Prothoracicotropic hormone-producing neurosecretory

Turkish Journal of Biology
http://journals.tubitak.gov.tr/biology/
Research Article
Turk J Biol
(2014) 38: 403-411
© TÜBİTAK
doi:10.3906/biy-1309-57
Prothoracicotropic hormone-producing neurosecretory neurons and antioxidative
defense in midgut of Lymantria dispar in trophic stress
Vesna PERIĆ-MATARUGA*, Milena VLAHOVIĆ, Marija MRDAKOVIĆ, Dajana TODOROVIĆ,
Dragana MATIĆ, Anja GAVRILOVIĆ, Larisa ILIJIN
Department of Insect Physiology and Biochemistry, University of Belgrade, Institute for Biological Research “Siniša Stanković”,
Belgrade, Serbia
Received: 30.09.2013
Accepted: 13.02.2014
Published Online: 14.04.2014
Printed: 12.05.2014
Abstract: As a very invasive insect species, Lymantria dispar is adaptable and sensitive to a changing environment. In insects the
neuroendocrine system first reacts to stress by production of prothoracicotropic neurohormones (PTTH) that control ecdysteroid
synthesis (morphogenetic and stress hormones). In this article, we report changes in the L2’ brain neurosecretory neurons that
synthesize PTTH in L. dispar larvae after feeding on locust tree leaves (Robinia pseudoacacia), an unsuitable host plant. Groups of
larvae (n = 20 per experimental group) were offered this in comparison with oak leaves (Quercus robur), a suitable control diet, for 3
days after molting into the fourth instar. L2’ neurons and their nuclei were enlarged and the amount of neurosecretory product in the
cytoplasm was increased (15.5%) after consumption of locust tree leaves in comparison to the control. Furthermore, activities of the
following antioxidative defense components were estimated: superoxide dismutase (SOD), catalase (CAT), and amount of glutathione in
the midgut. Higher SOD activity (13.85 ± 0.9 U/mg prot.) and glutathione amount (0.56 ± 0.06 µMGSH/g tissue) but unchanged CAT
activity was found in the midgut of larvae offered locust tree leaves when compared to the control.
Key words: Oak leaves, locust tree leaves, survival, superoxide dismutase, catalase, glutathione
1. Introduction
As a gradogenic polyphagous and invasive species, the
gypsy moth (Lymantria dispar L.) encounters different
environmental challenges. Considering that its host
range is estimated at more than 500 plant species within
73 families (Liebhold et al., 1995), the gypsy moth is
characterized by a variety of adaptations that ensure
survival and reproduction in heterogeneous and stressful
environments. Problems with gypsy moth overcrowding
are not solved yet (Demir et al., 2009, 2012). Plants from
the genus Quercus are accepted as the most suitable food
and the most susceptible to L. dispar population eruptions
(Maksimović, 1987; Liebhold et al., 1995). In the Republic
of Serbia, large areas of forest and orchards were attacked
(171,914 ha) by gypsy moths in 2012 according to the
State Enterprise for Forest Management of Serbia (http://
www.srbijasume.rs/). Other parts of Europe are also at
risk of gypsy moth invasion and degradation of forest
ecosystems. This pest insect is one of the most serious
insect defoliators of North American forests and urban
landscapes according to the US Forest Service (http://
www.fs.fed.us/). Gypsy moth defoliation results in loss
of growth, mortality of oak species, and shifts in species
*Correspondence: vesper@ibiss.bg.ac.rs
composition in mixed-oak forests (McGraw et al., 1990).
Young oak leaves contain low amounts of flavonoids
in spring when L. dispar attacks them (Salminen et
al., 2004). A high population density of gypsy moths
induces changes in leaf chemistry during defoliation, i.e.
flavonoid content increases, leading to suppression of the
insect immune response, and their resistance to viruses
is lowered (Martemyanov et al., 2012). One of the very
rare species of plant that is an unsuitable host for gypsy
moths is the locust tree, Robinia pseudoacacia (Barbosa
and Krischik, 1987). A nonfamiliar host plant for L.
