Larvicidal potency of selected xerophytic plant extracts on Culex pipiens (Diptera: Culicidae): Effect of Plant Extracts on Culex pipiens

Abstract

Chemical insecticides released into the environment may have adverse biological effects. Therefore, there is a need for ecofriendly insecticides for mosquito control. Xerophytic plant extracts that may provide more ecofriendly active component were evaluated against Culex pipiens 4th instars. Plant extracts prepared using different solvents with a Soxhlet apparatus and different concentrations were tested against Culex pipiens larvae. The effects were observed at 24 h and 72 h intervals and LD50 and LD90 values determined. Chloroform (CHCl3) and ethyl acetate (EtOAc) extracts of Althaea ludwigii were the most effective against Cx. pipiens 4th instars, but were highly dependent on extract concentrations and exposure time. Results suggest that A. ludwigii extracts contain bioactive compounds, such as phenols and saponins, that may provide effective Cx. pipiens larval control. However, the extract was found to be toxic to zebrafish larvae, and may be toxic to other aquatic fauna. Further studies to determine the active components and toxicity to other fauna are needed.

Full text

RESEARCH PAPER
Larvicidal potency of selected xerophytic plant extracts on
Culex pipiens (Diptera: Culicidae)
Nael ABUTAHA
1
, Fahd A. AL-MEKHLAFI
1,3
, Lamya Ahmed AL-KERIDIS
2
, Muhammad FAROOQ
1
,
Fahd A. NASR
1
and Muhammad AL-WADAAN
1
1
Bioproducts Research Chair, Department of Zoology, College of Science, King Saud University, Saudi Arabia
2
Department of Biology, Faculty of Science, Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia
3
Department of Agricultural Production, College of Agriculture and Veterinary Medicine, Thamar University, Yemen
Correspondence
Nael Abutaha, Bioproducts Research
Chair (BRC), Department of Zoology, College of
Science, King Saud University, P.O. Box 2455,
Riyadh 11451, Kingdom of Saudi Arabia.
Email: nabutaha@ksu.edu.sa
Received 3 May 2017;
accepted 20 December 2017.
d o i: 10 . 1111 / 174 8 - 5 9 67. 12 2 9 3
Abstract
Chemical insecticides released into the environment may have adverse biological
effects. Therefore, there is a need for ecofriendly insecticides for mosquito control.
Xerophytic plant extracts that may provide more ecofriendly active component were
evaluated against Culex pipiens 4th instars. Plant extracts prepared using different
solvents with a Soxhlet apparatus and different concentrations were tested against
Culex pipiens larvae. The effects were observed at 24 h and 72 h intervals and LD
50
and LD
90
values determined. Chloroform (CHCl
3
) and ethyl acetate (EtOAc) extracts
of Althaea ludwigii were the most effective against Cx. pipiens 4
th
instars, but were
highly dependent on extract concentrations and exposure time. Results suggest that
A. ludwigii extracts contain bioactive compounds, such as phenols and saponins, that
may provide effective Cx. pipiens larval control. However, the extract was found to be
toxic to zebrafish larvae, and may be toxic to other aquatic fauna. Further studies to
determine the active components and toxicity to other fauna are needed.
Key words: Althaea ludwigii,Culex pipiens, larvicidal activity, xerophytic plants, zebrafish.
Introduction
Insect-borne diseases pose serious health threats to human and
animal health worldwide (WHO 1996). Mosquitoes are
responsible for millions of deaths and hundreds of millions
of cases of mosquito-borne diseases annually (WHO 1996)
including malaria, dengue fever, chikungunya, zika virus,
yellow fever and Rift Valley fever (RVF) (Das et al.2007;
Ghosh et al.2012;Tantelyet al. 2017). The outbreak of
RVF in 2000 resulted in 2211 human cases with 290 deaths
in the Arabian Peninsula and an estimated 40,000 animals died
and 8,000–10,000 aborted (Al-Afaleq and Hussein 2011).
Mosquito-borne diseases also extend their impact on the
economy. During the RVF outbreaks, trading of cattle was
banned between the disease-associated countries, resulting in
ahugeeconomicloss.
Conventionally, chemical pesticides such as methoprene,
carbamates, pyriproxyfen, diflubenzuron, fenthion, malathion
and DDT have been employed for mosquito control (Su 1999).
