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Larvicidal Activities of Essential Oils Extracted from Five Algerian Medicinal Plants against Culiseta longiareolata Macquart. Larvae (Diptera: Culicidae).


Culicidae, or mosquitoes as commonly known, is a family of Diptera insects that reproduce quickly and abundantly. Simultaneously, this family includes major vectors for many deadly and dangerous diseases. Therefore, the importance of the mosquito family in terms of public health makes mosquito control an important initiative to minimize the negative effects of mosquito-born-diseases. Mosquito control may depend on various strategies; the most common in the past decades was the use of synthetic insecticides as inexpensive and available products. However, the use of synthetic insecticides has over time created environment pollution and resistance problems (1, 2).

Recently, eco-friendly methods were developed to control mosquitoes. For instance, the enhancement of behavior-based control tools and the development of repellent and toxic products based on botanic components can target different mosquito life stages (3, 4). Essential oils (EOs) extracted from different parts of plants were frequently tested for their mosquitocidal activity (5). These primary botanic materials present various biological activities. They can act as insecticides where they can affect the oviposition, survival, larval duration, pupation and insect emergence (6, 7). However, the larvae stage appears to be more appropriate to control mosquito populations because of the high reproduction rates and larvae food mechanisms that allow a high number of mosquito individuals to be targeted simultaneously. Therefore, the assessment of the larvicidal efficacy of various plant derivatives was the main objective of many research papers (8-11).

Culiseta longiareolata (Macquart 1838) constitutes with the Culex pipiens (Linnaeus 1758) complex the most abundant species in Algeria. It usually breeds near human habitations, however, the females prefer to feed on bird blood (12). Cs longiareolata has uniquely adaptive and survivor features. Kiflawi et al. (13) have confirmed that the females of this species showed an adaptive response against the risk of predation and negative density effects where they avoid laying their eggs in predator pools. Further, Cs longiareolata is considered as a primary vector of Plasmodium (Giovannolaia) circumflexum (Kikuth 1931), Plasmodium relictum (modified from Garnham 1966) and Plasmodium polare (Manwell 1934) in birds, and its capacity to transmit P. relictum in Algeria was proven experimentally (14, 15). In this context, we have assessed the larvicidal activity of EOs extracted from five aromatic medicinal plants, harvested from Northeastern Algeria, against Cs longiareolata larvae. The efficacy of the tested EOs will be evaluated by calculating the [LC.sub.50] and [LC.sub.90] values and by comparing them with the [LC.sub.50] and [LC.sub.90] values of the same EOs tested previously against other targeted mosquito species.


Mosquito Collection

Culiseta longiareolata larvae were collected regularly from three clean fixed and controlled pools in Algeria, where the mosquitoes were not exposed to any insecticides. Larvae of the third and fourth instar were used directly in the test; eggs, first and second instar larvae were reared in room temperature (27[degrees]C[+ or -]2[degrees]C), in a 12 h light: 12 h dark photoperiod, until the fourth instar was reached.

Essential Oils Extraction

The aerial parts of the tested plants were collected from different regions in the Mediterranean and semi-arid climate northeastern Algeria: Thymus vulgaris L. from Guelma, Artemisia herba-alba Asso from M'Sila, Juniperus phoenicea L. from Jijel, Rosmarinus officinalis Linn from Bouira and Eucalyptus globules L. from Batna. The plants' collection started at the beginning of the summer (June) in 2018. The samples were air-dried at room temperature. The dried plants were submitted to classical steam distillation for 3-6 h. The samples were exposed to the water vapor produced in the flask crosses, the vapor was charged with the EO, and then was condensed in the condenser. The EO floated on the water surface was then recuperated. The yield of the EOs was between 0.8 and 1.5%.

Larvicidal Bioassay

According to WHO guidelines for laboratory and field testing of mosquito larvicides (16), we tested the larvicidal activity of EOs extracted from the leaves of five aromatic medicinal plants T. vulgaris, A. herba-alba, J. phoenicea, R. officinalis ,E. globulus against Culiseta longiareolata larvae under laboratory conditions. The EOs were extracted by steam distillation, they were next serially diluted in ethanol to obtain 10%, 1%, 0.1% and 0.01% of stock solution, and 0.1-1ml of the previous dilutions were added to 100ml of water to obtain the final concentrations. A series of concentrations and controls were applied on 25 mosquito larvae distributed in five cups containing 100ml of water. A total of 8925 larvae were tested. We started the test with the lowest concentrations. The concentrations that showed less than 10% mortality were excluded. Concentrations that showed 10% mortality or more were replicated 4 times, and each test was run three times. After 24h of exposure, moribund and dead larvae were counted. We have chosen four concentrations which caused between 10% and 90% mortality to determine the [LC.sub.50] and [LC.sub.90] values. The data obtained from the four replicates in the three tests were pooled for analysis.

