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School of Applied Biosciences, Kyungpook National University, Daegu 702-701, Republic of Korea
Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea
Institute of Plant Medicine, Kyungpook National University, Daegu 702-701, Republic of Korea
d
Sustainable Agriculture Research Center, Kyungpook National University, Gunwi, Republic of Korea
b
c
a r t i c l e
i n f o
Article history:
Received 2 July 2015
Revised 30 September 2015
Accepted 25 October 2015
Available online 12 November 2015
Keywords:
Azadirachtin
Biopesticides
Gene expression
Oral ingestion
Whitey
a b s t r a c t
Azadirachtin is a plant-derived triterpenoid compound, which has adverse effects on growth, feeding, and
reproduction of insects. However, its action is not well understood at the molecular level. The effects of oral
ingestion of azadirachtin in whiteies, Bemisia tabaci, a serious pest of various agriculturally important plants,
were determined by assessing their mortality and changes in gene expression. Whiteies (0-day-old) were
allowed to ingest 20% sugar solution containing 0, 1, 5, or 10 ppm azadirachtin using a two-layered paralm
feeding chamber. Mortality gradually increased with time and increased dosage, with all individuals dead at 72
and 48 h at 5 and 10 ppm, respectively. Furthermore, the mRNA levels of 15 genes, which are associated with
development, metabolism, defenses, and stresses, were compared between whiteies which ingest either 0 or
5 ppm azadirachtin for 12 h by quantitative real-time RT-PCR. Most genes changed mRNA levels at less than 2
folds. However, the expression of thioredoxin peroxidase 1 and two ferritin genes which have protective roles
against oxidative stress was inhibited up to 36 folds more than that in untreated whiteies. These results
suggest that lethal toxicity of azadirachtin may be due to increased cellular oxidative stress of B. tabaci.
2015 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection
Society. Published by Elsevier B.V. All rights reserved.
Introduction
Azadirachtin is a triterpenoid compound, which is associated with
the seeds of the neem tree, Azadirachta indica A. Juss (Sapindales:
Meliaceae) (Mordue and Blackwell, 1993; Morgan, 2009). This
compound has pesticidal properties that are active against nearly 550
insects and pest species, including arthropods, nematodes, annelids,
and fungi (Govindachari and Gopalakrishnan, 1998; Veitch et al.,
2008; Hatti et al., 2014). It is a potent antifeedent, repellent, and
growth-regulating compound in a wide variety of phytophagous
insects. Its mode of action is known to inhibit ecdysteroid synthesis
resulting in the inhibition of insect growth and reproduction. Therefore,
the presence of azadirachtin in insects induces hormonal balances
resulting in the failure of molting or dramatic deformation at immature
stages of growth. In addition, azadirachtin has demonstrated cytotoxic,
antiproliferative, and antimitotic effects in insect cell lines (Salehzadeh
et al., 2003; Anuradha et al., 2007; Kumar and Poehling, 2007). For
http://dx.doi.org/10.1016/j.aspen.2015.10.011
1226-8615/ 2015 Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society. Published by Elsevier B.V. All rights reserved.
oviposition of adult females (Lynn et al., 2010). In addition, eld application of azadirachtin-A and its stable derivative tetrahydroazadirachtin-A
improved control efcacy against B. tabaci as well as leafhopper (Dhingra
et al., 2008). However, the mechanism of azadirachtin toxicity still
remains unclear at cellular and molecular levels.
Here, we determined the effects of the oral ingestion of azadirachtin
on the mortality of B. tabaci and investigated its effect on genes that
demonstrated early responses and were related to stress, immunity,
metabolism, cytoskeleton development, and reproduction in adult
B. tabaci.
Materials and methods
Insect rearing
A colony of Q-biotype Bemisia tabaci was maintained on tomato plants
(Lycopersicon esculentum Mill.) in insect-proof cages (45 60 90 cm).
The colony was reared in insect-rearing rooms at 25 1 C, with a
relative humidity of 60 5%, and a 16 h light/8 h dark (16 L:8D) photoperiodic cycle. For experiments, tomato leaves infested by mature
nymphs were prepared in Petri dishes with moisture the previous
evening. Adult eclosion was monitored daily, in the morning, and 0day-old adults (less than 12 h post-eclosion) were collected and used
in experiments.
Kit (Applied Biosystems, USA) in a PTC-200 thermal cycler (MJ Research, Watertown, MA, USA). Gene-specic primers were designed
for qRT-PCR according to Mahadav et al. (2009) (Table 2). The cDNA
samples (0.2 l) were run in triplicate in a 7300 Sequence Detection
System (Applied Biosystems, USA) using Power SYBR Green PCR Master
Mix (Applied Biosystems, USA). One cycle consisted of 95 C for 15 min,
followed by 45 cycles (95 C for 10 s, 60 C for 20 s, 72 C for 30 s) followed by a nal cycle for the dissociation stage (95 C for 15 s, 60 C for 30 s,
95 C for 15 s). The expression level of each gene was determined by
comparing the relative quantities of the cDNA to those of the respective
mRNA. The Ct (threshold cycles) values were used to calculate the
mRNA levels. The data were analyzed using the following formula:
2Ct = 2[Ct treatment Ct control] (Livak and Schmittggen, 2001).
The partial nucleotide sequence of the actin gene from B. tabaci
(AF071908) was identied from the Bemisia EST database in the NCBI
GenBank. Actin level was used as a reference to normalize the expression levels of the other genes.
