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Novel Histopathological and Molecular Effects of Natural Compound

Pellitorine on Larval Midgut Epithelium and Anal Gills of Aedes aegypti

Article in PLOS ONE · November 2013

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Novel Histopathological and Molecular Effects of Natural
Compound Pellitorine on Larval Midgut Epithelium and
Anal Gills of Aedes aegypti
Haribalan Perumalsamy1,2, Jun-Ran Kim1,2, Sang Mi Oh2, Je Won Jung2, Young-Joon Ahn2*,
Hyung Wook Kwon2*
1 Research Institute for Agriculture and Life Science, Seoul National University, Seoul, Republic of Korea, 2 WCU Biomodulation Major, Department of Agricultural
Biotechnology, Seoul National University, Seoul, Republic of Korea

Abstract
The yellow fever mosquito, Aedes aegypti, is a vector for transmitting dengue fever and yellow fever. In this study, we
assessed the histopathological and molecular effects of pellitorine, an isobutylamide alkaloid, on the third instar of Ae.
aegypti larvae. At 5 mg/l concentration of pellitorine, the whole body of the treated larvae became dark in color, particularly
damaged thorax and abdominal regions. Pellitorine was targeted mainly on midgut epithelium and anal gills, indicating
variably dramatic degenerative responses of the midgut through a sequential epithelial disorganization. The anterior and
posterior midgut was entirely necrosed, bearing only gut lumen residues inside the peritrophic membranes. Pellitorine
caused comprehensive damage of anal gill cells and branches of tracheole and debris was found in hemolymph of the anal
gills. RT-PCR analysis indicates that the compound inhibited gene expression encoding V-type H+-ATPase and aquaporine 4
after treatment with 2.21 mg/l pellitorine. These results verify that pellitorine merits further study as a potential larvicide
with a specific target site and a lead molecule for the control of mosquito populations.

Citation: Perumalsamy H, Kim J-R, Oh SM, Jung JW, Ahn Y-J, et al. (2013) Novel Histopathological and Molecular Effects of Natural Compound Pellitorine on
Larval Midgut Epithelium and Anal Gills of Aedes aegypti. PLoS ONE 8(11): e80226. doi:10.1371/journal.pone.0080226
Editor: George Dimopoulos, Johns Hopkins University, Bloomberg School of Public Health, United States of America
Received April 12, 2013; Accepted October 1, 2013; Published November 18, 2013
Copyright: ß 2013 Perumalsamy et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was carried out with the support of WCU (World Class University) Program (R31–10056) through the National Research Foundation of Korea
funded by the Ministry of Education, Science and Technology to YJA and HWK. The funding agency had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected] (Y-JA); [email protected] (HWK)

