Indian Journal of Experimental Biology

Vol. 62, August 2024, pp. 687-692

NOTES
Larvicidal activity and chemical Serbia, Italy, Hungary, Greece, France, Spain,
compositions of Juniperus phoenicea L. leave Croatia, Belgium, Austria, Germany, Tunisia, Burkina
Faso, Uganda, Nigeria, Ivory Coast and Senegal1. Cx.
extract against Culex pipiens L. pipiens also transmit several other arboviruses, such
Fouzi Boulkenafet1, Samia Benzazia1, Lamia Mellahi1,
as the Sindbis virus, Japanese encephalitis virus, and
Fahd A. Al-Mekhlafi2*, Nael Abutaha2, Mohammed S. Rift Valley fever virus, and act as vectors for filarial
Al-Khalifa2 & Simonetta Lambiase3 worms and plasmodia that cause avian malaria2-4.
1
Department of Natural Sciences and Life, University of 20th
Vector control using insecticidal agents is one of the
August 1955 Skikda, Algéria standard methods to control disease transmission.
2
Department of Zoology, College of Science, Acetofenate, propoxur and dichlorvos are commonly
King Saud University, Riyadh 11451, Saudi Arabia used insecticidal agents. The intense use of these
3
Department of Public Health, Experimental and Forensic insecticides by household communities, private
Medicine, University of Pavia, 27100 Pavia, Italy companies, and the government has resulted in
Received 26 September 2023; Revised 20 January 2024 resistance in Culex populations5. Resistance to these
insecticidal agents has been recorded in many
Mosquitoes are of great concern in many countries. Culex
pipiens L. (Diptera:Culicidae) is the vector of the West Nile virus countries6-8.
and Usutu Viruses. Intense use of insecticidal agents has resulted The mosquitocidal resistance is increasing, and the
in increased mosquitocidal resistance, and thereby search for new
success rate of new insecticidal agents in biocontrol
effective mosquitocidal agents. In this context, we have explored
the larvicidal potential of Phoenician juniper [Juniperus phoenicea L. programs is low9,10. Therefore, this necessitates
(Fam. Cupressaceae)] against Cx. pipiens and studied the searching for new mosquitocidal agents. Botanical
chemical composition of the leaf extract. Fresh leaves of mosquitocides have a long history and are a safer
J. phoenicea were macerated in 70% methanol. The LC50 at 24, 48 alternative to controlling various insect pests.
and 72 h post-treatment with methanol extract were 7.14, 3.97 and
3.17 µg/mL, respectively, against Cx. pipiens larvae. In treated Secondary metabolites extracted from plants usually
larvae, the cells lost integrity, nucleus and cell membrane damage, have low toxicity, degrade rapidly (within 3-4 days),
basal lamina detached, and lost nuclear and cytoplasmic materials. such as Neem insecticides11, and act faster than
From GC–MS analysis, 19 compounds were detected. Some of biological control12. Juniperus phoenicea L.
the phytochemicals detected in J. phoenicea extract were 9,12-
(Cupressaceae) is a small tree with a wide geographic
octadecadienoic acid (Z,Z)-(50.2.%), podocarpa-8,11,13-trien-3-one,
14-isopropyl-13-methoxy-(13.2%), -Amino-7,10-dimethyldibenzo distribution and can be found from Portugal to Saudi
[b,f][1,4]oxazepin-11(10H)-one (8.4%), and n-hexadecanoic acid Arabia13. This wide geographical range allows genetic
(5.6%). J. phoenicea offered promising larvicidal activity against variability14. Decoctions from the leaves are used to
Cx. pipiens. treat diuretic diarrhea, rheumatism, bronco-pulmonary
illness, and diabetes15. A hypoglycemic agent is made
Keywords: Cupressaceae, Mosquitocidal resistance, 9,12-
Octadecadienoic, Phoenician juniper, Saint Louis encephalitis from the berries and leaves of this plant16. Here, we
virus, West Nile virus, Usutu virus evaluated the larvacidal efficacy and chemical
constituents of J. phoenicea leaf extract against
Culex pipiens (Linnaeus, 1758), the principal vector Cx. pipiens larvae.
of the West Nile virus and Usutu viruses (USUV) are
major health concerns in several nations. WNV is Materials and Methods
commonly found in Africa, Europe, West Asia, Collection and extraction
Australia, Canada, and Venezuela. The USA, Russia, Juniperus phoenicea fresh leaves were collected in
Greece, Romania and the Middle East faced the most February-March from Djerma Batna city, Algeria, and
severe outbreaks. (WHO, 2023). Similarly, USUV identified by Sakhraoui N, Professor of Botany,
was detected in Switzerland, the Czech Republic, Natural and Life Sciences Department of University
————————
20th August 1955 Skikda, Algeria. The voucher
*Correspondence: sample (ALJP22026) was deposited in the same
E-Mail: [email protected] department. The leaves were washed using distilled
688 INDIAN J EXP BIOL, AUGUST 2024

