations. Of the fractions prepared, the stem-ethanol extract was the most potent, with LD50 values of 0.007 mg/mg (plant extract/female Ae. aegypti body weight). The stem-ethanol extract demonstrated a synergistic effect with deltamethrin using a modified CDC bottle bioassay. Mortality increased from 22% ± 0.42 % with deltamethrin alone, at the dose of 10 µg active ingredient in 30 min to 96% ± 0.17 % with the application of stem-ethanol extract (LD25 = 2.2 μg/mg) followed by deltamethrin at the stated dose and time. GC-MS studies showed piperine to be the most abundant compound in the prepared extracts. The study demonstrates the use of Piper amalago var. amalago as an effective synergist against Ae. aegypti.

Introduction

Aedes aegypti is an important anthropophilic mosquito vector in Latin America and the Caribbean (LAC)1. Several factors that include access to adequate clean water supply, quality of public health services, changes in weather patterns, human movement, and trade have facilitated not only the distribution of this opportunistic vector, but also the distribution of the pathogens transmitted by the species2,3 across the region1,4. The vector indiscriminately oviposits in moisture-collecting receptacles in and around human dwellings, and their eggs are drought-resistant, allowing survival in harsh environmental conditions5. Ae. aegypti mosquitoes are temperature-adapted vectors with egg-hatching rates, blood feeding, and viral transmission shown to increase with increasing temperatures6,7,8,9. Ae. aegypti mosquitoes are capable of transmitting Flaviviruses (dengue, Zika, West Nile virus, yellow fever) and an Alphavirus (chikungunya)1, targeting human hosts in endemic countries. With the exception of yellow fever, vaccines for pathogens transmitted by Aedes spp. are still in the development or preclinical trial phases or not readily available10,11. The introduction of novel Aedes-borne viruses to the region between 2013 and 2016, along with increased observations of dengue cases since 201912,13,14, has precipitated the need for improved Aedes-vector management within LAC15.

The main control strategies for Ae. aegypti involve the use of synthetic insecticides. Commercial synthetics are used by public health agencies to control Ae. aegypti populations to reduce disease outbreaks16,17, by farmers in the agricultural industry to control pests of crops16,18,19, and by the general population in their homes18,20,21. These chemicals are usually non-specific and have a negative impact on the environment due to their non-selectivity. Overuse of these insecticides has resulted in the evolution of resistance in mosquitoes19. Insecticide resistance in this vector results in a never-ending cycle of increased use of the insecticides to obtain effectiveness19, unless interrupted22. The exploration of new and effective methods for controlling Aedes aegypti mosquito populations is important to prevent the transmission of their disease-causing pathogens23,24.

Plants have developed secondary metabolites that are either toxic to insects or deter feeding. Thus, plants and their metabolites present possible effective biorational chemicals that may provide novel alternatives to manage both phytophagous and hematophagous insect vectors23,25,26,27,28,29. The LAC region hosts a wide diversity of plants30, and communities within the region have a strong knowledge of plants and their healing properties31. More than 80% of individuals from developing countries rely on the use of plants to offset the cost of allopathic medicine32,33,34. Plants routinely used in traditional medicine are known to have unique secondary metabolites that have added value to both the pharmaceutical35 and the agricultural industries26,35. The biodiversity of the LAC basin30,31,36 lends itself to the discovery of unique plant metabolites that may provide alternatives to synthetic chemical insecticides, which may be less toxic to humans and other organisms26. Plant-based insecticides may reduce the dependence on commercial synthetic insecticides and, by extension, reduce selective pressures associated with the development of resistance to insecticides in mosquitoes.