dispar, R. pseudoacacia is a widespread species in its native
habitat in southeastern North America. It was introduced
to Europe in 1601 (Chapman, 1935). Today, it has spread
throughout western, central, eastern, and southern Europe
and has become a major invasive species with a significant
impact on native plant communities. Despite the large
number of polyphagous species of Lepidoptera and the
long period after the introduction of R. pseudoacacia to
Europe, so far only a few native lepidopteran species have
adapted to it (Kulfan, 2012). The unfavorable effects of
locust leaves on gypsy moth larvae can be attributed to
the presence of phenolic defensive compounds (Barbosa
403
PERIĆ-MATARUGA et al. / Turk J Biol
and Krischik, 1987). Chromatographic separation of
an ethanolic extract of R. pseudoacacia leaves enabled
isolation of much quercetin, robitin, myricetin, and other
allelochemicals (Nasir et al., 2005). These compounds,
especially flavonoids and tannins, can induce metabolic
stress, including production of free radicals and a state
of oxidative stress in the larval gut (Perić-Mataruga et al.,
1988, 1997, 2001a, 2006b; Mrdaković et al., 2011, 2013).
A dominant role for the neuroendocrine system
in mechanisms of stress response in insects has been
indicated (Janković-Hladni, 1991; Perić-Mataruga et al.,
2001b; Gruntenko et al., 2005; Krishnan and Kodrik, 2012).
Different stressors modify the activity of neurosecretory
neurons (NSNs). Moreover, their responses are selective
and depend on the type and intensity of stressor and
environmental conditions (Chernysh, 1991; JankovićHladni, 1991). Neurohormones influence the release
of lipids (adipokinetic hormones) and carbohydrates
(hypertrehalosemic hormone) from the fat body depots,
stimulate the uptake of carbohydrates (hypoglycemic
hormone), regulate carbohydrate homeostasis (small
form of prothoracicotropic neurohormones), stimulate
(diuretic hormone) or suppress (antidiuretic hormone)
water excretion, and regulate steroid production [large
form of prothoracicotropic neurohormone (PTTH)]
(Gade and Goldsworthy, 2003). Analysis of PTTH in
Lepidoptera revealed that these peptide neurohormones
appear in several isoforms (Ishizaki and Suzuki, 1994).
Two forms were described: a large PTTH of molecular
mass 24–30 kDa (Kawakami et al., 1990) and a small
form or bombyxin of Mr 5–19 kDa (Ishizaki and Suzuki,
1994). The large form of PTTH is synthesized in 2 pairs
of dorsolateral protocerebral NSNs and is released from
their axons into the corpus allatum (Dai et al., 1994; Ilijin
et al., 2012b). This neurohormone was identified in L.
dispar larvae by Kelly et al. (1991). The L2’ protocerebral
dorsolateral NSNs in L. dispar synthesize the large form
of PTTH (Ilijin et al., 2012b). The hormone regulates
synthesis of ecdysteroids through the prothoracic gland
by elevating cyclic AMP and the calcium-dependent
pathway (Agui et al., 1980). Ecdysone is synthesized from
dietary cholesterol derived from food and, after a number
metabolic steps, the substance is released from the gland.
Conversion of ecdysone to the physiologically active form,
20-hydroxyecdysone (20-HE), occurs primarily in fat
bodies or midgut cells, but many other tissues are known
to contain ecdysone 20-monooxygenase, a key enzyme for
conversion of ecdysone to 20-HE (Mizoguchi et al., 1990;
Dai et al., 1994; Gade and Goldsworthy, 2003; Gade and
Marco, 2006).
Ecdysteroids are known to be most responsible for
eliciting the molting process and modulating stress
responses in insects (Chernysh, 1991; Gruntenko et
404
al., 2005). PTTH contributes to the processes of stress
adaptation by regulating morphophysiological changes
and development. Different stressors, such as trophic stress,
temperature, intoxication, photoperiod, proprioceptive,
mechanoreceptive stimuli, and many others, alter the
ecdysteroid secretion regulated by PTTH (Chernysh, 1991;
Rauchenbach, 1991). One of the most important members
among insect steroid hormones, 20-HE shows potent
antioxidative activity and minimizes oxidative stress after
prooxidative stimuli (Krishnan, et al. 2007; Krishnan and
Kodrik, 2012).