Introduction bioactive chemicals that have been perceived by the general public
as relatively safe and with fewer risks to the environment, and with
The yellow fever mosquito, Aedes aegypti, is cosmopolitan, minimal impacts to human and animal health [6–8]. Unlike
abundant and a vector for transmitting several human diseases conventional insecticides, certain PSMs can act at multiple and
such as dengue fever, Chikungunya, and yellow fever [1]. More novel target sites [9–11], thereby reducing the potential for
than 2.5 billion people are at risk of dengue infection over in 100 resistance [12,13]. Histopathological studies revealed that the
countries worldwide. There may be 50,100 million dengue midgut of insects is one of the main target organs for many
infections every year, including 22,000 deaths annually, mostly xenobiotics, including PSMs [14–16] and bacterial endotoxins
among children [2]. A number of mosquitoes are distinctly (Bacillus thuringiensis and Bacillus sphaericus) [17,18]. In particular, it
increasing in incidence with a high occurrence of dengue fever was initially reported that the isobutylamide alkaloid pellitorine
worldwide due to global warming, increased international travel, (Fig. 1) had potent larvicidal activity against third instar Ae. aegypti
and tainted fresh water pools [1,3]. It is extremely difficult to larvae [12]. However, no information is available concerning the
control Ae. aegypti because they adapt well to the environment with histopathological effects of natural pellitorine on Ae. aegypti larvae.
high resilience or with the ability to rapidly bounce back to initial In our present study, an assessment is made of the histopatholog-
numbers after disturbances resulting from natural phenomena or ical alterations in midgut epithelial cells and anal gills in the third
human interventions [4]. In addition, a serious problem with the instar larvae of Ae. aegypti following exposure to pellitorine using a
mosquito species is their ability to rapidly evolve resistance to fluorescent microscopy, a confocal laser scanning microscopy, and
conventional insecticides such as acetylcholinesterase (AChE) a transmission electron microscopy.
inhibitors, axonic nerve poisons such as pyrethroids, and insect In order to deal with these insults to hemolymph homeostasis,
growth regulators [5]. Therefore, there is a critical need for the larval and adult mosquitoes rapidly respond and restore water and
development of selective control alternatives with novel target sites ion balance. Four anal gills surrounding the anal opening are the
in mosquitoes. primary sites of Na+ and Cl– absorption in mosquito larva with
Plant secondary metabolites (PSMs) have been suggested as which ion and water regulation in hemolymph remains stable [19].
alternative sources for conventional biocides [6–8]. This approach The osmotic uptake of water at the anal gills is the primary
is appealing largely because they constitute a potential source of external site of ion uptake, normally contributing to 33% of body

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 Toxicity of Pellitorine on Larval Aedes aegypti

reagents used in this study were of reagent-grade quality and

available commercially.

Mosquitoes
The stock cultures of the insecticide-susceptible Ae. aegypti were
maintained in the laboratory without exposure to any known
insecticide [25]. Larvae were reared in plastic trays (2463565 cm)
Figure 1. Structure of the isobutylamide alkaloid pellitorine.
doi:10.1371/journal.pone.0080226.g001 containing 0.5 g of sterilized diet (40-mesh chick chow powder/
yeast, 1/1 by weight). Adults were maintained on a 10% sucrose
solution and blood fed on live mice. All stages were held at
weight gain per day [20–22]. It is believed that the presence of
2761uC and 65–75% relative humidity under a 16:8 h light:dark
aquaporins (AQPs), especially Aquaporin 4 (AaAQP4) which acts cycle.
as water channels, may facilitate the movement of water across
these tissues [23]. In addition, the anal gills of larval A. aegypti serve
as the major site for Na+, Cl– and K+ uptake by H+-ATPase and
Treatment with Natural Pellitorine
Natural pellitorine was used for treatment of third instar Ae.
Na+/K+-ATPase [22,24].
aegypti larvae during histopathological testing. A 5 mg/l quantity of
In this study, we have observed the gene expression analysis of
the compound in methanol was suspended in distilled water with
both V-type-H+-ATPase and aquaporin 4 (AaAQP4) in anal gills
Triton X-100 (20 ml/l), which is equivalent to approximately
after treatment with pellitorine was employed to investigate a
twofold quantities of the LC50 value (2.21 mg/l) of the compound
possible target site of the alkaloid.
[12]. For gene expression level observation we have used LC50
value (2.21 mg/l), because the mosquito larvae should be active
Materials and Methods rather than having paralysis effect. Groups of 20 mosquito larvae
were put into paper cups (270 ml) containing the test solution
Chemicals and Reagents
(250 ml). Controls received methanol–Triton X-100 solution in
Pellitorine was obtained from the root of Asarum heterotropoides as
distilled water.
reported previously [12]. Triton X-100 was obtained from Shinyo
Treated and control (methanol–Triton X-100 solution only)
Pure Chemicals (Osaka, Japan). All of the other chemicals and
larvae were held under the same conditions as those used for
colony maintenance for 24 h. Larvae were considered dead if its