The insecticides are expensive, pose hazards to the environment,
interfere with the natural food chain and are toxic to non-target
organisms, and some persist for a long time in the environment
with deleterious effects on public health (Reynolds and Parfitt
1993). In addition, overuse or misuse of insecticides has led to
insecticide resistance (Ranson et al. 2001). Reports showed that
in endemic countries between 2005 and 2011, 471 million
artemisinin-based therapies were purchased by private and
public sectors for malaria alone. Plasmodium spp. drug
resistance to artemisinins has been reported in many countries,
while mosquitoes have developed resistance to at least one
insecticide in 64 countries worldwide (WHO 2012). However,
in Saudi Arabia, the extensive application of insecticides to
control mosquito vectors after the oubreak of RVF in the Arabian
Peninsula in 2000 (Himeidan et al. 2014) and dengue fever in
1997 and 2004 (Alhaeli et al. 2016) resulted in increased
resistance in mosquitoes.
The insecticide resistance study was conducted in Riyadh
city aganist three populations of Cx. pipiens from Wadi Namar
Entomological Research 48 (2018) 362–371
© 2018 The Entomological Society of Korea and John Wiley & Sons Australia, Ltd

and AL-Wadi districts. Results demonstrated that Cx. pipiens
was resistant to deltamethrin, lambda-cyhalothrin, beta-
cyfluthrin, and bifenthrin (Al-Sarar 2010).
The house mosquito, Cx. pipiens, plays a significant role in
disease transmission to man and animals and is widely
distributed in the world (Cui et al. 2006; Kasai et al.2008).
In Saudi Arabia, Cx. pipiens is abundant in Riyadh (El-Khereji
et al. 2007), and it is reported to be a potential vector of
bancroftian filariasis (Omer 1996). Based on the increased
levels of insecticide resistance to commonly used insecticides,
there has been an increased interest in evaluating pesticides
that are less expensive, effective, and more environment-
friendly. Plant extracts have recently been evaluated as
potential agents for mosquito control (Al-Sharook et al.
1991; Murugan and Jeyabalan 1999; Muthukrishnan and
Pushpalatha 2001; Nathan and Sehoon 2006; Olayemi et al.
2014; Cheng et al. 2013; Choochote et al. 1999; Mustafa
and Al Khazaraji 2008; Nanyonga et al. 2012; Promsiri
et al. 2006; Rajkumar and Jebanesan 2005; Shivakumar
et al. 2013). The potency of plant extracts in controlling
mosquitoes is dependent on the biologically active
compounds such as steroids, phenols, alkaloids, saponins,
tannins and terpenoids (Shaalan et al. 2005; Al-Mekhlafi
et al. 2013) that inhibit insect development (Sharma et al.
2006), metamorphosis (Mwangi and Rembold 1987; Sukumar
et al. 1991; El-Bokl 2016), or may act as repellents and
oviposition deterrents (Castillo-Sánchez et al.2010).
Although there are several phytocompounds that have been
used for mosquito control, there is still a wide scope for the
discovery of active plant products (Yadav 1986), particularly,
in the flora of lesser investigated countries like Saudi Arabia.
The aim of the present study was to: (i) assess the larvicidal
activity of 69 extracts obtained from 10 different xerophytic
plant species against Cx. Pipiens 4th instars; (ii) evaluate the
histopathological alterations caused by the most potent
extract; and (iii) assess the effect of the most promising extract
on zebrafish larvae.
Materials and methods
Plant collection
Ten medicinal plants Schimpera arabica Hochst & steudel,
Cakile arabica Velen & Bornm, Calendula arvensis L., Emex
spinosa (L.) Camod, Chenopodium glaucum, Echium
horridum, Salsola imbricate, Portulaca oleracea, Paronychia
arabica,andA. ludwigii (AL), were collected from the desert
region in Riyadh, Saudi Arabia, during July 2015, and
separated into leaves, stems, roots, flowers, and fruits. The
plants were identified to species and voucher specimens
deposited at the Department of Botany and Microbiology,
College of Science, King Saud University, Saudi Arabia.
Preparation of the extracts
The plants were washed with distilled water and left to dry in a
well ventilated area at 25°C. Each dried plant part was ground
using a commercial blender, and the dried powders (30–50 g)
of various plant parts were extracted successively with
chloroform (CHCl
3
, 450 mL), ethyl acetate (EtOAc,
450 mL), and methanol (MeOH, 450 mL) in a soxhlet
apparatus for 24 h. The extracts were then centrifuged and
evaporated using a rotary evaporator (Heidolph, Germany).