Oxidative stress is caused by an imbalance between
the production of reactive oxygen species (ROS) and the
ability of a biological system to detoxify readily (Halliwell
and Gutteridge, 2007). Generation of ROS accompanies
oxygen metabolism in aerobes in diverse biochemical
reactions. During the respiration process about 2% of
molecular oxygen is permanently transformed into the
superoxide anion radical (O2·–), the principal generator
of ROS and consequently other free radicals (Halliwell
and Gutteridge, 2007). Prooxidant plant allelochemicals
(phenolic compounds), such as flavonoids and tannins,
are an exogenous source of oxidative stress acting against
phytophagous pest insects. Phenolics can participate in 4
major types of bonds, hydrophobic, hydrogen, ionic, and
covalent (Halliwell, 1996), to create toxic compounds.
Almost any oxidation of phenolics in the gut can generate
O2·–, because the reactive semiquinone can donate an
electron to molecular oxygen. In the insect gut, tissue
toxic phenoxyl radicals are formed via oxidative processes
owing to their ability to initiate free radical chain reactions
in the membrane and the propensity to cross-link with a
variety of molecules. The amount of oxidation of phenolics
depends on the prevailing physicochemical conditions in
the gut, including pH, redox potential, and antioxidative
potential. The midgut of phytophagous insects, including
L. dispar, is a highly oxidizing environment. Various
indicators of oxidative stress in the gut tissue of the
lepidopteran larvae of Spodoptera litoralis (Krishnan and
Sehnal, 2006) and Helicoverpa zea (Felton et al., 1992) given
a diet with phenol allelochemicals were found to increase.
However, some insects, including L. dispar, possess a suite
of antioxidative enzymes and nonenzymatic components
that protect their cells from oxidative radicals (Ahmad,
1992; Perić-Mataruga et al., 1997; Mirčić et al., 2013).
Superoxide anion radicals generated during oxidative
stress in insects are rapidly converted by cytosol copper/
zinc-containing superoxide dismutase (CuZnSOD) to
H2O2, which can be decomposed by catalase activity to
water and oxygen (Ahmad and Pardini, 1990; Fridovich,
1995; Perić-Mataruga et al., 1997). Overproduction
of H2O2 is strongly oxidative and mediates hydroxy
radical (OH) generation via Fenton and/or Haber–Weiss
PERIĆ-MATARUGA et al. / Turk J Biol
reactions. A most important nonenzymatic cellular
antioxidant is glutathione (GSH) (Ahmad and Pardini,
1990; Perić-Mataruga et al., 1997; Blagojević and GruborLajšić, 2000). It is the most abundant nonprotein thiol
in insect cells (Halliwell, 1996) and has a number of
metabolic functions. One of the most important is
protection of cells against oxidants and other xenobiotics.
Protective antioxidative reactions in insects are regulated
in a complex manner. Lately there was new information
about a strong role for insect hormones in modulating the
antioxidative defense strategy: adipokinetic hormones,
glucagon, juvenile hormones, ecdysteroids, etc. (Krishnan
et al., 2007; Alquicer et al., 2009; Krishnan and Kodrík,
2012; Grubor-Lajšić et al., 2013).
The objective of the present study was to investigate
the responses of L2’ NSNs of L. dispar (synthesis of
PTTH) and correlations with the first line of enzymatic
antioxidative protection [superoxide dismutase (SOD) and
catalase (CAT) activity and GSH content] in the midgut
of fourth instar larvae that had been fed for 3 days on R.
pseudoacacia leaves, an unsuitable host plant.
2. Materials and methods
2.1. Insect rearing
Lymantria dispar egg masses were collected in an oak
(Quercus rubra) forest (locality “Kosmaj”, 80 km from
Belgrade) and kept in a refrigerator at 4 °C from October
to March, when they were set for hatching. After hatching,
Lymantria dispar caterpillars were reared on oak leaves in
transparent petri dishes (diameter of 10 cm, depth of 1.5
cm, volume of 117.8 cm3) at 23 °C with a 16 h light/8 h dark
photoperiod. Larvae were reared on suitable oak leaves
until hatching of the fourth instar and then caterpillars
were randomly assigned to the following experimental
groups: 1) Control group fed suitable oak leaves (n = 20)
and 2) group fed unsuitable locust tree leaves (n = 20).
Survival was evaluated daily in each experimental group.
2.2. Histochemistry preparation and analysis
On the third day, the fourth instar caterpillars (n = 20
per experimental group) were sacrificed by decapitation
and head capsules were fixed in Bouin’s fixative for
24 h (Merck, Darmstadt, Germany). The histological
procedure was carried out according to Panov (1980).