Numerous studies have demonstrated insecticidal activity of extracts prepared from Piper genus (Piperaceae), a genus known for its rich chemical diversity23,27,28,31,37,38. Piper amalago L., native to the LAC, with three intraspecific varieties39, two of which are found in Jamaica, are traditionally used to treat digestive maladies31,40,41; however, their potential as insecticides and or synergists of insecticides appears underexplored23,42,43. The hexane-acetone root extract of Piper amalago var. nigrinodum (C. DC) Yunck., a Jamaican endemic, provided a novel amide, 7-(3',4'-methylenedioxyphenyl)-2E,4E-heptadienoic acid pyrrolidide, which was toxic to the Ae. aegypti larvae42. The other variety, Piper amalago var. amalago, native to the Caribbean, including Jamaica, and South Tropical America39, leaf essential oil extract, demonstrated potential as a biological control to prevent the breeding of citrus-feeding butterflies43. This leaf oil extract may have potential in the agricultural industry. Ethanolic extract of the leaf and stem of Piper amalago var. amalago was moderately toxic to lab reared Anopheles gambiae23. Several compounds with a range of pharmacological values have been extracted from plant parts belonging to the Piper genus, with the eluted compounds varying as a function of the polarity of the extraction solvent and the type of extraction41,42,44,45,46,47,48,49. Building on active metabolites with insecticidal activities already obtained from Piper amalago27,28,50, we continue to explore the insecticidal activity of Piper amalago var. amalago. In this study, the effects of ethanol and hexane extracts of Piper amalago var. amalago were explored as an insecticide and synergist of deltamethrin against Ae. aegypti as well as the potential mechanism of action.

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Protocol

Preparation of plant extracts
An initial specimen of Piper amalago var. amalago was collected in February 2018, and left to air-dry over a 72-h period. The specimen was identified by the botanist and the herbarium curator. The specimen was mounted, given the voucher numbers 36616 and 36617, and deposited in the herbarium at the University of the West Indies, Mona, Jamaica. Subsequent plant samples were collected to prepare ethanol or hexane extracts. The extractions were conducted as follows: the dried leaves, stems, and leaves and stems were pulverized and then submerged in 350 mL of analytical-grade ethanol or hexane for 72 h. After the 72-h period, each extract was suction-filtered using a Büchner funnel lined with 90-mm filter paper, with a vacuum pump attached, to separate plant particles from the extract. The extract was then concentrated via rotary evaporation, decanted, and placed under a fume hood to remove excess solvent23. The final mass of each extract was obtained. To prevent possible oxidation of the extracts, plant material was routinely collected and extracted.

Mosquito rearing
Ae. aegypti larvae and pupae were collected from mosquito breeding sites in communities in the Kingston and St. Andrew parishes (KSA), Jamaica51, initially, in September 2019, and subsequent generations (F1- F12) were reared. Mosquitoes were maintained at a constant temperature of 27 ± 2 °C and 70% ± 10% humidity in an incubator. New Orleans (NO) Ae. aegypti strains were used as the control strain for all experiments. Routinely, larvae were maintained in trays and fed koi pellets until pupation. Pupae were removed and transferred to cups in insect rearing cages per strain. Adult mosquitoes were provided 10% sucrose ad libitum. Subsequent mosquito collections were conducted to maintain their populations close to that of field-caught mosquitoes.

Insecticide resistance (IR) testing
The susceptibility of the KSA strain to deltamethrin was assessed using the Centers for Disease Control and Prevention (CDC) bottle assay52,53,54. Bottle bioassays were carried out on adult females 3-5 days old. Non-blood-fed adults (n = 20-25) were transferred from their cage with an aspirator powered by suction from inhalation to 250 mL bottles, precoated with deltamethrin at the CDC diagnostic dose of 10 µg/bottle. The control bottles were pre-coated with acetone. Mortality was recorded every 15 min up to 2 h53,54. Mosquitoes were considered dead if unable to stand53,54. The experiment was repeated three times. The test mosquitoes were classified as susceptible or resistant using the CDC guideline53,54.

Insecticidal assay - Topical application
Each prepared plant extract was resuspended in ethanol to obtain an initial stock solution (1 mg/µL), and further serially diluted (0-0.25 mg/µL) using ethanol. The resuspended extract was topically applied to the thorax of 3-5-day-old non-blood-fed adult female mosquitoes (n = 10) following previously published methods23,24,55. Briefly, the female mosquitoes were transferred from their cage using an inhalation aspirator to cups and anaesthetized/ knocked down on ice. The resuspended extract, 0.2 µL, was applied to the dorsal thorax using a 10-µL 700 series syringe fitted to a repeating dispenser. For the control group, ethanol, 0.2 µL, was topically applied. After each treatment exposure, mosquitoes were transferred to paper cups and fed 10% sucrose solution. Changes in mosquito flight or gait, if any, were noted for the first 5 h following initial exposure. The mortality was recorded at 24 h under standard insectary conditions. Each assay concentration was conducted in triplicate. The experiment was conducted twice.

