# Effectiveness of the mixture of Cannabis sativa L and piper umbellatum leaves essential oils with temephos and permethrin on major vectors *Anopheles gambiae* S.L and *culex quinquefasciatus*

Henri Abé, Foko Dadji Gisele Aurelie, Elie Baudelaire Ndjantou, Dicko Amadou, Joseph Lebel Tamesse

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Henri Abé, Foko Dadji Gisele Aurelie, Elie Baudelaire Ndjantou, Dicko Amadou, Joseph Lebel Tamesse. Effectiveness of the mixture of Cannabis sativa L and piper umbellatum leaves essential oils with temephos and permethrin on major vectors *Anopheles gambiae* S.L and *culex quinquefasciatus*. International Journal of Entomology Research, 2021, 6 (6), pp.202-214. hal-05057206

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# Effectiveness of the mixture of Cannabis sativa L and piper umbellatum leaves essential oils with temephos and permethrin on major vectors *Anopheles gambiae* S.L and *Culex quinquefasciatus*

Henri Abé<fnref n="1" />, <fnref n="2" />*, Gisèle Aurélie Foko Dadji<fnref n="2" />, Elie Baudelaire Ndjantou<fnref n="3" />, <fnref n="4" />, Dicko Amadou<fnref n="4" />, Joseph Lebel Tamesse<fnref n="2" />

## Abstract

Most of Arthropods borne diseases are endemic in tropical regions and especially in Sub-Sahara Africa. Synthetic insecticides use for vector control remain the first line of defence but they are associated with high toxicity, vector resistance, environmental adverse and limited alternative. The mixture of conventional insecticide with plant essential oil can be an alternative. Our study was undertaken to evaluate the efficacy of the mixture of *Cannabis sativa* L and *Piper umbellatum* leaves essential oil with permethrin and temephos on two major vectors *Anopheles gambiae* S.L and *Culex quinquefasciatus* under laboratory conditions.

*Cannabis sativa* L and *Piper umbellatum* Leaves essential oils were extracted by hydro distillation with Clavenger type apparatus. The chemical analysis of essential oils was carried out by GC-FID and GC-MS respectively for *Cannabis sativa* L and *Piper umbellatum*. Based on literature the LC₅₀ for permethrin and temephos were found by serial dilution. These constant concentrations were mixed with increasing sublethal concentrations of *Cannabis sativa* L and *Piper umbellatum* leaves essential oil. Bioassays were performed on the larvae and female adults of field population of *Anopheles gambiae* S.L and *Culex quinquefasciatus* according to the WHO and CDC guidelines. Probit regression was used to calculate the LC₅₀ and LC₉₅ of each mixture. The Kruskal Wallis test was used to assess the significance of difference on the mortality rate between treatments.

The mixture of temephos with essential oils is effective on larvae while the mixture of essential oils with permethrin is toxic for adult female of *Anopheles gambiae* S.L and *Culex quinquefasciatus*. The mortality is essential oil dose-dependent. For larvicidal activity, the LC₅₀ and LC₉₅ after 24 hours exposure for the mixture of *Cannabis sativa* L leaf essential oil with temephos were: 0. 14%; 0.002% and 0. 35%; 0. 026% respectively for *Anopheles gambiae* S.L and *Culex quinquefasciatus*; while these values for the mixture of *Piper umbellatum* leaf essential oil with temephos were: 0.30%; 0. 0011% and 0. 64%; 0.024% respectively for *Anopheles gambiae* S.L and *Culex quinquefasciatus*. After 48 hours exposure the LC₅₀ and LC₉₅ for the mixture of *Cannabis sativa* L leaf essential oil with temephos were: 0.011%; 0.0013% and 0.17%; 0.013% while for the mixture of *Piper umbellatum* leaf essential oil with temephos these values were: 0.023%; 0.001% and 0.38%; 0.015% respectively for *Anopheles gambiae* S.L and *Culex quinquefasciatus*. For the knock down activity of the mixture of *Piper umbellatum* leaf essential oil with permethrin, time necessary for knocking down 50% and 95% of exposed mosquito are: 27±0.4min; 14±0.8min and 53±0.6min; 52±0.14min respectively for *Anopheles gambiae* S.L and *Culex quinquefasciatus*. For *Cannabis sativa* L leaf essential oil mixture, time necessary for knocking down 50% and 95% of exposed mosquitoes are: 16±0.75min; 8±0.15min and 53±0.94min; 53±0.6min respectively for *Anopheles gambiae* S.L and *Culex quinquefasciatus*. For adulticidal activity, the LC₅₀ and LC₉₅ for the mixture of *Piper umbellatum* leaf essential oil with permethrin were: 0.024%; 0.011% and 0.18%; 0.17% respectively for *Anopheles gambiae* S.L and *Culex quinquefasciatus*; while for the mixture of *Cannabis sativa* L leaf essential oil these values were: 0. 027%; 0.011% and 0.18%; 0.15% respectively for *Anopheles gambiae* S.L and *Culex quinquefasciatus*.

**Conclusion:** The mixtures of *Cannabis sativa* L and *Piper umbellatum* leaves essential oils with temephos and permethrin showed the efficacy with low concentrations against larvae and adult female of field population of *Anopheles gambiae* S.L and *Culex quinquefasciatus*.

**Keywords:** *Anopheles gambiae* S.L, *Culex quinquefasciatus*, *Cannabis sativa* L, *Piper umbellatum*, temephos, permethrin, essential oils mixture, insecticide

## Introduction

Arthropod borne diseases and particularly malaria remain a serious public health issue in Africa. The prevention of these vector-borne diseases is based on the use of synthetic insecticides (Zaim *et al.*, 2002) [1]. However, all efforts made to eradicate these diseases, although making progress in recent years, are limited. Indeed, the modification of the

environment by anthropic activities such as agriculture (Nkya *et al.*, 2014; Janko *et al.*, 2018) [2, 3] urbanization (Oladepo *et al.*, 2010; Pimnon *et al.*, 2018) [4, 5] industries are at the origin of vectors resistance to conventional insecticides. Other factors also contribute to the development of vector resistance: the application of the same insecticides in a locality where resistance to these

<footnote n="1">¹ Department of Animal Biology and Physiology, Faculty of Science, University of Yaoundé I, Cameroon</footnote>

<footnote n="2">² Higher Teachers' Training College of the University of Yaoundé I, Cameroon</footnote>

<footnote n="3">³ National School of AgroIndustrial Sciences (ENSAI), University of Ngaoundere, Adamawa, Cameroon</footnote>

<footnote n="4">⁴ Laboratory of Physic and chemistry, Jean Baritiot Institute, University of Lorraine Metz, France</footnote>
insecticides is proven, and the under dosage of insecticides (Diniz et al., 2015) [6]. This resistance is expressed in various ways in target insects: avoidance behaviour of the insecticide (Agossa et al., 2015; Carasso et al., 2019), rapid degradation of the insecticide by detoxification (Ibrahim et al., 2016) [9], modification of the site of fixation of the insecticide (Lynd et al., 2018; Alout et al., 2008) [11, 12], cuticular structures that prevent the penetration of insecticides (Wang et al., 2019; Ingham et al., 2018) [13, 14]. Based on the findings on the hazardous effects of synthetic insecticides on the environment and resistance of vectors, several authors have proposed alternatives including the use of bio pesticides (Gnankiné et al., 2017; Bolzonella et al., 2019) [15, 16] because they are non-toxic for non-target organisms, biodegradable and offer the advantage of being composed of several active ingredients. Unfortunately, all the tests proposed in this way remain limited to the tests carried out in the laboratory. Thus, the essential oils although effective are very volatile. A combination of raw plant extracts or essential oils with synthetic insecticides can be an alternative to the vector resistance and the adverse effect of synthetic insecticides.