Head capsules were dissected and brain complexes were
extracted and then rinsed in 70 % ethanol (Hemos,
Belgrade, Serbia), dehydrated in a graded series of ethanol
(Hemos), impregnated in xylene (Hemos), and embedded
in paraffin wax (59 °C, Merck). Serial 3.5-µm sections of
brain complexes were cut for histochemistry (microtome,
820 Spencer) and collected on 0.2% gelatin/0.05% chrome
alum (Sigma-Aldrich) coated slides (6 slides per brain
complex). After drying for 48 h at 37 °C, the sections
were deparaffinized in xylene, rehydrated with 10 mM
phosphate buffered saline (Sigma-Aldrich), and stained by
the paraldehyde fuchsin technique of Panov (1980). NSNs
were stained dark purple/paraldehyde fuchsin-positive,
and on the basis of morphological characteristics, L2’ NSNs
were easily selected (1 pair per brain). Neurosecretory
products in NSN cytoplasm were stained dark purple
and nucleoli were light pink (Panov, 1980). The activity
of protocerebral L2’ NSNs was determined by combined
monitoring of the following cytological parameters: the
relative size of NSNs and their nuclei (expressed as the
mean values of smallest and largest diameters, µm) and
the amount of neurosecretory product in the cytoplasm
of neurons. All parameters were analyzed using Leica
QWin image analysis software. The relative quantity of
neurosecretory product via cytoplasmic optical density in
the L2’ PTTH-secreting NSNs was analyzed with National
Institutes of Health (NIH) software Image J 1.42q (NIH,
Bethesda, MD, USA) by means of digital image processing
technique. The relative amounts of neurosecretory product
in cytoplasm of L2’ PTTH-secreting NSNs in caterpillars
fed locust tree leaves were estimated in comparison
with L2’ neurons in caterpillars fed oak leaves (control),
postulated as 100% intensity of optical density.
For further details, see our previous publications
(Perić-Mataruga et al., 2001, 2006b; Ilijin et al., 2012a,
2012b, 2014).
2.3. Preparation of homogenates
After the caterpillars were sacrificed, the midguts were
dissected on ice and washed several times with ice-cold
physiological saline (0.9% NaCl). Midguts were pooled
by weight (2 per homogenate) and homogenized in a
buffer of 0.25 M sucrose, 0.05 M Tris-HCl, 1 mM EDTA,
pH 7.4 (1:10 w/v) according to Rossi et al. (1983) and
were sonicated according to Takeda et al. (1982). For
determination of the total amount of GSH, part of the
sonicated homogenate used to precipitate proteins with
5% sulfosalicylic acid and the total amount of GSH was
measured after centrifugation at 5000 rpm for 10 min. The
rest of the sonicated homogenate was centrifuged at 10,500
× g for 90 min and the activities of SOD and CAT were
determined in the supernatant.
SOD activity was determined according to Misra and
Fridovich (1972). This method is based on the ability of SOD
to prevent adrenaline autoxidation in an alkaline medium.
The adrenaline conversion into adrenochrome is followed
by the release of superoxide anion radicals that lead to
the acceleration of the autoxidation reaction. Adrenaline
autoxidation rate was determined spectrophotometrically
through the absorption change at a wavelength of 480
nm at 25 °C. SOD activity was expressed as the amount
of enzyme causing a 50% inhibition of adrenaline
autoxidation in units per milligram protein.
405
PERIĆ-MATARUGA et al. / Turk J Biol
CAT activity was determined according to the method
of Beutler (1982) using spectrophotometric determination
of dissolution of the standard concentration of H2O2
(10 mM) at 230 nm. Activity was expressed in
nanomoles as the amount of dissolved H2O2 reduced per
minute per milligram protein.
Determination of the total concentrations of GSH
(reduced GSH and oxidized GSSG) was conducted
according to the method described by Griffith (1980).
Sample proteins were precipitated in the homogenates
by sulfosalicylic acid. The method is based on a recycling
procedure where the oxidation of GSH with DTNB
[producing 5.5-dithiobis(2-nitrobenzoic acid)] and its
reduction by glutathione reductase with NADPH are
conducted reciprocally. The rate of formation of 2-nitro-5thiobenzoic acid was monitored spectrophotometrically at
412 nm, and the concentration of total GSH was calculated
in accordance to the standard and expressed per grams of
wet mass of tissue. Protein concentration was determined
according to Bradford (1976) using bovine serum albumin
as the standard.