The dried extracts were weighed, their yields calculated, and
then kept at 20°C until used.
Total extraction yeild %ðÞ¼
Mass of the extract
Mass of sample 100
Mosquito colony
Culexs pipiens larvae from a susceptible laboratory colony
maintained at the entomological insectary at King Saud
University, Riyadh at 27 ± 3°C, under a 14:10 h (light: dark)
photoperiod were used. Larvae were reared in plastic
containers (25 × 35 × 6 cm) with tap water and supplemented
with Tetramin fish meal (Tetra GmbH, Melle, Germany).
Fresh tap water and diet were changed every two days. Pupae
were transferred in plastic cups that were then placed in
rearing cages (50 × 50 × 50 cm). Emerged adults were
provided with 10% glucose solution in a glass tube connected
to a paper wick. Three to five day-old female mosquitoes were
allowed to feed on anesthetized mice (Swiss albino) under an
approved animal use protocol. A Petri dish containing 100 ml
tap water was kept inside the adult rearing cages for
oviposition and eggs collected after 2–3days.
Dose–response bioassay
Plant product crude extracts (10 mg) were reconstituted
separately in 1 mL of chromatography grade MeOH (Fisher
Scientific, UK). Different concentrations of the stock solution,
i.e., 6.25, 12.5, 25, 50, 75, and 100 μg/mL were placed in 12-
well plates and air-dried. The bioassays were carried out on
Cx. pipiens 4th instars. The assays were replicated three times
using 10 larvae for each concentration with MeOH used as a
solvent control. Larvae were provided fish meal during the
testing period. Larval mortality was observed at intervals of
24, 48, and 72 h post-exposure and the numbers of dead larvae
counted. The percent mortality, LD
50
,andLD
90
was
calculated for each assay.
Histopathological studies
The protocols for histopathological studies followed that of
(Bancroft and Gamble 2007). The treated and control Cx.
Effect of Plant Extracts on Culex pipiens
Entomological Research 48 (2018) 362–371 363
© 2018 The Entomological Society of Korea and John Wiley & Sons Australia, Ltd

pipiens 4th instars were fixed in 10% formalin for 72 h.
Paraffin-embedded preparations of the larvae were sectioned
at 4-μm thickness using a rotary microtome (Leica-RM2245,
Wetzlar, Germany) and stained with hematoxylin and eosin
(H&E). Images were captured using an OMX1200C, Nikon
microscope (Tokyo, Japan).
Toxicity assay using zebrafish larvae
AB wild-type strain of zebrafish originally obtained from the
Zebrafish International Resource Center (University of
Oregon, Eugene, OR, USA) were reared at 28°C and 14:10
light:dark photoperiod at the Department of Zoology, King
Saud University. Zebrafish larvae were obtained by pairwise
mating. The embryos were grown until they hatched and the
hatched larvae were reared for 15 days post-eclosion. The
larvae were fed three times daily with larval food (Larval
AP100 <50 micron; Zeigler Bros, Gardners, PA, USA).
The toxicity assay was performed by placing 5–7larvaein
35-mm glass dishes with or without the tested extract (10,
15, 20, 30 and 60 μg/mL) overnight at 28°C in an embryo
medium (5.0 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl
2
,
0.33 mM MgSO
4
). After 24 h of incubation in an air incubator,
the mortality (%) was recorded for each concentration and the
LD
50
and LD
90
values calculated. Three independent
treatments were conducted in triplicates.
Phytochemical analysis
Test for alkaloids
Fifteen milligrams of the extract was stirred with 1% HCl
(5 mL) on a water bath for 5 min and filtered. This filtrate
was mixed with 1 mL Dragendorff’s reagent, and the presence
of alkaloids confirmed by the formation of an orange-red
precipitate (Joshi et al. 2013).
Test for Saponins
Four milliliters of 20 mg/mL of the plant extract was pipetted
into a tube and diluted with 4 mL of distilled water and stirred
vigorously for 2 min. Foam lasting 10 min confirmed the
presence of saponins (Harborne 1998).