In the present study, we evaluated the effectiveness of the combination of *Cannabis sativa* L and *Piper umbellatum* leaves essential oils with temephos and permethrin on two major vectors, *Anopheles gambiae* S.L and *Culex quinquefasciatus* under laboratory conditions. In our previous work, the essential oils from the leaves of the two plants were found to be effective on the larvae and adults of *Anopheles gambiae* S.L and *Culex quinquefasciatus* under laboratory conditions (Abé et al., 2018; Abé et al., 2019) [17, 18]. Besides, these two plants are used worldwide for several purposes. *Cannabis sativa* L is known not only for its psychotropic effects on human subjects, but also in chemotherapy against nausea, to improve the treatment of HIV / AIDS (Mechoulam et al., 2002) [9], the treatment of pain (Manzanares et al., 2006) [20], treatment of multiple sclerosis (Hagenbach et al., 2001) [21] and Alzheimer's disease (Milton, 2002) [22], the treatment of cancer due to its antiproliferative effect (Galve-Roperh et al., 2000). The crude extract of *Piper umbellatum* non-toxic for small mammals (Da Silva et al., 2014) [24] appears to be effective against phospholipase of myotoxic venom of snake (Núñez et al., 2005) [25], exhibits protective and healing properties against stomach ulcers on an experimental basis in rodents (IF da Silva Junior et al., 2016) [26], anti-cancer and anti-inflammatory in Brazil (Hespporte Iwamoto et al., 2015); its effective in treating injuries in Cuba (Salehi et al., 2019) [27] and in West Africa in several tribes (Setzer et al.; 1999) [28]; to treat fever in Peru and onchocerciasis in Cameroon (Cho-Ngwa et al., 2016) [29]. *Piper umbellatum* is also used to treat kidney diseases, skin, burns, diarrhea, rheumatism, malaria and intestinal parasites (Roersch, 2010) [30].

## Materials and Methods

### Plant material

The leaves of *Piper umbellatum* were harvested in the morning at the University of Yaounde I campus while the *Cannabis sativa* L leaves were harvested in the village of Salla in the district of Ayos. Before hydro distillation, plants were identified at National Herbarium in comparison with the numbers of the collectors. For *Piper umbellatum*: R. Letouzey 2499 of the specimen number 3614SRFK, the specimen was classified as belonging to *Piperarceae* family

and for *Cannabis sativa* L the specimen was identified under the number 3614SRFK and classified as belonging to *Cannabaceae* family.

### Essential oil extraction and synthetic insecticide collection

The plant leaves essential oil extraction took place in the laboratory of the department of Microbiology of the University of Yaounde I using a Clavenger type apparatus. This extraction was performed in several sessions, each session during 6 hours in order to allow a maximum extraction. The essential oils were collected in a dark bottle, dried using anhydrous magnesium sulphate and stored at 4°C, away from UV rays before use. The mass of plants used and extraction yield are presented in table 2. The synthetic insecticides for our bioassays (permethrin and temephos) were provided by OCEAC (Organisation de Coordination pour la lutte Contre les Endémies en Afrique Centrale) with reference of AL International industry (France).

### Chemical analysis of *Piper umbellatum* and *Cannabis sativa* L leaves essential oils

The chemical analysis took place at two different laboratories: At Lorraine University, France, for *P. umbellatum* and at the National Institute of the Analytic Science of Paris for *C. sativa* L. The chemical analysis of *Piper umbellatum* leaf essential oil was performed by Gas Chromatography, coupled with Mass Spectrometry. The analysis was performed with two Perkin Elmer instruments, a Clarus 500 GC gas chromatograph coupled to an MS Clarus 500 mass spectrometer.

The column used in chromatography was an Elite 5MS (5% Diphenyl / 95% DimethylPolysiloxane stationary phase) 30 m long, with an internal diameter of 0.25 mm and a film thickness of the 0.25 µm stationary phase. The carrier gas was helium with a flow rate of 0.75 mL / min.

The injector was brought to 300 °C, the program begins with a step of 3 min at 60 °C and the temperature increases from 10 °C / min to 300 °C where a step of 3 min is performed.

The acquisition of the mass spectrum was carried out on an m / z range of between 20 and 400 with 0.1 scan / s and an electron ionization energy of 70 eV. The temperature of the source and the transfer line were 250 °C.

The sample to be analysed was solubilized in a small volume of dichloromethane.

The data processing software was Turbomass 6.1 and the database for the comparison of mass spectra were NIST MS Search 2.0.

For *Cannabis sativa* L leaf essential oil, the determination of retention data and the area percentage of the identified compounds were carried out on a two GC-FID systems:

1. An Agilent 5890 system equipped with HP-1 (ref: 1909 1 Z-115) column (50m x 320µm; 0.5 µm film thickness). GC oven temperature was kept at 80 °C for 8 minutes and programmed to 220 °C at the rate of 2 °C/min;

2. An Agilent 6890 system equipped with HP-Innowax (ref: 1909 1 N-216, Agilent Technologies, Santa Clara, CA 95051, USA) column (60 m x320µm,0.5µm film thickness). GC temperature was kept at 60 °C and programmed to 245 °C at the rate of 2 °C/min, then constant at 250 °C for 20 min.

The split ratio was adjusted to 1/100. The injector
temperature was 250 °C and the FID detector was kept at 250 °C. The carrier gas was Helium (1.3ml/min). Gas chromatography- Mass spectrometry was carried out using the first system Agilent 5890 equipped with HP-1column. The mass spectra was recorded in the electron impact mode at 70 eV using the aforementioned chromatographic conditions. Individual components of *Cannabis sativa* L leaves essential oil were identified by their retention index as described in Adams (2012) and their mass spectra were interpreted using the WILEY L computer library

## Mosquito's collection

For our bioassays, we used the larvae and adult female of wild population of *Anopheles gambiae* S.L and *Culex quinquefasciatus*. The aquatic stages of *Anopheles gambiae* S.L were collected in Yaounde down town, near river Ewoé (N 03°51′34.9″ and E 011°31′3″). *Culex quinquefasciatus* were sampled at Melen pound behind the Faculty of medicine and Biomedical Sciences of the University of Yaounde I (N 03°85′63.1″ and E 011°48′49.4″). Mosquitoes collected were reared in the insectary of the

Higher Teachers' Training College of the University of Yaounde I at 25-27°C and 75-78% relative humidity under photoperiod 12L: 12D. Larvae were fed with Tetramin Baby Fish Food at a rate of 2.5mg for 100 larvae per day (Price *et al.*, 2015, Vantaux *et al.*, 2016). The pupae were collected in plastic cups and placed in emergence cages. Adults from the pupae were fed using a 10% glucose solution. *Anopheles gambiae* S.L and *Culex quinquefasciatus* third and early four instars larvae and female aged of 2-5 days old were used to carry out bioassays.

## Preparation of tests solutions

Based on literature, we have previously determined the discriminatory doses of temephos (WHO, 2016) and permethrin (WHO, 2013) causing less than 98% of larval and adult mortality respectively. Absolute alcohol has been used for a serial dilution of synthetic insecticides and essential oils according to the WHO Protocol, (2005). We then mixed the given concentrations of essential oils with constant concentrations (CL₅₀) of temephos (0.6%) and permethrin (0.375%) according to Pennetier Protocol (Pennetier *et al.*, 2008) (table 1).