Statistical Analyses

Data were subjected to probit analysis using SPSS software V25 (Using probit model because of the normal distribution of data); and final concentrations were transformed to log10. Lethal concentration [LC.sub.50] and [LC.sub.90] with a 95% confidence limit (CL) suspected of killing 50% and 90% of the population respectively, were calculated and presented with the regression equations (Y= a +b*x) and regression coefficients ([R.sup.2]).


Five plant EOs were tested to evaluate their larvicidal activity, and the tested oils revealed various mortality percentages at different concentrations (Table 1). The majority of the tested oils showed 100% mortality at 200ppm final concentration, except for T. vulgaris that showed 100% mortality at 80ppm. Further, the oils started to affect the larvae life at different concentrations; the lowest concentration that caused equal or more than 10% mortality was 20ppm for T. vulgaris , 40ppm for J. phoenicea, 50ppm for A. herba-alba and R. officinalis and 70ppm for E. globules (Table1). The 24h [LC.sub.50] and [LC.sub.90] estimate, upper and lower values obtained from the larvicidal activity test of EOs extracted from the five plants in addition to the regression equations and regression coefficients are presented in Table 2. T. vulgaris was the most efficient with 25.64 (16.58-32.03) [LC.sub.50] and 50.53 (40.15-82.43) [LC.sub.90], while A. herba-alba was the least efficient. Likewise, the influence degree of increasing one unit of EOs concentration on their larvicidal activity was different. Among the tested EOs, the augmentation of one unit of R. officinalis concentration showed the highest influence in increasing the [LC.sub.50] and [LC.sub.90] (b=7.16). The [R.sup.2] was close to 1 in all probit analysis, the minimal residuals obtained between the observed and expected values was shown by E. globulus EO ([R.sup.2]=0.99) (Table 2).


The current study has confirmed that the EOs extracted from the aromatic medicinal plants T. vulgaris, A. herba-alba, J. phoenicea, R. officinalis and E. globulus present an efficient larvicidal activity against the Culiseta longiareolata larvae; however, the mortality responses obtained were varying.

T. vulgaris is a flowering herb that has a worldwide distribution (17). From the total of the tested oils, the T. vulgaris EO was the most efficient. This EO was previously assessed by Knio et al. (18) against the Ochlerotatus caspius (Pallas 1771) larvae; however, its toxicity against Oc caspius ([LC.sub.50]=33.65ppm; [LC.sub.90] =50.85ppm) was less than that shown by our T. vulgaris EO. Likewise, the larvicidal activity of the EOs extracted from the Juniperus species was tested in previous studies: J. Phoenicea against Aede salbopictus (Skuse 1894) ([LC.sub.50]= 55.5ppm; [LC.sub.90]= 77ppm), and J. virginiana L. against Ae aegypti (Linnaeus 1762) and Cx pipiens (19, 20). Comparing our results, our J. phoenicea EO showed lower larvicidal activity against Cs longiareolata. Moreover, the larvicidal activity of R. officinalis EO was assessed against Ae albopictus ([LC.sub.50]<250ppm), Cx tritaeniorhynchus (Giles 1901) ([LC.sub.50]= 115.38ppm; [LC.sub.90]= 211.53ppm) and Anopheles subpictus Grassi ([LC.sub.50]= 64.5ppm; [LC.sub.90]= 113.74ppm) (21, 22). The R. officinalis EOs tested against Ae albopictus, Cx tritaeniorhynchus and An subpictus in the previous researches showed lower values than the toxicity results that we obtained by testing the same EO against Cs longiareolata.