2.4. Data analysis
After the normality of data distribution had been tested,
differences between groups were carried out by Student’s
t-test. P-values below 0.05 were regarded as significant.
Data analysis was done by Statistic 6.0 software.
3. Results
3.1. Larval survival
Fourth instar gypsy moth (L. dispar) larvae showed a
trend of lower survival (95%) when fed locust tree (R.
pseudoacacia) leaves in comparison to those (97.89%)
given an oak leaf diet (Quercus rubra).
3.2. L2’ PTTH-producing neurosecretory neurons
L2’ NSNs in the protocerebral part of the brain of fourth
instar L. dispar after 3 days of feeding with 2 different
diets are shown in Figure 1. The size of these dorsolateral
protocerebral NSNs was increased in larvae fed unsuitable
locust tree leaves (22.97 ± 0.33 µm) when compared with
those fed oak leaves (20.01 ± 0.9 µm) (P < 0.05). Moreover,
the nuclei in L2’ NSNs were larger in the group offered
locust tree leaves (11.93 ± 0.11 µm) than in the group fed
oak leaves (9.23 ± 0.55 µm) at P < 0.01 (Figure 1). These
neurons contained smaller amounts of a fine granulated
neurosecretory product in the group fed oak leaves than
in the group given locust tree leaves. The amount of the
neurosecretory product was lower by 15.5% in L2’ neurons
from larvae given oak leaves than in those offered locust
leaves (Figure 2). The nuclei of the L2’ neurons had large
centrally positioned and clearly visible nucleoli, which
suggested intensive synthetic processes (Figure 2).
406
25 μm Size of L2' neurosecretory neurons
*
20
Size of L2' neurosecretory
neuron's nuclei
**
15
10
5
0
1 oak
Fed
Fed2 locust
4 oak
Fed
5 locust
Fed
Figure 1. Size of L2’ neurosecretory neurons and their nuclei in
the brain of Lymantria dispar L. (fourth instar) that were fed on
oak (Quercus robur) or locust (Robinia pseudoacacia) tree leaves
for 3 days. *: P < 0.05, **: P < 0.01.
3.3. Antioxidative defense
SOD activity in the midgut of the larvae fed locust leaves
(13.85 ± 0.9 U/mg prot.) was significantly higher than
that in the control group fed oak leaves (9.08 ± 0.64 U/
mg prot.) at P < 0.05 (Figure 3). CAT activity in the larval
midgut tended to decrease (202.27 ± 20.45 nM H2O2
min–1 mg prot.–1) in larvae fed locust leaves in comparison
with larvae fed oak leaves (215.9 ± 0.88 nM H2O2 min–1
mg prot.–1), but the difference between the groups given
different diets was not statistically significant (Figure 4).
The amount of GSH in the midgut of the L. dispar fourth
instar larvae was significantly elevated in response to
the locust leaf diet (0.56 ± 0.06 µMGSH/g tissue) when
compared to the oak leaf diet (0.38 ± 0.06 µMGSH/g
tissue) at P < 0.01 (Figure 5).
4. Discussion
The nervous system/neurohormones and hormones
that regulate ecdysteroids have been emphasized lately
as important in balancing the redox status as well as
the potential for antioxidative defense in insect tissues.
Vertebrate steroid hormones such as estrogen and related
components with a phenolic A ring (Jellinck and Bradlow,
1990; Liehr and Roy, 1998; McHugh et al., 1998) inhibit
oxidative cascades by donating hydrogen radicals to the
A-ring. Steroids in arthropods synthesized after the tropic
effect of PTTH neurohormones have a similar function
(Rees, 1995; Cai et al., 2002). The activity of PTTH-secreting
NSNs, which regulate ecdysteroids, i.e. 20-HE content in
the hemolymph, was higher in larvae fed unsuitable locust
tree leaves (Figures 1 and 2). 20-Hydroxyecdysone is the
most active form of ecdysteroid. Stress induces ecdysone
20-monooxygenase, an enzyme that catalyzes conversion
of ecdysone to the physiologically active form, 20-HE, in
Drosophila virilis. The activity of this enzyme correlated
with the level of 20-HE (Chentsova et al., 2007), which
may have improved the defense against free radicals and
the prooxidative effects of plant compounds. Lower lipid
PERIĆ-MATARUGA et al. / Turk J Biol
150
%
Amount of
neurosecretory
product
*
100
50
1
Fed oak leaves
2
Fed locust tree
Figure 2. Transverse cross section of L. dispar brain (fourth instar) in the area of
L2’ protocerebral dorsolateral neurosecretory neurons. The relative amount of
neurosecretory product in cytoplasm of L2’ PTTH-secreting neurosecretory neurons in
larvae fed on locust tree leaves are presented in comparison with neurons in larvae fed
on oak leaves (control) (NIH software Image J 1.42q). *: P < 0.01.