<table><caption>Table 1: Protocol of mixing essential oils with temephos and permethrin (synthetic insecticides)</caption><thead><tr><th>Concentration of synthetic insecticide (%)</th><th>Essential oil concentration (%)</th><th>Volume of synthetic insecticide(ml)</th><th>Volume of essential oil(ml)</th><th>Final volume(ml)</th><th>Final concentration of essential oil (%)</th></tr></thead><tbody><tr><td colspan="6"><strong>Temephos</strong></td></tr><tr><td>0.6</td><td>0.4</td><td>3</td><td>3</td><td>6</td><td>0.2</td></tr><tr><td>0.6</td><td>0.2</td><td>3</td><td>3</td><td>6</td><td>0.1</td></tr><tr><td>0.6</td><td>0.1</td><td>3</td><td>3</td><td>6</td><td>0.05</td></tr><tr><td>0.6</td><td>0.05</td><td>3</td><td>3</td><td>6</td><td>0.025</td></tr><tr><td>0.6</td><td>0.025</td><td>3</td><td>3</td><td>6</td><td>0.0125</td></tr><tr><td>0.6</td><td>0.0125</td><td>3</td><td>3</td><td>6</td><td>0.00625</td></tr><tr><td>0.6</td><td>0.00625</td><td>3</td><td>3</td><td>6</td><td>0.003125</td></tr><tr><td>0.6</td><td>0.003125</td><td>3</td><td>3</td><td>6</td><td>0.0015625</td></tr><tr><td colspan="6"><strong>Temephos</strong></td></tr><tr><td>0.6</td><td>0.8</td><td>3</td><td>3</td><td>6</td><td>0.4</td></tr><tr><td>0.6</td><td>0.4</td><td>3</td><td>3</td><td>6</td><td>0.2</td></tr><tr><td>0.6</td><td>0.2</td><td>3</td><td>3</td><td>6</td><td>0.1</td></tr><tr><td>0.6</td><td>0.1</td><td>3</td><td>3</td><td>6</td><td>0.05</td></tr><tr><td>0.6</td><td>0.05</td><td>3</td><td>3</td><td>6</td><td>0.025</td></tr><tr><td>0.6</td><td>0.025</td><td>3</td><td>3</td><td>6</td><td>0.0125</td></tr><tr><td>0.6</td><td>0.0125</td><td>3</td><td>3</td><td>6</td><td>0.00625</td></tr><tr><td>0.6</td><td>0.00625</td><td>3</td><td>3</td><td>6</td><td>0.003125</td></tr><tr><td>0.6</td><td>0.003125</td><td>3</td><td>3</td><td>6</td><td>0.0015625</td></tr><tr><td colspan="6"><strong>Permethrin</strong></td></tr><tr><td>0.375</td><td>0.4</td><td>3</td><td>3</td><td>6</td><td>0.2</td></tr><tr><td>0.375</td><td>0.2</td><td>3</td><td>3</td><td>6</td><td>0.1</td></tr><tr><td>0.375</td><td>0.1</td><td>3</td><td>3</td><td>6</td><td>0.05</td></tr><tr><td>0.375</td><td>0.05</td><td>3</td><td>3</td><td>6</td><td>0.025</td></tr><tr><td>0.375</td><td>0.025</td><td>3</td><td>3</td><td>6</td><td>0.0125</td></tr><tr><td colspan="6"><strong>permethrin</strong></td></tr><tr><td>0.375</td><td>0.4</td><td>3</td><td>3</td><td>6</td><td>0.2</td></tr><tr><td>0.375</td><td>0.2</td><td>3</td><td>3</td><td>6</td><td>0.1</td></tr><tr><td>0.375</td><td>0.1</td><td>3</td><td>3</td><td>6</td><td>0.05</td></tr><tr><td>0.375</td><td>0.05</td><td>3</td><td>3</td><td>6</td><td>0.025</td></tr><tr><td>0.375</td><td>0.025</td><td>3</td><td>3</td><td>6</td><td>0.0125</td></tr></tbody></table>

Temephos and permethrin provided by Al International industry (France)

## Bioassays

### Bioassay with larvae

The larvicidal potential of the essential oil mixture of each plant with temephos was evaluated on field mosquito larvae for 24 hours and 48 hours exposure period.

Larvae of third and early four instars were used to assess the

larvicidal activity of the mixture of *Piper umbellatum* and *Cannabis sativa* L leaves essential oil with temephos, following WHO Guidelines for Laboratory and Field Testing of Mosquito Larvicides (WHO, 2005). Before running the test, larvae were maintained during one hour in distilled water for observation. Tests concentrations were
prepared by adding 1ml of appropriate concentration of the mixture to disposable test cups containing 99 ml of spring water. Batches of 25 larvae of third and early four instars were transferred from observation cups to test cups. Four replicates were run for each concentration and an equal number of controls were run as well. The positive control was prepared by adding 1ml of temephos (0.6%) to 99 ml of spring water. Maintaining the constant concentration of temephos (0.6%) for the mixture, eight different concentrations of *Cannabis sativa* L leaf essential oils were used ( 0.2%; 0.1%; 0.05%; 0.025%; 0.0125%; 0.00625%; 0.003125% and 0.00156%). For *Piper umbellatum* leaf essential oil, nine different concentrations (0.4%; 0.2%; 0.1%; 0.05%; 0.025%; 0.0125%; 0.00625%; 0.00325% and 0.00125%) were also used against the larvae of *Anopheles gambiae* S.L and *Culex quinquefasciatus*. Each test was conducted three times on different days. No food was added to cups during the exposure period. Larvae were considered dead, when they were incapable of any movement or not swimming actively when touch. The mortality rate was recorded after 24 and 48 hours of exposure. The lethal concentrations of essential oil killing 50% and 95% of exposed larvae (LC₅₀ and LC₉₅) to the mixture were calculated using a log probit approach with STATISTICA software version 6.0.

## Bioassay with adult

Adult bioassays were performed with 2-5 days- old non-blood- fed females following the WHO(2013) and CDC(2013) Guidelines with cone using impregnated bed net with the mixture at the following concentrations of essential oils: 0.0125%, 0.025%, 0.05%; 0.1% and 0.2% for *Anopheles gambiae* S.L and *Culex quinquefasciatus*. The concentration of permethrin was constant in the mixture (0.375%). Absolute alcohol was used to dilute the essential oil at the various concentrations before mixing and 3ml of each concentration was used to impregnate a portion of bed net (98.47cm²). Due to the high volatility of essential oils, pieces of net prepared as indicated above were dried at room temperature away from sun light for 15 minutes before carrying susceptibility assays (Wang *et al.*, 2014, Faraone *et al.*, 2015) [³⁹]. Before each test, female mosquitoes were transferred from the cages to the cup for one hour and specimens with broken legs or unable to fly were discarded and replaced. Twenty replicates of batches of 5 female mosquitoes per cone were exposed to each concentration of the mixture for 1hour. Five replicates were run as control using a portion of net impregnated only with permethrin at

0.375%. Considering only the higher concentration of each essential oil, the number of knock down (KD) mosquitoes was recorded at 10 minutes intervals during 1 hour exposure period and the time required for knocking down 50% and 95% of the individuals(KDT₅₀ and KDT₉₅) estimated with 95% of confident interval. After the exposure period, mosquitoes were transferred back to recovery cups and provided with 10% of glucose solution soaked on cotton pad. Mosquito mortality was recorded 24 hours post-exposure; individuals with broken legs or not able to fly were also considered as dead. After the tests, adult mosquitoes of *Anopheles gambiae* S.L from field population and *Culex quinquefasciatus* were kept in Eppendorf tubes at -4°C for identification.

## Adult mosquito identification

Adults female of the field population of *Anopheles gambiae* S.L were morphologically identified using the Gillies and Coetzee key (1987) while *Culex quinquefasciatus* mosquito were identified using Peter Gupp (1996) and Reuben (1994) keys. Genomic DNA of *An.gambiae* S.L was extracted according to the Livak protocol (1984) and molecular identification of females was conducted according to Santolammazza *et al* protocol (2008). As for the adult females of *Culex quinquefasciatus*, their molecular identification was not possible because the TAG MANN device used did not allow it.