The other EOs E. globules and A. herba-alba were less efficient; however, their lethal concentrations were notable. E. grandis L. EO and its major components were assessed for their larvicidal activity against Aedes aegypti by Lucia et al. (23). The EO showed 32.4ppm [LC.sub.50] and the principal components [alpha]-pinene (52.71%) and 1,8-cineole (18.38%) showed 15.4ppm and 57.2ppm [LC.sub.50] respectively. The principal leaf oil components of E. globules harvested from Algeria are [alpha]-pinene and 1,8-cineole, according to Samir et al. (24). However, our E. globules EO tested against Cs longiareolata was less efficient ([LC.sub.50]= 95.83ppm). Furthermore, EOs extracted from Artemisia genus were assessed for their larvicidal activity against various mosquito species. Our A. herba-alba EO tested against Cs longiareolata larvae was more efficient ([LC.sub.50]= 86.67ppm) than A. vulgaris L. that was tested by Ilahi and Ullah (25) against Cx quinquefasciatus ([LC.sub.50]= 803.2ppm), but less efficient than A. absinthium L. tested by Govindarajan and Benelli (26) against An stephensi (Liston 1901), An subpictus, Ae aegypti, Ae albopictus, Cx quinquefasciatus (Say 1823), and Cx tritaeniorhynchus ([LC.sub.50]=41.85, 52.02, 46.33, 57.57, 50.57, and 62.16 ppm respectively). Various mosquito species were targeted in the previous researches to assess the larvicidal activity of EOs. However, Cs longiareolata was not previously targeted by EOs, but by the lichen metabolites evaluated by Cetin et al. (27), that showed high larvicidal activity against Cs longiareolata.

The results obtained confirm the previous studies; the use of EOs can serve as an eco-friendly method to control mosquito larvae. However, the noted variability in the efficacy level of the tested oils may be due to their chemical composition and the percentages of their principal components as [alpha]-Pinene, Camphor and 1,8-Cineole (Table 3); whereas, the direct use of the principal components of EOs may produce a higher efficacy in mosquito control. This hypothesis was proven in the study conducted by Lucia, Gonzalez-Audino (23), where the principal components of Turpentineand E. grandis EO showed lower [LC.sub.50] than that obtained by the use of the entire E. grandis EO. Moreover, the repellency effect of the thyme EO compounds against Culex pipiens mosquito evaluated by Park et al. (28) showed higher repellent efficacy of [alpha]-Terpinene and Carvacrol than the commercial formulation diethyltoluamide (DEET), and an equal efficacy between the Thymol component and the DEET.


The EOs extracted from the aromatic medicinal plants and their principal components may serve as safe products to control the Culiseta longiareolata larvae in Algeria; nevertheless, their practical application remains a fundamental step to evaluate their field efficacy and to note their possible secondary effects on non-targeted organisms.

Peer-review: Externally peer-reviewed.

Author Contributions: Conception/Design of study: I.N., M.B.; Data Acquisition: I.N.; Data Analysis/Interpretation: I.N.; Drafting Manuscript: I.N.; Critical Revision of Manuscript: I.N., M.B.; Final Approval and Accountability: I.N., M.B.; Supervision: M.B.

Conflict of Interest: The authors declare that they have no conflicts of interest.

Financial Disclosure: There are no funders to report for this submission.


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Ismahane Nabti (1) (iD), Mustapha Bounechada (1) (iD)

(1) University Ferhat Abbas, Faculty of Nature and Life Sciences, Department of Biology and Animal Physiology, Laboratory Research LADPVA, Setif Algeria

ORCID IDs of the authors: I.N. 0000-0001-5192-5626; M.B. 0000-0002-3237-5769

Please cite this article as: Nabti I, Bounechada M. Larvicidal Activities of Essential Oils Extracted from Five Algerian Medicinal Plants against Culiseta longiareolata Macquart. Larvae (Diptera: Culicidae). Eur J Biol 2019; 78(2): 133-138. DOI: 10.26650/EurJBiol.2019.0015

Address for Correspondence: Ismahane Nabti


Submitted: 17.09.2019

Revision Requested: 01.10.2019

Last Revision Received: 06.10.2019

Accepted: 16.10.2019

DOI: 10.26650/EurJBiol.2019.0015
Table 1: The mortality observed to the Culiseta longiareolata larvae,
caused by the application of the tested essential oils at different
concentrations, with the arithmetic mean (AM) and standard error (SE).

     Dead in a total of 300 larvae (AM[+ or -]SE)
IC   Aliquot  FC     Thymus                   Juniperus
(%)  (ml)     (ppm)  vulgaris                 phoenicea

     0,2       20     93 (7.75[+ or -]1.53)     -
     0,4       40    253 (21.08[+ or -]1.97)   94 (7.83[+ or -]1.23)
     0,5       50    257 (21.42[+ or -]1.59)  103 (8.58[+ or -]1.23)
 1   0,6       60    286 (23.83[+ or -]0.42)  156 (13[+ or -]1.58)
     0,7       70      -                        -
     0,8       80    300 (7.75[+ or -]1.53)   176 (14.67[+ or -]1.91)
     0,9       90      -                        -
     1        100    300 (25[+ or -]0.0)      255 (21.25[+ or -]1.55)
10   0,2      200    300 (25[+ or -]0.0)      300 (25[+ or -]0.0)