Figure 2. Light micrographs of midgut, thorax, and anal gill parts of third instar Ae. aegypti larvae without (A) and with treatment
with 5 mg/l of natural pellitorine (B) 635 magnification. Pellotorine treatment induced toxic effects on many different regions of the body
including thorax, abdomen, and anal gills. All experiments were conducted in triplicate in which 20 mosquito larvae were used in each replicate. More
than 10 live larvae from control and treated groups were randomly collected and used for histological analysis. All histological observations showed
similar results throughout experiments same in other results.
doi:10.1371/journal.pone.0080226.g002

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 Toxicity of Pellitorine on Larval Aedes aegypti

Figure 3. Histology of thorax and midgut regions of third instar Ae. aegypti larvae. (A) Control mosquito. A1. anterior midgut region of
control larvae has well-developed gastric caeca (GC) and single-layered midgut epithelium. A2. Central midgut region of control larvae consisted of
well-developed lumen content (LC), peritrophic space (PS), and peritrophic membrane (PM). A3. Posterior midgut region of control larvae consisted of
distinguished midgut epithelial layer, lumen contents, and peritrophic membrane. (B) Treated mosquito with 5 mg/l natural pellitorine showed
undistinguished enlarged portion of gastric caeca and damaged single-layered epithelial cells. B1. Pellitorine-treated larvae had an undistinguished
enlarged portion of gastric caeca and damaged single-layered epithelial cells (asterisks). B2. Central midgut region of pellitorine-treated larvae
showed demolished epithelial layer residues mixed with a few LC (asterisks). B3. Complete damaged residue of epithelial and peritrophic membranes
was observed in pellitorine-treated larvae (asterisk).
doi:10.1371/journal.pone.0080226.g003

body and appendages did not move when it was prodded with a larvae were sectioned at 10 mm thickness by using a Thermo
fine wooden dowel. All treatments were replicated three times Scientific Microm HM 340E rotary microcotome (Walldorf,
using 20 larvae per replicate. Germany). Sections were dried at 40uC overnight and subse-
quently dewaxed with Fisher Scientific CitriSolv (Fair Lawn, NJ)
Light Microscopic Analysis and rehydrated with a series of ethanol to phosphate-buffered
The pellitorine-treated and -untreated (control) third instar Ae. saline (PBS) solution as described previously [26]. Triple color
aegypti larvae were put on microscope slides at room temperature staining was carried out using Cason’s trichrome staining
for light microscopy. Morphological observations were made with procedures [27]. In brief, rehydrated paraffin sections were soaked
a Leica EZ4 HD equipped with an Integrated 3.0 Mega-Pixel into Cason’s trichrome stain for 15 min, and slides were gently
CMOS camera (Heerbrugg, Switzerland). swashed in tap water and subsequently distilled water three times.
Excess of water was removed and samples were mounted with
Histological Analysis by Cason’s Trichome Staining EMS permount (Hatfield, PA). Images were observed and
The treated and control third instar Ae. aegypti larvae were captured using an Olympus BX43 fluorescent microscope (Tokyo,
immediately fixed in 4% paraformaldehyde (PFA) buffer solution Japan).
(pH, 7.4) at 4uC overnight. Paraffin-embedded preparations of the

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 Toxicity of Pellitorine on Larval Aedes aegypti

Figure 4. Histology of anal gill region of third instar Ae. aegypti larvae. (A) Control larvae showed that the anal gill has inner lined epithelium
layer (EP) with well-organized anal gill cells (AGC) surrounded by thick cuticle layer. (B) Undistinguished damaged cuticle leading to completely
destroyed anal gill cells and shrinked anal gill of the treated larvae with 5 mg/l natural pellitorine compared to the anal gill of the control larvae.
doi:10.1371/journal.pone.0080226.g004