## Data analysis for Bioassays

The lethal concentrations inducing 50% and 95% (LC₅₀ and LC₉₅) larval mortality were calculated using log probit approach with WINDL CIRAD-CA software version 2.0 and STATISTICA software version 6.0 to plot the graphs. The relation between the exposure time, the mortality and the doses was assessed using probit regression.

For the adulticidal activity, the time at which 50% and 95% of adult mosquitoes were knocked down (Knock down time, KTD₅₀ and KDT₉₅) and the LC₅₀ and LC₉₅ were calculated using WINDL CIRAD-CA software version 2.0. The relation between the knock down time, mortality and the doses were assessed using probit regression.

## Results

### Yield of *Piper umbellatum* and *Cannabis sativa* L Essential oil

The table 2 below provides characteristics of *Piper umbellatum* and *Cannabis sativa* L leaves

<table><caption>Table 2: The oil yield of *Piper umbellatum* and *Cannabis sativa* L</caption><thead><tr><td>Plant name</td><td>Family</td><td>Certification number</td><td>Leaves weight(g)</td><td>Essential oil weight(g)</td><td>Extraction yield</td></tr></thead><tbody><tr><td>Piper umbellatum</td><td>Piperacea</td><td>3614SRFK</td><td>7390.8g</td><td>0.9g</td><td>0.012%</td></tr><tr><td>Cannabis sativa L</td><td>Cannabacea</td><td>25967SRF/Cam</td><td>3,375g</td><td>1.5g</td><td>0.044%</td></tr></tbody></table>

The extraction yield of *Piper umbellatum* and *Cannabis sativa* L leaves essential oils were very low and therefore require a huge mass of plant to obtain a volume of essential oils sufficient for biological assays. A better method of extraction, the judicious choice of the period of harvesting and the control of some edaphic factors could allow a good yield.

### Chemical composition of *Piper umbellatum* and *Cannabis sativa* L leaves essential oils

The chemical analysis of *Piper umbellatum* leaf essential oil revealed up to 24 different compounds comprising 89.60% of the essential oil. Major compounds were sesquiterpenes: copaene (9.63%) β-elemene (10.56%) and γ-murolene (11.37%). Unidentified components comprise 10.40% of the essential oil.
<table><caption>**Table 3:** Chemical composition of *Piper umbellatum* leaf essential oil (Abé *et al.*, 2019)</caption><thead><tr><th rowspan="2">Elution order</th><th rowspan="2">Compounds</th><th rowspan="2">Percentage</th><th colspan="2">Kovat retention index</th></tr><tr><th>Npo.</th><th>Po.</th></tr></thead><tbody><tr><td>1.</td><td>α-pinene</td><td>6.23</td><td>931</td><td>1035</td></tr><tr><td>2.</td><td>camphene</td><td>1.83</td><td>944</td><td>1063</td></tr><tr><td>3.</td><td>β-pinene</td><td>1.83</td><td>971</td><td>1132</td></tr><tr><td>4.</td><td>β-myrcene</td><td>4.60</td><td>981</td><td>1161</td></tr><tr><td>5.</td><td>limonene</td><td>1.56</td><td>1022</td><td>1203</td></tr><tr><td>6.</td><td>Z-β-ocimene</td><td>1.33</td><td>1025</td><td>1242</td></tr><tr><td>7.</td><td>E-β-ocimene</td><td>2.63</td><td>1037</td><td>1247</td></tr><tr><td>8.</td><td>linalool</td><td>1.27</td><td>1086</td><td>1533</td></tr><tr><td>9.</td><td>L-borneol</td><td>2.45</td><td>1153</td><td>1719</td></tr><tr><td>10.</td><td>terpinen-4-ol</td><td>2.96</td><td>1163</td><td>1611</td></tr><tr><td>11.</td><td>p-menth-1-en-8-ol</td><td>5.32</td><td>1280</td><td>-</td></tr><tr><td>12.</td><td>copaene</td><td>9.63</td><td>1375</td><td>1491</td></tr><tr><td>13.</td><td>β-elemene</td><td>10.56</td><td>1386</td><td>1577</td></tr><tr><td>14.</td><td>caryophyllene</td><td>1.37</td><td>1419</td><td>1612</td></tr><tr><td>15.</td><td>α-caryophyllene</td><td>1.18</td><td>1453</td><td>-</td></tr><tr><td>16.</td><td>γ-muurolene</td><td>11.37</td><td>1473</td><td>1689</td></tr><tr><td>17.</td><td>germacrene D</td><td>3.92</td><td>1480</td><td>1708</td></tr><tr><td>18.</td><td>α-farnesene</td><td>1.90</td><td>1496</td><td>1744</td></tr><tr><td>19.</td><td>δ-cadinene</td><td>2.30</td><td>1513</td><td>1765</td></tr><tr><td>20.</td><td>calamenene</td><td>1.14</td><td>1532</td><td>-</td></tr><tr><td>21.</td><td>z-nerolidol</td><td>4.56</td><td>1550</td><td>2036</td></tr><tr><td>22.</td><td>caryophyllene oxyde</td><td>1.95</td><td>1567</td><td>2001</td></tr><tr><td>23.</td><td>α-eudesnol</td><td>1.6</td><td>1652</td><td>2227</td></tr><tr><td>24.</td><td>phytol</td><td>6.1</td><td>2116</td><td>2613</td></tr><tr><td colspan="2">Total</td><td>89.6%</td><td>-</td><td>-</td></tr></tbody></table>

Analysis performed by gas chromatography with two Perkin Elmer instruments, a Clarus 500 GC gas chromatograph coupled to an MS Clarus 500 mass spectrometer. Individual components were identified by their retention index as described in Adams (2012) and their mass spectra are interpreted using the WILEY L computer library. Constituents are presented in the order of elution from the columns. Npo: non polar; Po: polar
Among these 24 identified compounds of *P.umbellatum*, α-

pinene; β-pinene; β-myrcene; linalool and α-caryophyllene were reported effective on various insects and pest.

The chemical analysis of *Cannabis sativa* L leaf essential oil revealed up to 81 components unlike that of *Piper umbellatum* (table 4). The essential oil of *Cannabis sativa* L is also composed of monoterpenes and sesquiterpenes, the most abundant of which were α-pinene, β-pinene, myrcene, E-β-Ocimene, terpinolene, E-β-Caryophyllene and α-Humulene.