     Dead in a total of 300 larvae (AM[+ or -]SE)
IC   Artemisia               Rosmarinus
(%)  herba-alba              officinalis

       -                       -
       -                       -
      24 (2[+ or -]0.75)      75 (6.25[+ or -]1.54)
 1    57 (4.75[+ or -]1.52)  106 (8.83[+ or -]1.56)
       -                       -
      89 (8.17[+ or -]1.36)  236 (19.67[+ or -]0.85)
       -                       -
     216 (18[+ or -]2.03)    274 (22.83[+ or -]1.21)
10   300 (25[+ or -]0.0)     300 (25[+ or -]0.0)

     Dead in a total of 300 larvae (AM[+ or -]SE)
IC   Eucalyptus
(%)  globules

 1     -
      68 (5.67[+ or -]1.1)
     107 (8.92[+ or -]1.02)
     134 (11.17[+ or -]1.6)
     159 (13.25[+ or -]1.69)
10   300 (25[+ or -]0.0)

IC(initial concentration), FC (final concentration)

Table 2: The [LC.sub.50] and [LC.sub.90] values of essential oils
extracted from T. vulgaris, A. herba-alba, J. phoenicea, R. officinalis
and E. globules against the 3rd and 4th instar larvae of the Culiseta
longiareolata, after 24 hours exposure period; with regression
equations and regression coefficients (R2).

               [LC.sub.50] (ppm) 95% CI  [LC.sub.90] (ppm) 95% CI
Essentialoils  Estimate  Lower  Upper    Estimate  Lower   Upper

Thymus         25.64     16.58    32.03   50.53     40,15    82.43
Juniperus      59.83     45.36    75.81  137.68     97.21  <250
Artemisia      86.67     66.59  <250     139.55     98.03  <250
Rosmarinus     64.18     55.41    72.56   96.55     82.73   139.84
Eucalyptus     95.83     92.27   101.09  168.25    146.59   201.87

Essentialoils  Sig (df)    Regression       R2

Thymus         p>0.05 (2)  y=-6.15+4.36*x   0.97
Juniperus      p>0.05 (3)  y=-649+3.66*x    0.9
Artemisia      p>0.05 (2)  y=-11.77+6.08*x  0.93
Rosmarinus     p>0.05 (2)  y=-12.93+7.16*x  0.98
Eucalyptus     p>0.05 (2)  y=-1045+5.28*x   0.99

Sig (significance level), df (degrees of freedom)

Table 3: Principal component percentages of T. vulgaris, A. herba-alba,
J. Phoenicea, R. officinalis and E. globules harvested from Algeria,
according to previous works.

Principalcomponents      T. vulgaris (29)  J. phoenicea (30)

Carvacrol                11.41              -
Thymol                   25.57              -
[alpha]-Pinene           12.1              34.5
[alpha]-Terpinylacetate   -                14.7
p-Cymene                 26.36              -
Thymoquinone             10.5               -
[beta]-Phellandrene       -                22.4
Camphor                   -                 -
1,8-Cineole               -                 -
[beta] -Caryophyllene     -                 -
Borneol                   -                 -
?-terpinene               -                 -
[beta]-thujone            -                 -
chrysanthenone            -                 -
trans-pinocarveol         -                 -

Principalcomponents      A. herba-alba (31)  R. officinalis (32)

Carvacrol                -                    -
Thymol                   -                    -
[alpha]-Pinene           Tr                   5.4
[alpha]-Terpinylacetate  -                    -
p-Cymene                 -                    -
Thymoquinone             -                    -
[beta]-Phellandrene      -                    -
Camphor                  19.4                14.6
1,8-Cineole              Tr                  12.2
[beta] -Caryophyllene    -                   10.9
Borneol                  -                   10.6
?-terpinene              23.8                 -
[beta]-thujone           15.0                 -
chrysanthenone           15.8                 -
trans-pinocarveol        16.9                 -

Principalcomponents      E. globules (24)

Carvacrol                 -
Thymol                    -
[alpha]-Pinene            8.8
[alpha]-Terpinylacetate   -
p-Cymene                  -
Thymoquinone              -
[beta]-Phellandrene       -
Camphor                   -
1,8-Cineole              71.3
[beta] -Caryophyllene     -
Borneol                   -
?-terpinene               -
[beta]-thujone            -
chrysanthenone            -
trans-pinocarveol         -
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Author:Nabti, Ismahane; Bounechada, Mustapha
Publication:IUFS Journal of Biology
Geographic Code:6ALGE
Date:Dec 1, 2019
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