Synergistic assays
To determine the potential of the extract as a synergist, a modified bottle bioassay with a synergist was used23. Non-blood-fed female mosquitoes, 3-5 days old (n = 20-25), were topically exposed to plant extract at the Lethal Dose 25% (LD25) concentration while anaesthetized/knocked down, as described above. The mosquitoes were transferred to a netted cup and observed for 1 h. After 1 h of active flying, mosquitoes were gently removed from the cups with an aspirator powered by suction from inhalation55 and then transferred to bottles precoated with 10 µg of deltamethrin (total coated bottles = 4). Ethanol was used as the control agent for topical application. The control bottles were coated with acetone. The rate of mortality was observed every 15 min up to 2 h or until 100% mortality was achieved. The efficacy of the extract as a synergist was compared with that of a known synergist, piperonyl butoxide (PBO), as suggested previously53,54. Initially, mosquitoes were exposed to bottles treated with 400 µg of PBO for 1 h. After the 1 h exposure, the mosquitoes were then transferred to netted cups and fed a 10% sugar solution. After 1 h, the mosquitoes were gently transferred with the aid of the aspirator to bottles coated with 10 µg of deltamethrin (total coated bottles = 4)53,54. The mosquitoes were observed every 15 min up to 2 h or until 100% mortality was achieved. The experiment was conducted three times.

Characterization of plant extracts by Gas chromatography - mass spectrometry (GC - MS) and high-performance liquid chromatography (HPLC)
The extract of P. amalago var. amalago was analyzed by GC-MS system coupled to a mass-selective detector with DB-5 ms column (60 m × 0.25 mm; 0.25-µm film thickness). Pure helium was utilized as the carrier gas at a flow rate of 1 mL/min. The temperature program was initiated at 45 °C and increased at a rate of 1 °C/min to 280 °C, where the rate was then increased to 2 °C/min. Injector and detector temperatures were maintained at 210 °C and 220 °C, respectively. The extract injection volume was 1 µL. Retention indices were calculated using Kovats' procedure. The compounds within the extract were identified via a comparison of mass spectral data with those of the National Institute of Standards and Technology Mass Spectral Database (NIST) 17 library, as described previously56.

Identification of the compound present was achieved by comparison of the extract with a reference standard using HPLC. Separation of the extract was undertaken using a stationary-phase HPLC. The column used was C-18, with 75% acetonitrile and 25% water as solvents. The extract was standardized against the key phytochemical identified with 99% confidence by the GC-MS analysis.

In vitro mosquito CYP assays
The Michaelis-Menten kinetics were initially calculated for CYP9J32 (90 pmol/mL; Km 19.52 µM) enzyme, the concentration used in the inhibitory assay. CYP assays were conducted using the purified piperine extract dissolved in 0.1% final acetonitrile concentration. The fluorogenic substrate, DEF, was dissolved in DMSO. The final concentration of DMSO was 0.5%. A solvent control prepared with 0.5% DMSO and 0.1% final acetonitrile was tested to correct for any effects of the solvents across the dilution range. As a reference to compare the inhibitory property of piperine, deltamethrin, and PBO were used as standard controls. The procedure followed similar methods as described previously23. The enzymatic reactions were conducted in 96-well plates, in 50 mM potassium phosphate buffer solution with 5 mM MgCl2, adjusted to pH 7.4, while varying the concentration of the test compound. All reactions were preincubated at 37 °C for 5 min, while maintaining 420 rpm on a shaker. A NADPH-generating system, which consisted of NADP+ (0.001 mM), glucose-6-phosphate (0.025 mM), and glucose-6-phosphate dehydrogenase (5 units/mL), was added to initiate the reaction. The final volume in each well was 200 µL. The reactions were monitored at 485 nm/530 nm (excitation/emission) using a fluorescence spectrophotometer with an incubator and shaker. The inhibitory data are presented as the mean ± standard error of the mean (SEM). The assays were performed in triplicate.