water, dried at 25ºC in a well-ventilated area, and immediately fixed in formaldehyde (10%).
ground using a commercial grinder. A hundred Afterwards, sections with a thickness of 5 µm were
grams of plant powder was macerated in 70% cut from the embedded larval preparations. The
methanol (300 mL) and filtered, and the filtrate was sections were deparaffinized and rehydrated with
rotary evaporated (Heidolph, Germany), and the yield ethanol in phosphate-buffered saline (PBS) solutions.
was calculated. The sections were stained with hematoxylin and eosin
Test mosquitoes using a Leica Biosystems autostainer (Wetzlar,
Larvae of Cx. pipiens were collected from Natural Germany). A microscope was used to view and record
and Life Sciences Department, University of 20th images (Olympus BX53, Japan).
August 1955 Skikda, Algeria. They were housed in GC–MS analysis
35, 20 and 15 cm plastic trays and fed fish flakes GC–MS analysis was carried out using a Perkin
(Aquafin max feeding) at 30°C. With a sieve, the Elmer GC Calarus 600T combined with a Clarus
pupae were manually separated into a 500 mL beaker 600T single quadrupole mass spectrometer attached to
of tap water. Adult mosquitoes were fed glucose using an autosampler and gas chromatography interfaced to
tissue paper soaked in a 10% glucose solution and a mass spectrometer (GC–MS) instrument. The
occasionally blood-fed using trapped mice. The laid following parameters were used: helium (99.999%)
eggs were used for control and bioassay tests. was used as the carrier gas at a constant flow of
Larvicidal bioassay
1.491 mL/min and injection volume of 1.0 mL, injector
The larvicidal bioassay was conducted based on temperature was 140°C, and ion source temperature
WHO guidelines (WHO, 2005). Different concentrations was 200°C. The capillary column was 624 ms (30 m
of J. phoenicea extract (0.5, 1, 2, 4 and 8 mg/mL) 0.32 mm 1.8 m) operating in an electronic mode
were prepared. The prepared concentrations were at 70 eV. The oven was set to a temperature of 45°C.
used to investigate the larvicidal potential against At 70 eV, mass spectra were recorded.
Cx. pipiens (3rd instars). Fifty millilitres of dechlorinated Statistical analyses
tap water were placed in plastic boxes, to which 10 Each assay’s means and standard deviation were
larvae were added. Control (0.01% methanol) and computed after being carried out in triplicate. Tukey's
treated larvae were maintained under similar test was used after one-way ANOVA to determine the
laboratory conditions for colony maintenance without larval mortality rate. The analyses were done using
providing fish flake. Five replicates of each SPSS (SPSS version 15, USA). Statistical significance
concentration were used in the experiment. The dead was indicated by a P-value less than 0.05.
larvae were counted after 24, 48 and 72 h of
treatment. A larva was considered dead if it did not Result and Discussion
move when touched with a fine wooden rod. The After the extraction process, 100 g of J. phoenicea
observed mortality of the larvae is calculated using extract yielded 12 g of extract. The results show a
the formula: remarkable mortality rate based on the concentration
Observed mortality % ൌ and duration of larvae treated with J. phoenicea
Total number of dead mosquitoes extract. The death rate increased when the dose was
ൈ 100 increased; for the 0.5 dose, mortality was not noted
Total sample size
Observed mortalities were corrected by the Abbott until 72 h after treatment. After 24 h, mortality
(1925) formula: reached 34.33% for the 8 mg/mL dose against 21.33%
for the 4 mg/mL doses. After 48 h of treatment, the
Corrected mortality % =
ሺ% ୭ୠୱୣ୰୴ୣୢ ୫୭୰୲ୟ୪୧୲୷ – % ୡ୭୬୲୰୭୪ ୫୭୰୲ୟ୪୧୲୷ሻ
8 mg/mL dose gave 68.66% mortality. Finally, 100%
ൈ 100 mortality was reported with the 8 mg/mL dose after
ሺଵ଴଴ – % ୡ୭୬୲୰୭୪ ୫୭୰୲ୟ୪୧୲୷
72 h of exposure. The analysis of variance (ANOVA)
which allows natural mortality to be eliminated.
displayed a highly significant difference (P ≤0.001)
Histologic analysis between the different concentrations of J. phoenicea
Cx. pipiens third-instar larvae were subjected to post-24, 48 and 72 h (Table 1) of exposure. Different
treatment with the 24 h LC50 value (7.14 µg/mL) of concentrations had different larvicidal effects, as
J. phoenicea extract. At 24 h post-treatment, evidenced by the numerous comparisons of mortality
larvae treated and untreated with the extract were at 24, 48 and 72 h.
 BOULKENAFET et al.: LARVICIDAL POTENTIAL OF PHOENICIAN JUNIPER AGAINST CX. PIPIENS 689