20
Superoxide dismutase activity
U/mg prot.
15
*
10
5
0
Fed oak leaves
Fed locust tree leaves
Figure 3. Activity of superoxide dismutase in the midgut of
Lymantria dispar (fourth instar) fed on oak (Quercus robur) or
locust (Robinia pseudoacacia) tree leaves for 3 days. *: P < 0.01.
nM H2O2 min–1 mg prot.–1
350
Catalase
300
250
200
150
100
Fed oak leaves
Fed locust tree leaves
Figure 4. Activity of catalase in the midgut of Lymantria dispar
(fourth instar) fed oak (Quercus robur) or locust (Robinia
pseudoacacia) tree leaves for 3 days.
407
PERIĆ-MATARUGA et al. / Turk J Biol
μMGSH/g tissue
0.9
Amount of glutathione
*
0.6
0.3
0
Fed oak leaves
Fed locust tree leaves
Figure 5. Glutathione content in the midgut of Lymantria dispar
(fourth instar) fed on oak (Quercus robur) or locust (Robinia
pseudoacacia) tree leaves for 3 days. *: P < 0.01.
peroxidation and protein carbonylation following 20-HE
injection has been found with a subsequent decrease in
oxidative radicals and minimization of oxidative stress
due to paraquat in Pyrrhocoris apterus (Krishnan et al.,
2007). The hormone affects the GSH redox shuttle and
γ-glutamyl transpeptidase (GGT) activity, i.e. transfer of
the γ-glutamyl moiety of GSH to an acceptor that may be
an amino acid, a peptide, or water (forming glutamate).
The γ-glutamyl cycle is thought to sustain the cellular
GSH concentration. GGT plays a key role in this cycle,
as a pathway for the synthesis and degradation of GSH
and drug and xenobiotic detoxification (Karp et al.,
2001; Roesijadi et al., 2007). It is interesting that, besides
functional changes in PTTH-producing NSNs, our results
showed more GSH in the midgut tissue of locust leaf-fed
caterpillars than in the control group (Figure 5). GSH is
an important antioxidant that acts by several mechanisms,
including scavenging free radicals (Sies, 1999). Recovery
of GSH following 20-HE injection is only partially due to
the abrogation of oxidative radicals. The concentration of
intracellular GSH depends on the availability of substrates
for its synthesis and on the rate of depletion into the
extracellular space, as there is only small or no uptake
into cells. It is metabolized by membrane-bound γ-GTP,
which removes the γ-glutamyl group, thereby rendering
the remaining cysteinylglycine susceptible to cleavage by
a membrane dipeptidase. The released cysteine can be
transported into the cell and used as a substrate for GSH
synthesis. The ameliorating effect of 20-HE on γ-GTP
activity may be based on stabilization of its anchor in
the plasma membrane (reduced lipid peroxidation) or
on protection against direct oxidative damage, which
contributes to the GSH recovery.
Higher mortality, prolonged development time
including the fourth larval instar, lower pupal and larval
mass, and digestive, antioxidative, and neuroendocrine
408
reorganization were demonstrated previously as chronic
effects of a locust tree diet on L. dispar (Perić-Mataruga et
al., 1997; Lazarević et al., 2002). Here we showed a trend
of higher mortality of L. dispar larvae given locust leaves
for just 3 days. Ingested phenolic compounds become
extensively oxidized, which produces ROS (Canada et
al., 1990; Barbehenn et al., 2001; Barbehenn et al., 2009).
Moreover, quinones, the oxidation products of phenols
(Zheng et al., 1997; Barbehenn et al., 2006) may also be toxic
and can cause the formation of gut lesions (Thiboldeaux et
al., 1998; Perić-Mataruga et al., 2006a) and oxidative stress
in the midgut tissues of L. dispar (Pecci, 2011).