Homology modeling
Preliminary structural models for CYP9J32 (Ae. aegypti) were constructed using AlphaFold57. The heme group was positioned following a structural alignment with Homo sapiens CYP3A4 (PDB ID: 6DAA)58. A molecular dynamics simulation of the structural coordinates for CYP9J32 in water was conducted using previously described parameters23.

Molecular docking
The final coordinates of the above-described molecular dynamics simulation were used to conduct molecular docking analyses with piperine using a previously described protocol23.

Data analysis
The toxicity data with the use of insecticides are presented as % mortality ± standard deviation (SD). The plant extract toxicity assays were conducted in triplicate. The data of the plant extract toxicity assays are presented as lethal doses (concentration of the plant extract per body weight of female Ae. Aegypti) at 50% and 95% mortality ± 95% fiducial confidence interval (95% CI) per population (R version 3.6.2). The mortality in the control group for all toxicity assays was less than 20%. Abbott's formula was used to correct the mortality rate in each treated group when necessary72. Statistical analyses were completed using SPSS for Windows (version 17.0). One-way analysis of variance (ANOVA) was used to determine significant differences (p < 0.05) between means where possible.

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Results

The susceptibility of Ae. aegypti collected across Kingston and St. Andrew (KSA), Jamaica, to the CDC bottle bioassay; discriminating dose of deltamethrin was assessed by measuring the mortality of mosquitoes to deltamethrin (10 µg active ingredient) at the diagnostic time of 30 min. Mortality to deltamethrin was observed up to 2 h. The wild-caught mosquitoes were resistant to deltamethrin, with mortality ranging from 0% to 22%, whereas the susceptible New Orleans (NO) strain all died at this dose (n = ...

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Discussion

We explored the extracts of Piper amalago var. amalago, a plant native to the Caribbean and Latin America, as an insecticide and synergist of deltamethrin against Ae. aegypti. Ethanolic and hexane extracts of the aerial parts of the plant were prepared. All ethanolic extracts resulted in 100% mortality within 24 h (Table 1). The stem preparation had the greatest toxic effect against the Aedes aegypti, albeit much less effective than with the similar application of delt...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