<table><caption>**Table 4:** The chemical composition of *Cannabis sativa* L leaf essential oil (Abé *et al.*, 2018)</caption><thead><tr><th rowspan="2">Elution order</th><th rowspan="2">Compound</th><th rowspan="2">Percentage</th><th colspan="2">Kovat indices</th></tr><tr><th>HP-1</th><th>Innowax</th></tr></thead><tbody><tr><td>1.</td><td>Z-3-Hexenol</td><td>0.03</td><td>842</td><td>1380</td></tr><tr><td>2.</td><td>E-2-Hexenol</td><td>0.02</td><td>852</td><td>1408</td></tr><tr><td>3.</td><td>Heptanone-2</td><td>0.03</td><td>864</td><td>1182</td></tr><tr><td>4.</td><td>Hexenol</td><td>0.03</td><td>869</td><td>1351</td></tr><tr><td>5.</td><td>Heptanal</td><td>0.03</td><td>880</td><td>1185</td></tr><tr><td>6.</td><td>2-Amyl furan</td><td>0.01</td><td>884</td><td>1124</td></tr><tr><td>7.</td><td>α-Thujene</td><td>0.12</td><td>923</td><td>1032</td></tr><tr><td>8.</td><td>α-Pinene</td><td>8.12</td><td>931</td><td>1035</td></tr><tr><td>9.</td><td>Camphene</td><td>0.013</td><td>944</td><td>1063</td></tr><tr><td>10.</td><td>Octene-1-ol-3</td><td>0.03</td><td>961</td><td>1427</td></tr><tr><td>11.</td><td>6-Methyl-5-heptenone-2</td><td>0.005</td><td>964</td><td>1116</td></tr><tr><td>12.</td><td>Sabinene</td><td>0.12</td><td>967</td><td>1118</td></tr><tr><td>13.</td><td>β-Pinene</td><td>3.64</td><td>971</td><td>1132</td></tr><tr><td>14.</td><td>6-Methyl-5-hepten-2-ol</td><td>0.02</td><td>974</td><td>1163</td></tr><tr><td>15.</td><td>Myrcene</td><td>31.75</td><td>981</td><td>1159</td></tr><tr><td>16.</td><td>χ-3-Carene</td><td>0.92</td><td>997</td><td>1011</td></tr><tr><td>17.</td><td>α-Phellandrene</td><td>0.42</td><td>998</td><td>1175</td></tr><tr><td>18.</td><td>α-Terpinene</td><td>0.31</td><td>1010</td><td>1188</td></tr><tr><td>19.</td><td>Hexylacetate</td><td>0.02</td><td>1010</td><td>1272</td></tr><tr><td>20.</td><td>P-Cymene</td><td>0.11</td><td>1014</td><td>1200</td></tr><tr><td>21.</td><td>Limonene</td><td>1.3</td><td>1022</td><td>1203</td></tr><tr><td>22.</td><td>1,8-Cineole+β-phellandrene</td><td>1.29</td><td>1022</td><td>1213</td></tr><tr><td>23.</td><td>Z-β-Ocimene</td><td>1.10</td><td>1025</td><td>1242</td></tr><tr><td>24.</td><td>E-β-Ocimene</td><td>9.65</td><td>1037</td><td>1247</td></tr><tr><td>25.</td><td>γ-Terpinene</td><td>0.23</td><td>1049</td><td>1255</td></tr><tr><td>26.</td><td>E-Thujan-4-ol</td><td>0.03</td><td>1053</td><td>1463</td></tr></tbody></table>
<table><tr><td>27.</td><td>Fenchone+methyl benzoate</td><td>0.02</td><td>1072</td><td>-</td></tr><tr><td>28.</td><td>p-Cymenene</td><td>0.09</td><td>1072</td><td>1437</td></tr><tr><td>29.</td><td>Terpinolene</td><td>14.76</td><td>1079</td><td>1290</td></tr><tr><td>30.</td><td>Nonanal</td><td>0.03</td><td>1083</td><td>1391</td></tr><tr><td>31.</td><td>Linalool</td><td>0.09</td><td>1086</td><td>1533</td></tr><tr><td>32.</td><td>Z-Thujan-4-ol</td><td>0.03</td><td>1092</td><td>-</td></tr><tr><td>33.</td><td>Perillen</td><td>0.02</td><td>1098</td><td>1429</td></tr><tr><td>34.</td><td>α-Fenchol</td><td>0.07</td><td>1100</td><td>1570</td></tr><tr><td>35.</td><td>Z-p-Menth-2-en-1-ol</td><td>0.03</td><td>1114</td><td>1570</td></tr><tr><td>36.</td><td>E-p-Menth-2-en-1-ol</td><td>0.07</td><td>1114</td><td>1571</td></tr><tr><td>37.</td><td>4E, 6Z-Allo-ocymene</td><td>0.01</td><td>1119</td><td>1375</td></tr><tr><td>38.</td><td>Ipsdienol</td><td>0.03</td><td>1125</td><td>-</td></tr><tr><td>39.</td><td>Epoxy terpinolene</td><td>0.05</td><td>1130</td><td>-</td></tr><tr><td>40.</td><td>Menthone</td><td>0.04</td><td>1136</td><td>1465</td></tr><tr><td>41.</td><td>Borneol</td><td>0.04</td><td>1153</td><td>1719</td></tr><tr><td>42.</td><td>P-Cymen-8-ol</td><td>0.12</td><td>1161</td><td>1864</td></tr><tr><td>43.</td><td>Terpinen-4-ol</td><td>0.07</td><td>1163</td><td>1611</td></tr><tr><td>44.</td><td>α-Terpineol</td><td>0.04</td><td>1175</td><td>1682</td></tr><tr><td>45.</td><td>Citronellol</td><td>0.02</td><td>1212</td><td>1764</td></tr><tr><td>46.</td><td>Hexyl butyrate +methylchavicol</td><td>0.06</td><td>1224</td><td>-</td></tr><tr><td>47.</td><td>E-Anethole</td><td>0.10</td><td>1264</td><td>1826</td></tr><tr><td>48.</td><td>α-Ylangene</td><td>0.20</td><td>1370</td><td>1490</td></tr><tr><td>49.</td><td>α-Copaene</td><td>0.01</td><td>1375</td><td>1491</td></tr><tr><td>50.</td><td>β-Elemene</td><td>0.02</td><td>1386</td><td>1577</td></tr><tr><td>51.</td><td>Isocaryophyllene</td><td>0.23</td><td>1408</td><td>-</td></tr><tr><td>52.</td><td>Z-α-Bergamotene</td><td>0.11</td><td>1410</td><td>1559</td></tr><tr><td>53.</td><td>α-Santalene</td><td>0.14</td><td>1415</td><td>1582</td></tr><tr><td>54.</td><td>E-β-Caryophyllene</td><td>10.72</td><td>1419</td><td>1612</td></tr><tr><td>55.</td><td>E-α-Bergamotene</td><td>0.85</td><td>1435</td><td>1575</td></tr><tr><td>56.</td><td>Allo-Aromadendrene</td><td>0.30</td><td>1439</td><td>1620</td></tr><tr><td>57.</td><td>α-Guaiene +unidentified compound</td><td>0.04</td><td>1442</td><td>-</td></tr><tr><td>58.</td><td>E-β-Farnesene</td><td>1.23</td><td>1449</td><td>1663</td></tr><tr><td>59.</td><td>γ-Elemene+unidentified compound</td><td>0.04</td><td>1449</td><td>-</td></tr><tr><td>60.</td><td>α-Humulene</td><td>3.28</td><td>1452</td><td>1654</td></tr><tr><td>61.</td><td>γ-Muurolene</td><td>0.08</td><td>1473</td><td>1689</td></tr><tr><td>62.</td><td>Selina-4,11-diene</td><td>0.22</td><td>1475</td><td>1674</td></tr><tr><td>63.</td><td>Selina-4,7(11)diene+E-α-Bisabolene</td><td>0.36</td><td>1475</td><td>1688</td></tr><tr><td>64.</td><td>β-Selinene</td><td>0.58</td><td>1480</td><td>1700</td></tr><tr><td>65.</td><td>α-Selinene</td><td>0.41</td><td>1491</td><td>1707</td></tr><tr><td>66.</td><td>E,E-α-Farnesene</td><td>0.20</td><td>1496</td><td>1744</td></tr><tr><td>67.</td><td>β-Bisabolene</td><td>0.21</td><td>1499</td><td>1727</td></tr><tr><td>68.</td><td>7-Epi-α-Selinene</td><td>0.21</td><td>1507</td><td>1764</td></tr><tr><td>69.</td><td>γ-Cadinene</td><td>0.05</td><td>1513</td><td>1740</td></tr><tr><td>70.</td><td>δ-Cadinene</td><td>0.09</td><td>1513</td><td>1765</td></tr><tr><td>71.</td><td>12-Nor-caryophyll-5-en-2-one</td><td>0.02</td><td>1515</td><td>-</td></tr><tr><td>72.</td><td>γ-Selinene</td><td>0.24</td><td>1526</td><td>-</td></tr><tr><td>73.</td><td>Germacrene B</td><td>0.14</td><td>1535</td><td>1823</td></tr><tr><td>74.</td><td>Selina-3,7(11)-diene</td><td>0.37</td><td>1537</td><td>1783</td></tr><tr><td>75.</td><td>E-Nerolidol</td><td>0.15</td><td>1550</td><td>2036</td></tr><tr><td>76.</td><td>Spathulenol</td><td>0.03</td><td>1565</td><td>2107</td></tr><tr><td>77.</td><td>Epoxy Caryophyllene</td><td>0.08</td><td>1563</td><td>-</td></tr><tr><td>78.</td><td>CaryophylleneOxide</td><td>1.25</td><td>1567</td><td>2001</td></tr><tr><td>79.</td><td>HumuleneOxide</td><td>0.30</td><td>1597</td><td>2047</td></tr><tr><td>80.</td><td>Not identified compound</td><td>0.14</td><td>-</td><td>-</td></tr><tr><td>81.</td><td>Not identified compound</td><td>0.18</td><td>-</td><td>-</td></tr><tr><td colspan="2">TOTAL</td><td>98.27</td><td></td><td></td></tr></table>