Immunostaining Analysis dures at 95uC for 5 min, followed by 35 cycles of 95uC for 30 s,
Rehydrated samples mentioned previously were subsequently 58uC for 30 s, 72uC for 1 min, and a final extension at 72uC for
subjected to immunostaining procedures [26]. To obtain neuron- 5 min using an Applied Bioscience Thermal Cycler (Foster City,
specific staining, unspecific binding sites in rehydrated samples CA). Aarps7 gene, an Ae. aegypti gene, was used as a control for
were blocked for 1 h in 3% Life Technologies normal goat serum normalization. All experiments were triplicate. The information of
(Grand Island, NY) in PBS solution. Afterwards, anti-horseradish primers used to amplify each gene is as follows: Aarps7: forward
peroxidase (HRP) antibody (Jackson ImmunoResearch laboratory, primer-59-CTGGAGGATCTGGTCTTC-39; reverse primer- 59-
West Grove, PA) conjugated to Alexa Fluor 488 (Jackson GTGTTCAATGGTGGTCTG-39 (Ref-ID_5572090), AaAQP4:
ImmunoResearch laboratory) was employed at 1:250 concentra- forward primer- 59-ATGCCACTGCTTGTCCCTAC-39; reverse
tion at 4uC overnight. Samples were washed with PBS solution primer 59-TTTCCGAAATGACCTTGGAG-39 [23], AaV-type
three times and mounted with Vector Laboratories Vectashield H- H+ ATPase: forward primer 59-GTTGTTCTGGCTCTGC-
1500 mounting medium with DAPI (49, 6-diamidino-2-phenylin- TGTTA-39; reverse primer- 59-GAGTGTTCTCGATAAGCCA-
dole) (Burlingame, CA). Fluorescent images were captured using a TAATC-39 [24]. In order to analyze the relative gene expression
Carl Zeiss LSM 700 confocal laser scanning microscope (Jena, levels, gene bands on agarose gels were visualized using Bio-Rad
Germany). Gel Doc XR + Imaging system (Hercules, CA). Subsequently, the
band intensity was automatically computed by densitometry
Transmission Electron Microscopic Analysis standard with Fuji Multi-Gauge version 3.0 software (Tokyo).
The midgut and anal gills of the pellitorine-treated and control The relative gene expression levels (%) in treatment groups were
larvae were fixed in Karnovsky’s fixative 2% (v/v) glutaraldehyde calculated as follows: the band intensity of pellitorine-treated
and 2% (v/v) PFA in 0.05 M sodium cacodylate buffer, pH 7.2 at group 4 the band intensity of control group 6100 [31]. Relative
4uC in darkness for 2–4 h, and washed with the same buffer three gene expression level of control groups was normalized to 100%
times [28]. The specimens were postfixed with 1% (w/v) osmium (Control/Control 6100). Statistical analysis for significant differ-
tetroxide in the same buffer at 4uC for 2 h, and washed with ence in gene expression patterns was tested using Student t-test
distilled water three times. The postfixed specimens were (SPSS, version 17, USA).
dehydrated through a graded series of ethanol increasing
concentrations up to 100% for 15 min. The specimens were Results
further treated with propylene oxide two times each for 15 min as
a transitional fluid, and embedded in Spurr’s resin [29]. Ultrathin Pathological Symptoms by Pellitorine
sections (approximately 50 nm thickness) were cut with a RMC The normal morphology for the whole body of the control third
MT-X ultramicrotome (Tucson, AZ), stained with 2% aqueous instar Ae. aegypti larvae showed the common appearance of the
uranyl acetate for 7 min at room temperature and with Reynolds typical structure with well-developed distinguished head, thorax,
lead citrate [30] for 7 min. The sections were mounted on copper and abdomen region (Fig. 2A). The whole body of the Ae. aegypti
grids, and the micrographs were obtained from a Carl Zeiss Libra larvae treated with pellitorine became dark in color, particularly in
120 Plus transmission electron microscope (TEM) (Jena, Germany) the damaged thorax and abdominal region (from 1st to 5th
at 80 kV. Observations were taken of 20 larvae under the TEM. segments). Damaged internal gastric caeca and dark black spots
were observed in the thorax region and the anal gills, respectively
Analysis of Gene Expression (Fig. 2B).
Total RNAs were isolated from the anal gills of fifty larvae using
a Qiagen RNeasy Mini Kit (Valencia, CA). Using 1 mg of total Histopathological Effect of Pellitorine by Cason’s Staining
RNA, cDNA was synthesized with oligo-dT with Invitrogen Well-developed gastric caeca (GC) and single-layered midgut
Superscript III enzyme (Grand Island, NY). Then, using a epithelia were observed in the anterior midgut regions of the
template of 1 ml of synthesized cDNA, polymerase chain reaction control Ae. aegypti larvae (Fig. 3A1), whereas undistinguished
(PCR) amplification was performed with gene specific primer sets enlarged portions of gastric caeca and damaged single-layered
for target genes, AaAQP4 (XM_001647996), and AaV-type-H+- epithelial cells were observed in the pellitorine-treated larvae
ATPase (AF092934). PCR conditions were performed by proce- (Fig. 3B1). The central midgut regions of the control larvae