Table 1 — Variations in mortality of Culex pipiens 3rd instar treated with Juniperus phoenicea as a function of concentration and time
Conc. (mg/mL) Time (h) df F
24 48 72
0.5 0.00±0.00aD 0.00±0.00aE 2.00±2.00aE 2 1.00
1 0.00±0.00bD 8.00±2.00aD 12.67±1.85aD 2 16.62
2 8.00±4.47cC 31.33±0.82bC 43.55±0.89aC 2 179.47
4 21.33±1.22cB 53.33±1.36bB 74.66±1.90aB 2 374.86
8 34.33±3.20cA 68.66±2.86bA 95.55±2.72aA 2 160.22
LC50 7.14 3.97 3.17
LC90 11.59 6.16 4.78
LC95 12.14 6.43 4.98
F 174.96 289.40 411.27
Df 4 4 4
[Identical capital letters in columns and identical small letters in rows denote differences that are insignificant at P ≤0.001]

Fig. 1 — Photomicrograph of midgut histology of Cx pipiens larvae 3rd instar. (A & B) Control; and (C & D) Treated (with Juniperus
phoenicea extract). [In the extract-treated group, the larvae showed peritrophic membrane degradation, displacement of the epithelial layer,
irregular blebbing, and sloughing. blebbing (Bc), Degraded microvilli (DMV), degenerating epithelial cells (DEC), degenerating nuclei (DN),
degenerating peritrophic membrane (DPM), extrusion (E), food bolus (FB), and nuclei (N). Magnification: A & C 200X, B & D 400X]
Histopathological study larvae, the midgut cells were swollen, and vesicles of
In the control group, the cytoplasm in gut epithelial different sizes were detected (Fig. 1 C & D). The
cells (cuboidal/flattened cells) was clear with a J. phoenicea extract-treated larvae had deteriorated
rounded nucleus (Fig. 1 A & B). However, in treated microvilli, blebbing and degenerating nuclei,
690 INDIAN J EXP BIOL, AUGUST 2024