Table 1 Extract yields for chloroform, ethyl acetate and methanol solvents extracts of leaf, stem, root and fruit for ten xerophytic plants
Mass of Extract (g) Yield (%)
Species Family Mass of Plant Part (g) Chloroform (CHCl
3
) Ethyl acetate (EtOAC) Methanol (MeOH)
Schimpera arabica Brassicaceae Leaves (38 g) 0.547 (1.4%) 0.553 (1.45%) 1.16 (3%)
Stem (50 g) 0.511 (1%) 0.124 (0.25%) 2.6 (5.4%)
Fruit (27 g) 0.474 (1.75%) 0.11 (0.4%) 3 (11%)
Root (7 g) 0.07 (1%) 0.03 (0.4%) 0.46 (8.4%)
Cakile arabica Brassicaceae Leaves (29 g) 0.4 (1.4%) 0.35 (1.2%) 1.45 (5%)
Stem (40 g) 0.4 (1%) 0.25 (0.64%) 2.4 (6%)
Calendula arvensis Asteraceae Leaves (8 g) 0.228 (2.8%) 0 (0%) 0.923 (11.5%)
Stem (28 g) 0.14 (0.5%) 0.01 (0.03%) 3.25 (11.6%)
Fruit (16 g) 0.182 (1.1%) 0.023 (0.14%) 2.31 (14.4%)
Emex spinosa Polygonaceae Leaves (23 g) 0.886 (3.8%) 0.09 (0. 4%) 1.52 (2.4%)
Stem (27 g) 0.205 (0.75%) 0.047 (0.1%) 2 (7.4%)
Chenopodium glaucum Chenopodiaceae Leaves (50 g) 0.58 (1.16%) 0.041 (0.08%) 2.67 (5.34%)
Stem (37.5 g) 0.154 (0.41%) 0.073 (0.19%) 3.48 (9.3%)
Fruit (23 g) 0.416 (1.8%) 0.067 (0.3%) 2.37 (10.3%)
Echium horridum Boraginaceae Leaves (18 g) 0.268 (1.5%) 0 (0%) 2.29 (12.7%)
Stem (35 g) 0.375 (1%) 0.059 (0.16%) 2.52 (7.2%)
Salsola imbricata Chenopodiaceae Whole plant (27 g) 0.351 (1.3%) 0.0653 (0.24%) 2.0879 (7.4%)
Stem (22 g) 0.0514 (0.23%) 0.0125 (0.05%) 2.11 (9.6%)
Portulace oleracea Portulacaceae Leaves (32 g) 0.51 (1.6%) 0.084 (0.26%) 1.66 (5.2%)
Stem (36 g) 0.309 (0.85%) 0.335 (0.93%) 2.86 (7.94%)
Paronychia Arabica Caryophyllaceae Whole plant (50 g) 0.22 (0.44%) 0.12 (0.24%) 3.86 (7.72%)
Althaea ludwigii Malvaceae Stem (38 g) 0.128 (0.33%) 0.0315 (0.08%) 3.186 (8.4%)
Flower (22.5 g) 2.254 (10%) 0.2655 (1.2%) 5.784 (25.7%)
N. Abutaha et al.
364 Entomological Research 48 (2018) 362–371
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Phenol estimation
Fifty microliters of distilled water was mixed with 12.5 μLof
sample (1 mg/mL) and 12.5 μL of 25% Folin–Ciocalteu
reagent. The reaction mixture was allowed to stand for
5 min, followed by the addition of 7% Na
2
CO
3
(125 μL).
The mixture was then placed for 1.5 h in darkness and the
absorbance measured at 760 nm using a UV/visible
spectrophotometer. The total phenolic content was calculated
using a gallic acid standard curve (Joshi et al. 2013).
Flavonoid estimation
The total flavonoid content was quantified following the
method of Ghosh et al. (2013), with slight modifications. In
brief, 100 μL of 2% aluminum chloride was mixed with
100 μL of sample (1 mg/mL), followed by incubation for
10 min at room temperature. Absorbance was measured at
368 nm and the flavonoid content calculated using the
calibration curve of a standard flavonoid, quercetin.
High performance liquid chromatography (HPLC)
analysis
Extracts were filtered using a 0.20 μm polyvinylidene
difluoride (PVDF) filter (Macharey-Nagel, Düren, Germany)
prior to the HPLC analysis. The HPLC instrument (Perkin
Elmer Series 200, Norwalk, CT, USA) was equipped with
UV/visible detectors. Ten microliters of the sample were
injected onto a C18 column (250 mm × 4.6 mm) at room
temperature (25°C). A mobile phase consisting of water (A)
and acetonitrile (B) was employed as the solvent system and
the wavelength measured at 245 nm.