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 Toxicity of Pellitorine on Larval Aedes aegypti

control larvae (Fig. 3A3), whereas completely damaged residues of

epithelial and peritrophic membranes were observed in the
pellitorine-treated larvae (Fig. 3B3).
Well-developed glandular anal gill cells which were connected
with rectum through the anal canal were observed in the control
Ae. aegypti larvae. The anal gills were surrounded by a thick
permeable cuticle layer consisted of anal gill cells (Fig. 4A).
However, undistinguished damaged cuticle was observed in the
pellitorine-treated larvae (Fig. 4B), leading to completely destroyed
anal gill cells (Fig. 4B2).

Histopathological Effect of Pellitorine by Immunostaining

Confocal laser scanning micrographs revealed well-developed
cardial nerves and subpopulations of neurons ended in rich
neuropils in thorax and anterior midgut regions of the control Ae.
aegypti larvae (Fig. 5A1–3). Particularly in the control larvae, the
gastric caeca consisted of well-developed neuropils (Fig. 5A1–3). In
contrast, cardial nerves in the thorax and anterior midgut regions
of the pellitorine-treated larvae showed disappearance of neuro-
pils, and also in the region of gastric caeca (Fig. 5A4–6). The
control larvae contained more neuronal cells and processes around
the epithelial layer of the posterior midgut region (Fig. 5B1–3).
Few neurons in damaged epithelial layer cells were observed in the
posterior midgut regions of the pellitorine-treated larvae (Fig. 5B4–
6). Well-developed neurons elongated throughout large swelling
anal gill cells were observed in the anal gill regions of the control
larvae (Fig. 5C1–3). The anal gill regions of the treated larvae
showed damaged and disappearance of neurons (Fig. 5C4–6).