displacement of the epithelial layer, and partial loss of 98% mortality, followed by R. stricta (91%)
the peritrophic membrane. compared to azadirachtin.
GC-MS analysis
The current study evaluated the histological
Preliminary investigation of J. phoenicea extract changes that J. phoenicea extracts caused in the
showed the presence of phytochemicals of diverse midgut of Cx. pipiens third instars. Some
chemical nature. Some of the main phytochemicals modifications include enlarged intercellular gaps,
identified in the extract are 9,12-octadecadienoic acid cytoplasmic vacuolization, fragmented nuclei and a
(Z,Z)- (50.2%), podocarpa-8,11,13-trien-3-one, 14-iso distorted epithelial layer. Moreover, the separation of
propyl-13-methoxy- (13.2%), -amino-7,10-dimethyl the peritrophic membrane and alteration of microvilli
dibenzo[b,f][1,4]oxazepin-11(10H)-one (8.4%), n-hexa led to the destruction of midgut architecture. These
decanoic acid (5.6%) and Furo[2,3-b]quinoline, 4,6,7- histopathological changes caused by the J. phoenicea
methanol extract on the Cx. pipiens midgut
trimethoxy (3.4%) (Table 2).
architecture were in agreement with several
Untapped bioactive secondary metabolites from researchers using different plants20-25. Al-Mehmadi
plants have intriguing phytochemicals that can replace and Al-Khalaf26 reported that treatment of Culex
synthetic insecticides. Natural products such as plants quinquefasciatus with Melia azedarach L. resulted in
are rich in novel bioactive compounds, as drugs significant damage to the larvae's midgut epithelium,
derived from plants have significantly impacted leading to cell vacuolization, microvilli damage, and
human health. The efficacy of phytochemicals cell death. Abutaha et al.27 also reported
on mosquito larvae has been demonstrated in morphological alternations in the larval midgut of Cx.
several research. Phytochemicals are promising pipiens when treated with Cinnamomum burmannii
biodegradable larvicidal agents and have less harmful and Syzygium aromaticum combined extract. They
effects on non-target organisms17. The larvicidal observed different morphological alternations, such as
potentials of 32 oils from various plants against microvilli degradation, peritrophic membrane
the third instar of Cx. pipiens were reported by damage, and loss of nuclei. From GC–MS analysis,
Baz et al.18 All oils extracted showed larvicidal 19 compounds were detected. Compounds, such as
activity, and their LT50 values ranged from 9.67 to 9,12-octadecadienoic acid (Z,Z)- (50.2%) and n-
37.64 h. Al-Solami19 reported the efficacy of acetone hexadecanoic acid (5.6%) have been earlier reported
extract of Rhazya stricta, Ruta chalepensis, Lantana for their larvicidal potential28,29. However, the other
camara, and Amorpha fruticose against Cx. pipiens. compounds detected were also reported from different
The results showed that the L. camara extract caused plant extracts that showed larvicidal potential in
Table 2 — Compounds present in 70% methanol extract of Juniperus phoenicea leaves detected using GC-MS analysis
Compound R.T. (min) Area (%) Biological activity
4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl- 9.76 2.19 Antioxidant, anti-inflammatory, and antimicrobial activities31,32
Phenol, 2,5-bis(1,1-dimethylethyl)- 14.46 0.96 Antifungal6
Azacyclohexane, 3-[1-pyrrolidyl]- 17.51 0.75 -
Pentadecanoic acid, 14-methyl-, methyl ester 18.52 1.78 Antimicrobial, antifungal33
n-Hexadecanoic acid 18.89 0.72 Larvicidal27
5-[3,4-Methylenedioxybenzyl]-2-thiohydantoin 19.32 5.64 -
9,12-Octadecadienoic acid, methyl ester 20.15 1.72 -
9,12-Octadecadienoic acid (Z,Z)- 20.53 1.77 -
10-Octadecenoic acid, methyl ester 20.59 2.38 -
9,12-Octadecadienoic acid (Z,Z)- 21.01 50.21
Octadecanoic acid 21.19 0.98 Immunoregulatory and anti-inflammatory34
2-Amino-7,10-dimethyldibenzo[b,f][1,4]oxazepin- -
11(10H)-one 21.50 8.47
Methyl 9,12-heptadecadienoate 21.79 0.62 -
9,17-Octadecadienal, (Z)- 23.61 0.63 -
Furo[2,3-b]quinoline, 4,6,7-trimethoxy- 24.28 3.42 Anti-parasitic and antitumor activity35
1,3,12-Nonadecatriene 25.80 0.59 Antidiabetic and antilipidemic properties36
Podocarpa-8,11,13-trien-3-one, 14-isopropyl-13-methoxy- 25.91 13.16 -
3H-3a-Azacyclopenta[a]indene-2-carbonitrile, 3-oxo-1- -
(piperidin-1-yl)-4,5,6,7-tetrahydro- 26.83 3.21
beta-Sitosterol 27.311 0.83 Larvicide37
 BOULKENAFET et al.: LARVICIDAL POTENTIAL OF PHOENICIAN JUNIPER AGAINST CX. PIPIENS 691

combination30. Thus, the larvicidal potential could be 7 Kioulos I, Kampouraki A, Morou E, Skavdis G & Vontas J,
related to the individual compound or synergistic Insecticide resistance status in the major West Nile virus
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