Statistical analysis
Mean larval mortalities were transferred into probit analyses.
The LC
50
,LC
90
, UCL (upper confidence limits) and LCL
(lower confidence limits) values, chi-square test and other
statistical parameters were calculated using Microsoft Excel
(Finney 1952).
Results
A total of 69 extracts from ten plant species were evaluated for
toxicity against Cx. pipiens 4th instars. Plant extract yields (%)
using CHCl
3
, EtOAc, and MeOH solvents were determined.
MeOH demonstrated the highest yield of all the solvents tested
(Table 1). None of the 69 extracts using MeOH solvent
showed an effect against Cx. pipiens 4th instars, except A.
ludwigii flower extracts. Plant parts extracted with CHCL
3
showed a higher efficiency against 4th instars when compared
Table 2 Larvicidal activity of different concentrations of Althaea ludwigii chloroform, ethyl acetate and methanol fruit extracts against Culex pipiens 4th instars. Each value represents the
mean ± standard deviation of three independent replicates
Extract type Time
% of Larval l mortality ± SD of mosquitoes
LC
50
(μg/mL)
(95%LCL-UCL)
LC
90
(μg/mL)
(95%LCL-UCL) X
2
Slope
Concentration (μg/mL)
6.25 12.5 25 50 75 100
Chloroform (CHCl
3
) 24 0±00 0±00 0±00 23.33±3.33 46.67±5.77 100±0 (79.1)59.727–104.635 (177.1) 133.798–234.398 0.961* 3.659
48 0 ± 00 33.33 ± 5.77 33.33 ± 5.77 43.33 ± 3.33 73.33 ± 6.67 100 ± 0 (49.8) 37.456–66.113 (121.4) 91.405–161.339 0.974* 3.308
72 10 ± 3.33 16.33 ± 6.67 63.33 ± 5.77 73.33 ± 12.01 100 ± 00 100 ± 0 (42.6) 27.834–65.260 (212.6) 138.812–325.462 0.860* 1.836
Ethylacetate(EtOAC) 24 0±00 0±00 0±00 0±00 0±00 0±00 ND†ND†ND†ND†
48 0±00 0±00 0±00 0±00 0±00 0±00 ND†ND†ND†ND†
72 0±00 0±00 0±00 13.33±12.01 33.33±3.33 60±5.7 (85.4)68.441–106.517 (153.6) 123.088–191.566 0.997* 5.028
Methanol (Methanol) 24 0 ± 00 0 ± 00 0 ± 00 0 ± 00 0 ± 00 0 ± 00 ND†ND†ND†ND†
48 0±00 0±00 0±00 0±00 0±00 0±00 ND†ND†ND†ND†
72 0±00 0±00 0±00 0±00 0±00 0±00 ND†ND†ND†ND†
LC
50
, Lethal concentration 50% mortality; LC
90
, Lethal concentration 90% mortality; LCL, lower confidence limits; UCL, upper confidence limits; χ
2
, chi-square.
*Significant at P<0.05 level.
†ND, not determined.
Effect of Plant Extracts on Culex pipiens
Entomological Research 48 (2018) 362–371 365
© 2018 The Entomological Society of Korea and John Wiley & Sons Australia, Ltd

with either EtOAc and MeOH extracts at the same
concentrations tested. The A. ludwigii extract at all tested
concentrations resulted in mortality of Cx. pipiens at 72 h
against 4th instars. However, the susceptibility of the
mosquito larvae was positively correlated with the
concentrations tested and the period of exposure. Thus,
the LC
50
values were 79.1, 49.8 and 42.6 μg/mL after 24 h,
48 and 72 h, respectively. On the other hand, the EtOAc
extract of A. ludwigii exhibited LC
50
value of 85.4 μg/mL only
after 72 h, whereas no inhibitory effect was noted for the
MeOH extract (Table 2).
Cross-sections of the midgut of control and extract-treated
Cx. pipiens 4th instars were investigated. The mid-gut of the
control group consisted of a single layer of columnar
epithelium cells with microvilli surrounded by a peritrophic
membrane; each cell containing a large granular nucleus
located in the middle of the cell (Fig. 1). After 24 h, the
cross-section of the midgut showed deformation and
detachment of epithelial cells from the basal lamina of Cx.
pipiens 4th instars treated with A. ludwigii CHCL
3
flower
extract at 79.1 μg/mL. The midgut also showed the
degeneration of the microvilli in some regions and lysis of
the midgut showed swollen cells along with cytoplasmic and
nuclear lysis.