Histopathological Effect of Pellitorine on Anterior and

Posterior Midgut
A transmission electron (TE) micrographs revealed that a
peritrophic membrane (PM) in the anterior midgut regions of the
control Ae. aegypti larvae enclosed the midgut lumen contents (LC)
and is separated from outer surface by midgut epithelia by a
narrow peritrophic space (PS) (Fig. 3A2, Fig. 6A1). The anterior
midgut regions of the control larvae were composed of well
speared cellular contents and also possessed a prominent nucleus
Figure 5. Confocal laser scanning micrograph histology
(Fig. 6A1). In addition, the anterior midgut epitheial layer
observations by immunostaining. (A1–3) Anterior midgut region
of the control larvae showed well-developed cardial nerves and consisted of well-developed epithelial cells having a large nucleus
subpopulation of neuron ends in rich neuropils. (A4–6) Cardial nerves (Fig. 6A2). The lumen contents of the anterior midgut regions also
in thorax and anterior midgut regions of larvae treated with 5 mg/l contain numerous well-developed fat body tissues (Fig. 6A3).
alkaloid pellitorine showed disappearance of neural processes. (B1–3) In All of the cellular contents in the anterior midgut regions of the
posterior midgut region, neuronal cells and processes near the pellitorine-treated larvae were devastated compared to the control
epithelial layer of the posterior midgut region were present. (B4–6)
Posterior midgut region in the pellitorine-treated larvae also showed larvae. In particular, the nucleus and cells surrounding the nucleus
the lack of cells in the epithelial layers. (C1–3) Anal gill region of the were completely destroyed (Fig 6B1). The anterior midgut
control larval mosquito showed well-developed neurons elongated epithelial cells showed completely damaged and undistinguished
throughout large swelling anal gill cells. (C4–6) Neurons were damaged cellular residues (Fig. 6B2). In addition, the fat body tissues in the
and disappeared in treated larvae. Arrowheads shown in (A) represent anterior midgut lumen contents were demolished by pellitorine
cardial nerves, gastric caeca, and neural processes. Arrowheads in (B)
represent neuronal cells in epithelial layers of the gut. Arrowheads in (C) treatment (Fig. 6B3).
indicate neuronal elongation and branches to anal gills. Scale bars The posterior midgut of the control larvae was characterized by
depict 40 mm. a few epithelial cells (Fig. 3A3) with electron-dense cytoplasm
doi:10.1371/journal.pone.0080226.g005 (Figs. 7A1 and A2) as reported previously [15,32]. The posterior
midgut regions showed numerous dark cells within the nucleus and
consisted of well-developed lumen contents (LC), and were also showed some polysomes in well-developed lumen contents as
surrounded by an inner transparent peritrophic membrane (PM) in control larvae sections (Figs. 7A2). Pellitorine destroyed all
and an outer midgut epithelial layer. Both inner and outer posterior midgut epithelial cell layers. In particular, cells in the
membranes were separated by peritrophic space (PS) (Fig. 3A2). In luminal contents region were completely destroyed (Fig. 7B1) and
the central midgut regions of the treated larvae, demolished caused the degeneration of dark cells and polysomes (PS) (Fig. 7B2).
epithelial layer residues were observed (Fig. 3B2). Distinguished An undistinguished cellular layer bearing only residues inside the
midgut epithelial layers, lumen contents, and peritrophic mem- gut lumen was also observed in the posterior midgut (Fig. 7B2).
branes were observed in the posterior midgut regions of the

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 Toxicity of Pellitorine on Larval Aedes aegypti

Figure 6. Transmission electronic micrographs of anterior midgut regions of third instar Ae. aegypti larvae without (A) and with
treatment with 5 mg/l of natural pellitorine (B). A1. A control larva showed well-developed peritrophic membrane (PM) enclosing midgut
lumen contents. The midgut lumen cells consist of prominent nucleus and other cellular content present in the cytoplasm. A2. Organization of
anterior midgut epithelial cells with large nucleus in a control larva. A3. Anterior midgut lumen content possesses numerous fat body (FB) tissue. B1-
2. In the anterior midgut region of larvae treated with 5 mg/l natural pellitorine, all cellular contents including the nucleus were destroyed and cell
masses in the cytoplasm were extruded. B3. Fat body tissues of lumen content were demolished by pellitorine treatment. N, nucleus;
doi:10.1371/journal.pone.0080226.g006

Histopathological Effect of Pellitorine on Anal Gills pellitorine-treated larvae as compared to the control larvae.
The TEM study revealed that the anal gill of the control larvae Similarly, aquaporin protein gene expression level was inhibited in
consisted of an epithelia and covered by a thin and relatively the treated larvae as compared to the control larvae. The
permeable cuticle [20,33]. The epithelium is extensively trache- expression level of rps7 gene of Ae. aegypti (Aarps7) was not
ated with tracheolar anal gill cells, and the anal gill lumen is filled affected by both treated and control larvae.
with hemolymph and is continuous with the hemocoel [33]
(Fig. 8A). In contrast, the damaged outer membrane was Discussion
surrounded by a permeable cuticle layer of anal gill in
The current microscopic analysis clearly indicates that pellitor-
pellitorine-treated larvae, which led to internal cytoplasmic
ine caused histopathological alterations in thorax, midgut, and
destruction, particularly degeneration of all tracheolar anal gill
anal gill regions in the third instar larvae of Ae. aegypti.
cells (Fig. 8B).
Investigations on the modes of action and the resistance
mechanisms of plant-based biocides are of practical importance
Pellitorine-induced Target Gene Expression because they may provide useful information on the most
The transcript expression patterns of V-type H+-ATPase that is appropriate formulations to be adapted for future commercializa-
involved in the Na+, Cl–, and K+ uptake co-transport process and tion and future resistance management. Also, they may contribute
a putative aquaporin protein in anal gills were observed in both to the development of selective mosquito control alternatives with
control and pellitorine-treated Ae. aegypti larvae (Fig. 9). The gene novel target sites and low toxicity [6,7]. It has been reported that
expression of V-type H+-ATPase was significantly inhibited in the pellitorine is effective against Cules pipiens pallens larvae with high