Analysis performed by gas chromatography with FID detection on 2 columns of different polarity: HP1 and INNOWAX Individual components are identified by their retention index as described in Adams (2012) and their mass spectra are interpreted using the WILEY L computer library. Constituents are presented in the order of elution from the columns.

**Larvicidal activity of the mixture of *Piper umbellatum* and *Cannabis sativa* L leaves essential oil with temephos**
**Larvicidal activity of the mixture of *Piper umbellatum* with temephos on *Anopheles gambiae s.l* and *Culex quinquefasciatus* in 24 hours

*Anopheles gambiae* S.L and *Culex quinquefasciatus* larvae were exposed to the mixture of *Piper umbellatum* leaf essential oil with temephos for 48 hours exposure period. Dead larvae were recorded after 24 hours and 48 hours.
<figure><img src="image_3.png" /></figure>

gamb.: *Anopheles gambiae* S.L; Cx.: *Culex quinquefasciatus*

<caption>**Fig 1:** Larvicidal activity of the mixture of *Piper umbellatum* leaf essential oil with temephos on *Anopheles gambiae* S.L and *Culex quinquefasciatus* after 24 h</caption>

The different regression line equations of the effect of the mixture of *Piper umbellatum* leaf essential oil with temephos on *Anopheles gambiae s.l* and *Culex quinquefasciatus* for 24 hours exposure are: Yg=9.91+132.58X (H= 3.12; p= 0.014) and YCx=54.42+2071.32X(H=23.2; p<0.05) respectively. Lethal concentrations for 50% and 95% mortality (LC50 and LC95) are: 0.30%; 0.0011% and 0.64%; 0.024% respectively for *Anopheles gambiae* S.L and *Culex quinquefasciatus*.

Larvicidal activity of *Piper umbellatum* mixture on *Anopheles gambiae* S.L and *Culex quinquefasciatus* in 48hours

<figure><img src="image_4.png" /></figure>

gamb.: *Anopheles gambiae* S.L; Cx.: *Culex quinquefasciatus*

<caption>**Fig 2:** Larvicidal activity of the mixture of *P.umbellatum* leaf essential oil with temephos on *Anopheles gambiae* S.L and *Culex quinquefasciatus* after 48 h</caption>

The observation was extended to 48 hours. The efficacy increases with the duration of the exposure and the concentrations of the essential oil. The regression line equations generated are: Yg=89.39+90.75X (H=9; p=0.0008) and YCx=73.75+1412.28X (H=10; p<0.05) respectively for *Anopheles gambiae* S.L and *Culex quinquefasciatus*.

Thus, lethal concentrations for killing 50% and 95% of the exposed larvae (CL50) after 48 hours are: 0.023% and 0.001% respectively for *Anopheles gambiae* S.L and for *Culex quinquefasciatus*. While the lethal concentrations for 95% (LC95) mortality are: 0.38% and 0.015% respectively.

Larvicidal activity of *Cannabis sativa* L leaf essential oil mixture on *Anopheles gambiae* S.L and *Culex quinquefasciatus*

Larvicidal activity of *Cannabis sativa* L leaf essential oil mixture on *Anopheles gambiae* S.L and *Culex quinquefasciatus* in 24h

<figure><img src="image_5.png" /></figure>

Gamb.: *Anopheles gambiae* S.L; Cx.: *Culex quinquefasciatus*

<caption>**Fig 3:** Larvicidal activity of the mixture of *Cannabis sativa* L leaf essential oil with temephos on *Anopheles gambiae* S.L and *Culex quinquefasciatus* after 24 h</caption>

The regression line equations for larvicidal activity of the mixture of *Cannabis sativa* L leaf essential oil with temephos generated are: Yg=42.71+50.91X (H=3.34; p=0.012) and YCx=68.55+217.30X (H=10.17; p<0.05) respectively for *Anopheles gambiae* S.L and *Culex quinquefasciatus*. Lethal
concentrations for 50% and 95% mortality of the larvae are: 0.14%; 0.002% and 0.35% and 0.026% respectively for *Anopheles gambiae* s.l and *Culex quinquefasciatus*. *Culex quinquefasciatus* larvae are more susceptible to the mixture of *Cannabis sativa* L leaf essential oil with temephos than those of *Anopheles gambiae* S.L.

## Larvicidal activity of *Cannabis sativa* L leaf essential oil mixture with temephos on *Anopheles gambiae* S.L and *Culex quinquefasciatus* after 48h

<figure><img src="image_6.png" /><figcaption>gamb.: *Anopheles gambiae* S.L; Cx.: *Culex quinquefasciatus*</figcaption></figure>

<caption>**Fig 4:** Larvicidal activity of the mixture of *Cannabis sativa* L leaf essential oil with temephos on *An.gambiae* S.L and *Cx.quinquefasciatus* after 48h</caption>

Larvicidal activity of the mixture increases with dose and the duration of exposure. The regression line equations of the adjuvant effect of *Cannabis sativa* Leaf essential oil for effectiveness of temephos on the larvae of *Anopheles gambiae* S.L and *Culex quinquefasciatus* after 48 hours exposure are: Yg=49.39+260.55X (H=3.2; p=0.014) and YCx=81.06+134.27X (H=15.52; p<0.05) respectively for *Anopheles gambiae* S.L and *Culex quinquefasciatus*. The lethal concentrations for 50% and 95% mortality of the exposed larvae are: 0, 011%; 0.0013% and 0.17%; 0.013% respectively for *Anopheles gambiae* S.L and for *Culex quinquefasciatus*.

Lethal concentrations of the mixture efficient after 48 hours exposure are tenfold lower than those necessary for 24 hours exposure, both for the larvae of *Anopheles gambiae* S.L and *Culex quinquefasciatus*.

## Adjuvant property of *Cannabis sativa* L and *Piper umbellatum* leaves essential oil for effectiveness of permethrin on adult female of *Anopheles gambiae* S.Land *Culex quinquefasciatus*

### Knock down activity of the mixture of *Piper umbellatum* leaf essential oil with permethrin on *Anopheles gambiae* S.L and *Culex quinquefasciatus*

According to WHO(2003) and CDC(2013) procedures, adult female of *Anopheles gambiae* S.L and *Culex quinquefasciatus* were exposed to the impregnated material for 1 hour; at 10 minutes interval, individuals feld on their back or on the side were recorded.