The A. ludwigii flower extract resulted in dose dependent
activity against zebrafish larvae. The higher the concentration,
the greater the larval toxicity. The A. ludwigii extract was
highly toxic with LC
50
and LC
90
values of 10.98 and
17.65 μg/mL respectively, indicating that the extract is
potentially toxic to vertebrates at a concentration far lower
than the concentration that kills C. pipiens larvae (Table 3).
The preliminary phytochemical test showed that the plant
extracts contain phenols and saponins, but not alkaloids or
flavonoids. The phenolic content of the CHCL
3
extract
calculated was found to be 20.8 μg/mL.
The HPLC chromatogram was used to detect the peaks of
bioactive extract components at different retention times (t
R
).
The HPLC fingerprint of A. ludwigii CHCl
3
extract showed
the presence of a few major peaks, as evidenced by the
different retention times in the chromatogram (Fig. 2).
Discussion
Mosquito larval stages are an important target for vector
control. The use of synthetic larvicidal agents to control
mosquito larvae presents a potential hazard to other organisms
and the environment. Extracts derived from plants often show
promising results as larvicidal agents, as they are often
inexpensive, biodegradable, and highly effective (Alkofahi
et al. 1989; Jang and Ahn 2002; Ghosh et al. 2012).
This is the first report of the larvicidal activity of A. ludwigii
against mosquitoes. However, some plant species belonging
to the same Family (Malvaceae) have been reported to
A
BD
C
Figure 1 Photomicrograph of the mi dguts
of Culex pipiens 4th instars treated with
chloroform flower extract of Althaea
ludwigii at 24 h post-treatment. (A, B)
Cross-sections of the midgut of untreated
larvae. (C, D) Cross-sections of the midgut
of treated larvae. Degraded microvilli
(DMV); degenerating epithelial cells (DEC),
degenerating peritrophic membrane
(DPM); degenerating nuclei (DN).
N. Abutaha et al.
366 Entomological Research 48 (2018) 362–371
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demonstrate larvicidal activity against Anopheles stephensi,
Culex quinquefasciatus,andAedes aegypti larvae
(Govindarajan 2010). The larvicidal activity against Cx.
pipiens, showed a reliable potency only for the A. ludwigii
CHCL
3
flower extract. The larvicidal activity of plant extracts
does not depend only on species, plant parts used,
environment and time of cultivation, but also on the type of
the solvents polarity. Our present study showed that the
most active extract was CHCL
3
extract of the flower parts of
the A. ludwigii.LikewiseChoreet al. (2014) and Kovendan
et al. (2012) observed that the solvent polarities used for
extraction have influence larval mortality, in addition to the
plant part used.
Plant secondary metabolites can result in physiological and
cellular disorders that include inhibition of
acetylcholinesterase, interference of mitochondrial respiration
and malpighian tubules, and also may affect midgut
epithelium or gastric caecae in mosquito larvae (Rattan
2010). Our results showed several histopathological
alterations in the midgut, including separation of the epithelial
cells from the basement membrane, as well as swelling,
elongation, and deformation of the epithelial cells in larvae
treated with CHCl
3
fruit exact of A. ludwigii.Inaddition,
disruption of the brush border and absence of microvilli were
also seen in some areas. These observations are in agreement
with previous reports of other plants that demonstrated
phytotoxicity (Al-Mehmadi and Al-Khalaf 2010;
Jiraungkoorskul and Jiraungkoorskul 2015; Jiraungkoorskul
2015; Kjanijou et al. 2012; Pavananundt et al. 2013), which
suggest that plant extracts may result in morphological
changes in mosquito larvae midgut epithelial cells. Regardless
of the extracts used, the similarity of the alterations in the
mosquito gut indicates that these changes are a common
response to cellular toxicity, which indicates that the midgut
epithelium may be the primary site of action of most
phytotoxic plants (Perumalsamy et al.2013).Manyplants
extracts manifest toxicity to different mosquito species
(Sukumar et al. 1991; Shaalan et al. 2005; Kishore et al.
2014) and have been suggested as potential substitutes to
synthetic larvicides. Numerous compounds derived from
plants demonstrate larvicidal activity include alkaloids, e.g.,
pellitorine, guineensine, pipercide, and retrofractamide,
isolated from Piper nigrum fruit (Lee 2000; Park et al.