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 Toxicity of Pellitorine on Larval Aedes aegypti

Figure 7. Transmission electronic micrographs of posterior midgut regions of third instar Ae. aegypti larvae without (A) and with
treatment with 5 mg/l of natural pellitorine (B). A1–2. The posterior midgut region of the control larva was characterized by few epithelial cells
with electron-dense cytoplasm. Also, posterior midgut lumen has shown numerous dark cells with nucleus and polysomes. B1. Posterior midgut
region of a larva treated with 5 mg/l natural pellitorine showed the compound destroyed all posterior midgut epithelial cells layer and also caused
degeneration of dark cells and polysomes. B2. Residual tissues inside the gut lumen were observed in the posterior midgut in a pellitorine-treated
larva. PM, peritrophic membrane; LC, lumen contents; PS, polysomes; DC, dark cell
doi:10.1371/journal.pone.0080226.g007

levels of resistance to AChE inhibitors such as chlorpyrifos, duration of the treatment, and the taxon [16,34]. Nasiruddin and
fenitrothion, and fenthion as well as axonic nerve poisons such as Mordue [34] reported that azadirachtin caused some of the initial
a-cypermethrin and deltamethrin [12]. These results suggest that effects on necrosis, particularly associated with the swelling of the
the alkaloid pellitorine and the pyrethroid and organophosphate cell and organelles, vesiculation of membranes, and dilation of
(OP) insecticides do not share a common mode of action. In rough endoplasmic reticulum in locusts. It has been also reported
addition, histopathological investigations indicate that the midgut that tannic acid caused dramatic degenerative response of the
epithelium is the site of action of plant preparations and PSMs in midgut through a sequential epithelial disorganization in C. pipiens
Papilio polyxenes and Papilio glaucus [14], some aquatic dipteran larvae [15]. Our current study revealed that pellitorine caused
larvae [15,16], and Schistocerca gregaria and Locusta migratoria [34], dramatic degenerative responses in thorax and anterior and
Rhodnius prolixus [35], and several species of Acridoidea [36]. posterior midgut regions of Ae. aegypti larvae by targeting ion
Midgut epithelium is known to have functions such as ionic and transporting cells in gastric caeca of the thorax region and
osmotic regulation [32], lipid and carbohydrate storage epithelial cells of the anterior and posterior midgut region where
[32,37,38], control of the midgut lumen pH, and the secretion osmoregulation-related machineries such as, H+V-ATPase are
of digestive enzymes and absorption of nutrients [39,40]. The highly expressed in the anterior midgut of Ae. aegypti larvae
histopathological effects differ qualitatively according to the [24,41,42].
localization of organs along the midgut and quantitatively The anal gills of mosquito larvae are important primary sites of
according to the concentration of test material examined, the NaCl uptake, thereby acting to offset the dilution of the

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 Toxicity of Pellitorine on Larval Aedes aegypti