Statistical analysis
Data were analyzed by Sigma Plot 8.0. All data are expressed as the
mean SE (standard error). Statistical data analysis was conducted
with SAS software version 9.0 (2003). The treatment methods were
compared using the least signicant difference (LSD) test at p 0.05.
Preparation of azadirachtin
Table 1
Comparison of ingested amount of B. tabaci between 20% sucrose solution with or without
5 ppm azadirachtin.
Treatments
20
20
12 h
9.0 3.5a
0b
41.6 11.5a
15.0 5.1b
Ingested amounts were calculated for 6 h and 12 h feeding durations of a single 0-dayold (less than 12 h post-eclosion) adult female.
Means (SE) followed by the same letter are not signicantly different by DMRT's
test at p 0.05.
PCR (Table 3). Our results showed that whiteies that ingested
azadirachtin did not signicantly change the expression levels of most
genes although their rates were decreased 12 folds than those of
non-treated whiteies. However, 3 genes, including ferritin 2, ferritin
17, and thioredoxin peroxidase 1, decreased by 6.47, 6.73, and 3.23
folds, respectively, revealing signicant differences when compared to
those in untreated whiteies (Table 3). Thus, this suggests that ingested
azadirachtin differentially regulates specic genes at the transcription
levels within cells. Zhao et al. (2014) reported that azadirachtin binds
proteins within the nucleus of the cells. This suggests that azadirachtin
can act as regulators for the transcription of specic genes within the
nucleus.
It is interesting that whiteies that ingested azadirachtin showed
signicantly inhibited transcript levels of two ferritin genes as well as
thioredoxin peroxidase-1, which have protective roles within the cells
against oxidative stress. Ferritin is a complex consisting of 24 subunits
including 12 heavy chain and 12 light chain subunits and has a role in
iron storage and transport within the cells (Pham and Winzerling,
2010). Iron is an essential element present in limited supply in the
Table 2
Gene-specic primer sequences of Bemisia tabaci for real-time RT-PCR, as described in
Mahadav et al. (2009).
Target genes
Sequences (5 3)
GenBank
accession
numbers
hsp40
AJ509088
hsp70
hsp90
knottin 3
ferritin 2
ferritin 17
thioredoxin peroxidase 1
p8 protein
cytochrome P450 (CYP6CM1)
ecdysone receptor
paramyosin
tropomyosin
myosin H chain
myolin
prolin
actin
Fig. 1. Effect of azadirachtin ingestion on the mortality of Bemisia tabaci. Azadirachtin was
diluted to 0, 1.5, and 10 ppm in a 20% sucrose solution and fed to adult whiteies (n = 50)
in the paralm sandwich chamber. Numbers of dead whiteies were counted every 6 h.
Each point represents mean SE of three replications from different sets of experiments
of identical environmental conditions.
cells (Tang and Zhou, 2013). However, it is also a highly toxic precursor
of reactive oxygen species (ROS), such as superoxide anions (O2),
hydrogen peroxide (H2O2), and hydroxyl radicals (OH), which induce
oxidative stress, disrupting normal cellular processes. Ferritin expression is also essential for resistance against oxidative stresses in the bacterium, Listeria monocytogenes (Dussurget et al., 2005). In addition,
thioredoxin peroxidase, together with thioredoxin and thioredoxin
reductase, is a component of the thioredoxin system, which is an important and conserved system implicated in protection against oxidative
stress since it reduces peroxides such as H2O2 to harmless products.
The expression of genes encoding the components of the thioredoxin
system is essential for protection against damage caused by oxidative
stress. Thus, our results suggest that whiteies that have ingested
azadirachtin may have signicant oxidative stress due to the
disturbance of antioxidative system in their cells. Recently, Dere et al.
DQ093377
DQ093381
EE597369
Table 3
Effect of azadirachtin ingestion (5 ppm) on the expression of various genes of Bemisia
tabaci.
Genes
EE603117
EE597196
EE598240
EE600305
EU344879
EF174329
EE597453
EE601593
EE599189
EE674613
EE597329
AF071908
hsp70
hsp90
cytochrome P450
knottin 3
ferritin 2
ferritin 17
thioredoxin peroxidase 1
p8 protein
myolin
tropomyosin
myosin H chain
prolin
paramyosin
hsp40
ecdysone receptor
mRNA levels
Without
azadirachtin
With
azadirachtin
4.6 0.7 a
4.5 0.85 a
2.7 0.65 a
3.3 0.36 a
3.6 0.68 a
3.2 0.67 a
3.6 0.20 a
2.6 0.81 a
4.6 0.41 a
3.7 1.38 a
2.5 0.70 a
3.0 0.48 a
3.0 0.49 a
2.6 0.4 a
3.0 0.70 a
3.60 0.68 a
3.30 0.6 a
1.96 0.34 a
2.40 0.18 a
0.55 0.05 b
0.48 0.22 b
1.56 0.31 b
1.80 0.36 a
2.42 0.39 a
3.36 0.40 a
1.42 0.06 a
1.96 0.13 a
1.96 0.13 a
2.90 0.5 a
5.03 0.8 a
Fold
differences
1.28
1.37
1.37
1.37
6.47
6.73
3.23
1.44
1.92
1.11
1.79
1.54
1.54
+1.12
+1.65
Adult females (less than 12 h post-eclosion) (n = 50) were allowed to ingest a 20%
sucrose solution with or without 5 ppm azadirachtin for 12 h, then total RNA was extracted for the analysis of quantitative real-time PCR.
Means (SE) followed by the same letter are not signicantly different by DMRT's
test at p 0.05.
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