<figure><img src="image_7.png" /><figcaption>**Fig 5:** Knock down activity of the mixture of *Piper umbellatum* leaf essential oil with permethrin on *Anopheles gambiae* S.L and *Culex quinquefasciatus*</figcaption></figure>

For 1 hour exposure, the mixture of *Piper umbellatum* leaf essential oil with permethrine knock down activity on adult female of mosquito was assessed with 95% confident interval. The regression equations generated for each mosquito are:

Yg= 2.66+1.74X (H=5.051; p=0.003) and Ycx= 32.46+1.20X (H=7.98; p=0.0005) respectively for *Anopheles gambiae* S.L and *Culex quinquefasciatus*. Time necessary for knocking down 50% and 95% (KDT₅₀ and KDT₉₅) of exposed mosquito are: 27±0.4min; 14±0.8min and 53±0.6min; 52±0.11min respectively for *Anopheles gambiae* S.L and *Culex quinquefasciatus*. The females of *Culex quinquefasciatus* succumb more quickly to the toxic effect of the mixture compared to those of *Anopheles gambiae* S.L.

### Knock down activity of the mixture of *Cannabis sativa* L leaf essential oil with permethrin on *Anopheles gambiae* S.L and *Culex quinquefasciatus*

Considering only the higher concentration of the mixture, adult female of *Anopheles gambiae* S.L and *Culex quinquefasciatus* were exposed to the mixture of *Cannabis sativa* L leaf essential oil with permethrin for 1 hour. At 10 minutes interval, individuals knocked down were recorded.

<figure><img src="image_8.png" /><figcaption>gamb.: *Anophles gambiae* S.L Cx.: *Culex quinquefasciatus*</figcaption></figure>

<caption>**Fig 6:** Knock down activity of *Cannabis sativa* L leaf essential oil mixture with permethrin on adult female of *Anopheles gambiae* S.L and *Culex quinquefasciatus*</caption>
The regression line equations generated are: Yg=29.73+1.21X (H=8.22; p=0.0004) and YCx=41.93+0.99X (H=9.52; p=0.0002) respectively for *Anopheles gambiae* S.L and *Culex quinquefasciatus*. Thus, time necessary for knocking down 50% and 95% of exposed mosquitoes are: 16±0.75min; 8±0.15min and 53±0.94min; 53±0.6 min respectively for *Anopheles gambiae* S.L and *Culex quinquefasciatus*. After one hour of contact no difference of

susceptibility appears between the two mosquitoes species (H=1.88; p=0.82).

**Adulticidal activity of the mixture of *Piper umbellatum* leaf essential oil with permethrin on *Anopheles gambiae* S.L and *Culex quinquefasciatus***

After the exposure, 24h late, dead mosquito were recorded and the curve below was plotted.

<figure><img src="image_9.png" /><figcaption>**Fig 7:** Adulticidal property of the mixture of *Piper umbellatum* leaf essential oil with permethrin on *Anopheles gambiae* S.L and *Culex quinquefasciatus*</figcaption></figure>

The regression line equations generated are: Yg=55.40+211.64X (H=8.14; p=0.0012) and YCx=69.76+141.76X (H=11.22; p=0.0003). From these equations, lethal doses for 50% and 95% mortality (LC50 and LC95) are: 0.024%; 0.011% and 0.18%; 0.17% respectively for *Anopheles gambiae* S.L and *Culex quinquefasciatus*. Adult of *Anopheles gambiae* S.L seem to be less susceptible to the toxicity of the mixture of permethrin with *Piper umbellatum* leaf essential oil than

those of *Culex quinquefasciatus* (H=4; p=0.03).

**Adulticidal activity of the mixture of *Cannabis sativa* L leaf essential oil with permethrin on *Anopheles gambiae* S.L and *Culex quinquefasciatus***

After the contact of adult mosquitoes with increasing concentrations of the mixture and 24 hours post exposure, dead individuals were counted and the curve below was plotted with 95% confident interval.

<figure><img src="image_10.png" /><figcaption>gam: *Anopheles gambiae* S.L; Cx: *Culex quinquefasciatus*</figcaption></figure>

<caption>**Fig 8:** Adulticidal activity of the mixture of *Cannabis sativa* L leaf essential oil with permethrin on *Anopheles gambiae* S.L and *Culex quinquefasciatus*</caption>
The different regression line equations of the activity of the mixture of permethrin and *Cannabis sativa* L leaf essential oil are: $Y_{An}=41.79+294.3X(H=6.2; p=0.003)$ and $Y_{Cx}=56.45+244.4X (H=8.4; p=0.001)$ respectively for *Anopheles gambiae* S.L and *Culex quinquefasciatus*. Lethal concentrations for 50% and 95% ($LC_{50}$ and $LC_{95}$) mortality are: 0.027%; 0.01% and 0.18%; 0.15% respectively for *Anopheles gambiae* S.L and *Culex quinquefasciatus*. Adult of *Anopheles gambiae* S.L seem to be less susceptible the toxicity of the mixture of *Cannabis sativa* L leaf essential oil than those of *Culex quinquefasciatus* (H=3.9; p=0.016).

## Identification of adult mosquitoes

Molecular analysis carried out with DNA extracted from 71 adult individuals of the *gambiae* complex collected near the Ewoé River and the morphological determination of individuals collected from Melen pond revealed the results given in the table 5 below.

<table><caption>**Table 5:** Specific composition of adult mosquitoes collected at Melen pond and on the banks of the Ewoé River (Abé *et al.*, 2019)</caption><thead><tr><th>Harvest site</th><th>gender</th><th>Species</th><th>Number</th><th>%</th></tr></thead><tbody><tr><td rowspan="3">Melen Pond</td><td>**Anopheles**</td><td>An. gambiae s.l</td><td>4</td><td>2%</td></tr><tr><td rowspan="2">**Culex**</td><td>Cx.perfuscus</td><td>14</td><td>7%</td></tr><tr><td>Cx.quinquefasciatus</td><td>182</td><td>91%</td></tr><tr><td colspan="2">Total</td><td>-</td><td>200</td><td>100%</td></tr><tr><td rowspan="2">Ewoe river</td><td rowspan="2">**Anopheles**</td><td>An. gambiae s.s</td><td>3</td><td>4,23%</td></tr><tr><td>An. coluzzii</td><td>68</td><td>95,77%</td></tr><tr><td colspan="2">Total</td><td>-</td><td>71</td><td>100%</td></tr></tbody></table>

## Discussion

The aim of our study was to evaluate the effectiveness of the combination of *Piper umbellatum* and *Cannabis sativa* L leaves with temephos and permethrin on *Anopheles gambiae* S.L and *Culex quinquefasciatus*, two major vectors under Laboratory conditions.

The chemical analysis of essential oils revealed 81 compounds for *Cannabis sativa* L and 24 compounds for *Piper umbellatum*. These compounds constitute 98.27% and 89.6% of the mass of essential oil analysed (respectively for *Cannabis sativa* L and for *Piper umbellatum*) and are mostly terpenes. In addition, during this chemical analysis of essential oils, several compounds remained undetermined. More sophisticated equipment and software will allow their subsequent determination

Comparing the results of chemical analysis, there are more compounds in *Cannabis sativa* L than in *Piper umbellatum* leaf essential oil. This difference would depend on the specificity of each plant and also on the type of equipment used for the chemical analysis of essential oils. Terpenic compounds such as pinenes, terpinolene, 1-8 cineol found in these plants have been reported to have insecticidal, bactericidal, acaricidal, antiviral and fungicidal activity by several authors (Brochot *et al.*, 2017).

The morphological and molecular identification of adult mosquitoes from wild individuals of *Anopheles gambiae* S.L collected near Ewoe River revealed a composition of 95.77% of *Anopheles coluzzii* and 4.23% of *Anopheles gambiae* S.L; while the only morphological identification of adult mosquitoes from Melen's pond revealed: 2% of *Anopheles coluzzii*; 7% *Culex perfuscus* and 91% *Culex quinquefasciatus*. Thus, according to the CDC (2013), the results of biological assays carried out on mosquitoes from Ewoé River and Melen pond are attributed to *Anopheles*

coluzzii (*Anopheles gambiae* S.L) and *Culex quinquefasciatus* respectively.