Figure 8. Transmission electronic micrographs of anal gill regions of third instar Ae. aegypti larvae. (A) The anal gill of the control larva
was surrounded by thick cuticle (CU) and inner surface covered with epithelial layer (EP) having large swelling anal gill cells (AGC) filled with
hemolymph. (B) The anal gill of a larva treated with 5 mg/l natural pellitorine showed damaged outer membrane surrounded by a thick cuticle, which
led to internal lumen content destruction, particularly degeneration of all anal gill cells.
doi:10.1371/journal.pone.0080226.g008

hemolymph by the dilute habitat [21,43,44]. Donini and serve as the major site for Na+, Cl–, and K+ uptake, also
O’Donnell [22] confirmed with the use of self-referencing ion- complementing the role of the Malpighian tubules and rectum.
selective microelectrodes that the anal gills of Ae. aegypti larvae Additionally, Marusalin et al. [23] proved that putative aquaporin
homologs, especially AaAQP4, play an important role in
mediating water movement across the anal gill epithelia of the
mosquito larvae. However, few information is available with
respect to the histopathological as well as gene expression patterns
of V-type H+-ATPase and AaAQP4 by insecticides or PSMs on
anal gills of mosquito larvae. Our present study has revealed that
pellitorine inhibited AaAQP4 expression levels. This natural
compound may disturb the Na+, Cl–, and K+ co-transport system
mainly by the degeneration of anal gill cells and the damage of
outer thick permeable cuticle membranes of Ae. aegypti larvae. In
contrast, the gene expression encoding V-type H+-ATPase protein
in the Na+, Cl–, and K+ ion co-transport system has shown slight
decrease in our study, even though it has shown significant effects
on the gene expression level by pellitorine treatment. More
detailed examination on ion exchange effects in anal gills by
natural pellitorine compound remains to be investigated, even
though it has been reported that it is difficult to measure the ion
exchange in place due to their morphological characteristics [19].
These original findings indicate that pellitorine caused the
histopathological alterations and inhibition of gene expression of
V-type H+-ATPase and aquaporin protein in the anal gills. The
findings may also contribute to a better larvicide mode of action
understanding of an alkaloid against A. aegypti.
In conclusion, pellitorine caused degenerative responses in the
cell organelles of the thorax, midgut regions, and anal gills,
possibly by targeting with the osmoregulation system. The alkaloid
acts as a potential mosquito larvicide with a specific target site for
the control of mosquito populations. Further research is needed to
Figure 9. Gene expression patterns by pellitorine treatment. (A)
establish the toxicity of aquatic non-target organisms.
RT-PCR of AaAQP4, AaV-H+-ATPase and Aarps7 genes in control and
treatment groups. (B) Relative gene expression level of AaV-type H+- Acknowledgments
ATPase and aquaporin 4 (AaAQP4), compared to the expression level of
Aarps7 gene. The gene expression of AaV-type H+-ATPase and We appreciate Drs. Ahn and Kwon’s lab members for rearing mosquitoes.
aquaporin 4 (AaAQP4) in the anal gills was inhibited in a larva treated We also thank staff members in NICEM (National Instrumentation Center
with 5 mg/l natural pellitorine as compared to the control larvae. * for Environmental Management) at Seoul National University for TEM
indicates significant difference in gene expression levels in control and assistance. Our special thanks go to a PLoS One editor Dr. G. Dimopoulos
pellitorine treated groups (Student’s t-test, N = 3, P,0.01). and two anonymous reviewers for shepherding our manuscript into more
doi:10.1371/journal.pone.0080226.g009 advanced level of a science paper through their feedback, comments, and

PLOS ONE | www.plosone.org 8 November 2013 | Volume 8 | Issue 11 | e80226

 Toxicity of Pellitorine on Larval Aedes aegypti

suggestions. Finally, we thank Seoul National University and WCU Author Contributions
program launched by the National Research Foundation and the Ministry
of Education, Science and Technology of Korea, which mostly made this Conceived and designed the experiments: HP HWK Y-JA. Performed the
research possible. experiments: J-RK SMO JWJ. Analyzed the data: HP HWK Y-JA.
Contributed reagents/materials/analysis tools: HP HWK Y-JA. Wrote the
paper: HP HWK Y-JA.

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