We are grateful to Mr. M.T. Babumon for support with the GC-MS and HPLC assays.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
250 mL bottlesWheaton Industries, DWK Life SciencesN/AToxicity assay
700 series syringe Hamilton, NV, USAN/AToxicity assay
90-mm filter paperWhatmanN/AExtraction Assay
Acetone (99%) Fisher Bioreagent (NJ, USA)https://www.fishersci.com/shop/
products/acetone-99-thermo-
scientific/AAL104070F#?
keyword=Acetone%2099%		Dissolving solution, control reagent
Acetonitrile (99.8%)Sigma-Aldrich (St. Louis, MO, USA)https://www.sigmaaldrich.com/
GB/en/search/acetonitrile-(99.8%25)
?focus=products&page=1&perpage
=30&sort=relevance&term=
acetonitrile%20%2899.8%
25%29&type=product		Dissolving solution
Black 96-well plates Thermo Labsystems, Basingstoke, UKN/AEnzyme assay
DB-5 ms column  (60m × 0.25 mm; 0.25 μm film thickness)Hewlett-PackardN/AIdentification of active secondary metabolite in the plant
Deltamethrin (98%)Sigma-Aldrich (St. Louis, MO, USA)https://www.sigmaaldrich.com
/GB/en/search/deltamethrin-
(98%25)?focus=products&page
=1&perpage=30&sort=relevance
&term=deltamethrin%20%2898
%25%29&type=product		Standard insecticide
Diethoxyfluorescein (DEF; 98.1%)Cypex Ltd. (Dundee, UK)531001-10Enzyme assay
Dimethyl sulfoxide (DMSO) (99.9%)Sigma-Aldrich (St. Louis, MO, USA)https://www.sigmaaldrich.com/
GB/en/search/dimethyl-sulfoxide
-(dmso)-(99.9%25)?focus=products
&page=1&perpage=30&sort=
relevance&term=dimethyl%20sulfoxide
%20%28DMSO%29%20%2899.9%
25%29&type=product		Dissolving solution
Ethanol (99%)Fisher Bioreagent (NJ, USA)https://www.fishersci.com/us/
en/catalog/search/products?
keyword=Ethanol+%2899%25%29		Dissolving solution, control reagent
GC–MS system Agilent Technologies, CA, USAAgilent 7820 AIdentification of active secondary metabolite in the plant
GC–MS system Agilent Technologies, CA, USAAgilent 7820 AIdentification of active secondary metabolite in the plant
Glucose-6-phosphateSigma-Aldrich (Gillingham, UK)https://www.sigmaaldrich.com/
GB/en/search/glucose-6-phosphate?
focus=products&page=1&perpage
=30&sort=relevance&term=glucose
-6-phosphate&type=product		Enzyme assay
Glucose-6-phosphate dehydrogenase Sigma-Aldrich (Gillingham, UK)https://www.sigmaaldrich.com/GB
/en/search/glucose-6-phosphate-
dehydrogenase?focus=products
&page=1&perpage=30&sort=
relevance&term=glucose-6-phosphate
%20dehydrogenase&type=product		Enzyme assay
Hexane (98%)Fisher Bioreagent (NJ, USA)https://www.fishersci.com/us/
en/catalog/search/products?
keyword=Hexane+%2898%25%29		Dissolving solution
IncubatorShel Lab, Sheldon Manufacturing, USAL127Insect Rearing
Insect rearing cagesBugdorm, TaiwanN/AInsect Rearing
Magnesium chlorideSigma-Aldrich (Gillingham, UK)https://www.sigmaaldrich.com/
GB/en/search/magnesium-
chloride?focus=products&page
=1&perpage=30&sort=relevance
&term=Magnesium%20chloride&type=product		Enzyme assay
Mass selective detector Agilent Technologies, CA, USAAgilent 5975Identification of active secondary metabolite in the plant
Nicotinamide adenine dinucleotide phosphate (NADP+)Sigma-Aldrich (Gillingham, UK)https://www.sigmaaldrich.com/
GB/en/search/nicotinamide-
adenine-dinucleotide-phosphate
-(nadp%2B)?focus=products&
page=1&perpage=30&sort=relevance
&term=nicotinamide%20adenine%
20dinucleotide%20phosphate%20
%28NADP%2B%29&type=product		Enzyme assay
PB600 repeating dispenser Hamilton, NV, USAToxicity assay
Piperine (97%) Sigma-Aldrich (St. Louis, MO, USA)https://www.sigmaaldrich.com/
GB/en/search/piperine-(97%25)?dym
=piperidine%20%2897%25%
29%2Cpiperazine%20%2897%
25%29&focus=products&page=1
&perpage=30&sort=relevance&
term=piperine%20%2897%25%
29&type=product		Compound standard
Piperonyl butoxide (PBO; 90%)Fluka, Buchs (Switzerland)N/AStandard pyrethroid insecticide
Potassium phosphateSigma-Aldrich (Gillingham, UK)https://www.sigmaaldrich.com/
GB/en/search/potassium-phosphate?
focus=products&page=1&perpage
=30&sort=relevance&term=potassium
%20phosphate&type=product		Enzyme assay
Rotary evaporator Büchii (DE, USA)B-481Extraction Assay
SigmaPlot v. 10. Systat Software IncVersion 10.0Enzyme assay
Stationary-phase HPLC Agilent Technologies, CA, USAAgilent 1100 seriesIdentification of active secondary metabolite in the plant
Vacuum pump GAST (USA)0523-V4-G588DXExtraction Assay
Varioskan ThermoFisher, UKN/AEnzyme assay, fluorescence spectrophotometer with an incubator and shaker

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