Regarding the adjuvant effect of the essential oil of the leaves of *Cannabis sativa* L for the effectiveness of temephos on the larvae of the wild strain of *Anopheles gambiae* S.L after 24 hours, the mortality rate (46 ± 0.2%) is almost constant between essential oil concentrations from 0.00156% to 0.0062% and similar to the larval mortality rate for temephos acting alone. But from a concentration of 0.0125% of *Cannabis sativa* L leaf essential oil in the mixture, there is a drop in larval mortality from 46 ± 0.2% to 37 ± 0.2%. Unlike the larvae of *Anopheles gambiae* S.L, this drop in larval motility in *Culex quinquefasciatus* (44 ± 0.4%) is immediately observed from a concentration of 0.00125% from *Cannabis sativa* L leaf essential oil in the mixture with temephos. Larvae mortality then increases for each of the species with the concentration of *Cannabis sativa* Lleaf essential oil in the mixture with temephos. Likewise, mixing temephos with *Piper umbellatum* laef essential oil gives almost similar larval mortality rates for *Anopheles gambiae* S.L (52 ± 1.2%) between the essential oil concentrations of 0.00125% to 0.0125% in the mixture with temephos. But from a concentration of 0.025% of P.umbellatum leaf essential oil, there is a drop in larval mortality of the field strain of *Anopheles gambiae* S.L from 52 ± 1.2% to 38 ± 1%. Subsequently, the effectiveness of the combination against the larvae of the field strain of *Anopheles gambiae* S.L increases with the doses of *Piper umbellatum* leaf essential oil in the mixture with teemphos. For *Culex quinquefasciatus* larvae, the mortality rate (68 ± 0.7%) immediately increases with the concentration of essential oil in the mixture. It thus appears, an effect of partial neutralization of the toxicity of temephos by the essential oils of *Cannabis sativa* L and *Piper umbellatum* leaves essential oil.

As for the activity of mixing the essential oils each plant leaf, *Cannabis sativa* L and *Piper umbellatum* with permethrin on wild adults of *Anopheles gambiae* S.L and *Culex quinquefasciatus*, the comparison of the average knock down rates on the effect of the mixture of *Cannabis sativa* L leaf with permethrin does not show a significant difference between adults of the two species (H = 1.88; p = 0.12). For the permethrin-essential oil combination of *Piper umbellatum* leaf essential oil on the contrary, the comparison of the means of the Knock down rates of the two species shows a significant difference (H = 4; p = 0.042). *Culex quinquefasciatus* succumbing faster than *Anopheles gambiae* S.L Knock down although being a preliminary phase of mortality does not lead to mortality with the same proportions for the mixture permethrin-essential of *Cannabis sativa* L leaf. Thus, comparison of the average mortality rates of the two species shows that adults of *Culex quinquefasciatus* are more susceptible to the mixture than those of the field strain of *Anopheles gambiae* S.L Comparison of average mortality rates of adults of the field strain of *Anopheles gambiae* S.L and of *Culex quinquefasciatus* shows that the individuals of *Culex quinquefasciatus* are more sensitive as well for the mixture permethrin-essential oil of *Cannabis sativa* L leaf (H = 3.9; p = 0.016) than for the permethrin - essential oil mixture of *Piper umbellatum* leaf (H = 4; p = 0.03). This behavior would be linked to the specificity of each of the two species. There is also a drop in the mortality rate of adults of the field strain of *Anopheles gambiae* S.L compared to the
control in the lowest concentrations of essential oil in the permethrin-essential oil mixtures. These rates thus go from 49 ± 0.5% for permethrin acting alone to 38 ± 0.6% in the mixture of permethrine with 0.0125% from *Cannabis sativa* L leaf essential oil and from 51 ± 0, 7% for permethrin acting alone to 46 ± 1% in the mixture of permethrin with a concentration of 0.0125% from *Piper umbellatum* leaf essential oil. This drop in the mortality rate, on the contrary, is not observed in *Culex quinquefasciatus*; their mortality rate immediately increases with the concentrations of essential oil in the mixture.

Indeed, whether for the larvicidal or adulticidal activity of mixtures of synthetic insecticides with essential oils on *Anopheles gambiae* S.L and *Culex quinquefasciatus*, there appears to be a partial attenuation of the toxicity of synthetic insecticides used in this work (temephos and permethrin). This partial neutralization or attenuation of the toxicity of temephos and permethrin by *Cannabis sativa* L and of *Piper umbellatum* leaves essential oil could be due to a blocking or partial inhibition of the active principles of temephos and permethrin. This partial antagonism has been observed by some authors when combining linalol-thymol with spirotetramat against *Myzus persicae* (Faraone et al., 2015) [39] or when combining several essential oils with permethrin against adults of *Aedes aegypti* and of *Anopheles gambiae* (Gross et al., 2017) [46]. This activity of partial inhibition of some of the active ingredients of temephos and permethrin without permanently altering their effectiveness, would offer an advantage in the fight against vectors and other harmful insects. Because the impact of reference insecticides on non-target organisms could thus be limited. In addition, the rebound effect observed for the activity of temephos and permethrin thereafter with increasing doses of essential oils would allow their effectiveness to be improved compared to resistant insects. The work carried out by several authors to assess the effectiveness of the combination of essential oils with reference insecticides has shown the effectiveness of the essential oil-permethrin combinations against adults of *Aedes aegypti* (Chansang et al., 2018) [47] and flies domestic (Joffe et al., 2011) [48]. In the work of these authors, by mixing with essential oils, the concentrations of synthetic insecticides are reduced while preserving their effectiveness due to the low concentrations of essential oils. This is the case in this work; the essential oils from *Cannabis sativa* L and *Piper umbellatum* leaves increase the effectiveness of temephos and permethrin, just like studies carried out in Ghana on a strain of *Anopheles gambie* S.L resistant to deltametrin and permethrin (Dadzie et al., 2017) [49] and in Iran on the effectiveness of the combination of the essential oils of *Eucalyptus globulus* and *Rosmarinus officinalis* on cockroaches, *Culex pipiens*, *Anopheles stephensi* and house flies (Zibaee and Khorram; 2015) [50].

## Conclusion

From this study, it appears that low concentrations of essential oil from *Cannabis sativa* L and *Piper umbellatum* partially inhibits the effectiveness of temephos and permethrin on adult and larvae of *Anopheles gambiae* S.L and *Culex quinquefasciatus*. The effectiveness of the mixture then increases with the concentrations of essential oil.

To properly establish the effectiveness of the mixture of *Cannabis sativa* L and *Piper umbellatum* leaves essential oil

with synthetic insecticides on *Anopheles gambiae* S.L and *Culex quinquefasciatus*, trials must be carried out in the field. More appropriate apparatus and methods will allow an improvement in the extraction yields of essential oils and the identification of the compounds which have hitherto been unknown. Other in-depth studies may explore how to resolve the problem of the extreme volatility of essential oils; determine the essential oil responsible for the partial inhibition of the toxicity of temephos and permethrin and the mechanisms involved. The exploration of the adjuvant property of local plants essential oil must continue, in view of the fact that the combination of essential oil with synthetic insecticides would reduce their hazardous effect to non-target organisms and enhance their effectiveness against resistant insects.

## Authors Declaration

The authors declare no competing interests.

## Authors' participation

All authors participated equality in this work.

## Acknowledgments

We thank Dr. Antonio Ch Christopher -Nkondjio and Dr. Parfait Awono Ambene for their support and councils; Dr. Catherine Rupp Jacques and Hervé Casabianca for Chemical analysis of plant essential oil; Dr. Elysée Nchoutpouen for morphological identification of adult mosquitoes.

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