2002), coumarins isolated from Cnidium monnieri fruit (Wang
et al. 2012), ethyl cinnamate and ethyl p-methoxycinnamate
isolated from Kaempferia galanga rhizomes (Ahn et al.
2008), quassin isolated from Piper longum (Evans and Raj
1991), neolignans isolated from Piper decurrens (Chauret
et al. 1996), cyanogenic glycoside isolated from Sorghum
bicolour (Jackson et al.1990),lactonesisolatedfrom
Bryonopsis laciniosa (Kabir et al. 2003), phenols isolated
from Vanilla fragrans (Sun et al. 2001), and saponin isolated
from Balanites aegyptiaca (Chapagain et al. 2008). Thus, the
Table 3 Toxicity of different concentrations of Althaea ludwigii fruit extracts on zebrafish larvae. Each value represents the mean ± standard deviation of three independent experiments
Extract Type Time
% of Larval l mortality ± SD of Zebrafish
LC
50
(μg/mL)
(95%LCL-UCL)
LC
90
(μg/mL)
(95%LCL-UCL) X
2
Slope
Concentration (μg/mL)
10 15 20 30 60 100
Chloroform (CHCl
3
) 24 40 ± 0.7 80 ± 0.3 100 ± 00 100 ± 0 100 ± 0 100 ± 0 (10.98) (8.861–13.615 ) (17.6 5) 14.24 2–21.883 0.998* 6.218
Ethyl acetate (EtOAC) 24 100 ± 0 100 ± 0 100 ± 0 100 ± 0 100 ± 0 100 ± 0 ND†ND†ND†ND†
Methanol(MeoH) 24 100±0 100±0 100±0 100±0 100±0 100±0 ND†ND†ND†ND†
LC
50
, lethal concentration 50% mortality; LC
90
, lethal concentration 90% mortality; LCL, lower confidence limits; UCL, upper confidence limits; χ
2
, chi-square.
*Significant at P<0.05 level.
†ND, not determined.
Effect of Plant Extracts on Culex pipiens
Entomological Research 48 (2018) 362–371 367
© 2018 The Entomological Society of Korea and John Wiley & Sons Australia, Ltd

larvicidal activity of A. ludwigii might be attributed to the
presence of phenols or saponins.
Herbal sample fingerprint analysis is an important quality
control tool of growing interest in herbal products (Sarker
et al. 2005; Fan et al. 2006). Fingerprint construction is
helpful to detect adulterations, to control the process of
extraction, and to test the quality of a finished product. Herbal
sample fingerprints are a set of characteristic chromatographic
or spectroscopic signals that enable sample recognition
through comparison (Ciesla 2012). While many
chromatographic methods have been used for fingerprint
construction Springfield et al. (2005) suggested that
fingerprinting using HPLC is the best method for extract
characterization. Although the CHCl
3
extract is a crude
extract, it definitely has an effective larvicidal compound(s)
that could enable its use as a plant larvicidal agent.
Natural products may be more environmentally safe at
concentrations used than synthetic pesticides; however, this
does not eliminate the possibility that natural larvicides also
may negatively impact non-target organisms. While the
effective larvicidal extract used here had a promising
larvicidal effect, it demonstrated high toxicity to zebrafish
larvae, which indicates potential toxicity to non-target
organisms in aquatic environments. The isolation of active
compounds from A. ludwigii flower extracts could lead to
the development of natural insecticide products to replace
synthetic mosquitocides.
The present study demonstrated the use of A. ludwigii fruit
extract as a potential larvicidal agent against Cx. pipiens
larvae. Investigating the modes of action of plant-based
insecticides is of great importance because it could provide
useful information on the appropriate formulations to be taken
into consideration for future commercialization. In addition, it
might assist in the development of mosquito control
substitutes with novel target sites and minimum toxicity
(Ahn et al. 2006; Isman 2006).
Conclusions
The A. ludwigii flower extract showed larvicidal activity
against Cx. pipiens 4
th
instars. These effects are linked to gut
deformation, the detachment of epithelial cells, and
degeneration of microvilli. Phenols or saponins could be
responsible for the observed larvicidal activity of the extract.
This extract also demonstrated a high level of toxicity to
zebrafish larvae and may a pose significant environmental
hazard.
Acknowledgment
The authors are grateful to the Deanship of Scientific
Research, King Saud University for funding through Vice
Deanship of Scientific Research Chairs.
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