Our results confirmed that SOD activity (Figure 3) was
higher in the midgut of fourth larval instar fed locust tree
leaves for 3 days. It is interesting that CAT activity tended
to be lower in the individuals fed locust leaves (Figure
4), similar to chronic exposure of fourth larval instar to
trophic stress by locust tree feeding (Perić-Mataruga et
al., 1997). Considering that the phenol-induced stress
results in increased formation of O2•− and consequently
reactive oxygen species (Canada et al., 1990; Barbehenn
and Martin, 1994; Barbehenn et al., 2001), regulation by
SOD and CAT is necessary. SOD works continuously to
eliminate O2•− with production of H2O2. CAT catalyzes
the decomposition of H2O2 to water and oxygen. It is
possible that CAT could effectively reduce H2O2 to low
levels after the feeding of larvae with unsuitable locust tree
leaves. We presume that the enzyme ascorbate peroxidase
could be competing with the overproduction of H2O2
to compensate for the defect of CAT levels in order to
eliminate and detoxify the elevated levels of H2O2. PTTH
and ecdysteroids inhibit ROS and reactive nitrogen species
production and modulate oxidative stress by inducing
signal transduction pathways. They are also associated
with increased expression of gene products involved in
maintaining redox homeostasis, including methionine
sulfoxide reductase (an antioxidant repair enzyme that
reduces oxidized methionine to methionine), and may
regulate conversion of oxidized proteins to their reduced
state (Weissbach et al., 2005; Roesijadi et al., 2007). It is
interesting that the expression of the SOD gene in some
insects and mammals is responsive to ecdysone and is
regulated at both transcriptional levels, although the
molecular mechanisms of this regulation are poorly
understood. We could also suppose that higher mortality
was an aftermath of the prooxidant activity of phenols in
the unsuitable locust leaves and consequently allocation of
resources towards defense mechanisms and induction of
antioxidative components. These are exhausting processes
that can affect larval survival.
Our previous research demonstrated correlations
between the prooxidative effect of locust tree leaves in
chronic treatment (Perić-Mataruga et al., 2000) and
activation of L2’ NSNs (Perić-Mataruga et al., 1997,
PERIĆ-MATARUGA et al. / Turk J Biol
2001). These neurohormones regulate steroid synthesis
and their potential to alleviate oxidative damage through
reorganization od antioxidative strategies (Krishnan et al.,
2007).
Acknowledgment
This study was supported by the Serbian Ministry of
Education, Science and Technological Development,
Grant 173027.
References
Agui N, Bollenbacher WE, Granger NA, Gilbert LI (1980). Corpus
allatum is release site for insect prothoracicotropic hormone.
Nature 285: 609–670.
Chapman AG (1935). The effects of black locust on associated species
with special reference to forest trees. Ecological Monographs
5: 37–60.
Ahmad S (1992). Biochemical defence of pro-oxidant plant
allelochemicals by herbivorous insects. Biochem Syst Ecol 20:
269–296.
Chentsova NA, Gruntenko NE, Raushenbakh II (2007). Ecdysone
20-monooxygenase activity in Drosophila virilis strains varying
in ecdysteroid response to heat stress. Genetika 43: 999–1001.
Ahmad S, Pardini RS (1990). Mechanisms for regulating oxygen
toxicity in phytophagous insects. Free Radic Biol Med 8: 401–
413.
Chernysh SI (1991). Neuroendocrine system in insect stress. In:
Ivanović J, Janković-Hladni M, editors. Hormones and
Metabolism in Insect Stress. Boca Raton, FL, USA: CRC Press,
pp. 69–97.
Alquicer G, Kodrik D, Krishnan N (2009). Activation of insect
anti-oxidative mechanisms by mammalian glucagon. Comp
Biochem Physiol B 152: 226–227.
Barbehenn RV, Bumgarner SL, Roosen EF, Martin MM (2001).
Antioxidant defenses in caterpillars: role of the ascorbaterecycling system in the midgut lumen. J Insect Physiol 47:
349–357.
Barbehenn RV, Jaros A, Lee G, Mozola C, Weir Q, Salminen JP
(2009). Hydrolyzable tannins as “quantitative defenses”:
limited impact against Lymantria dispar caterpillars on hybrid
poplar. J Insect Physiol 55: 297–304.
Dai J, Mizoguchi A, Gilbert LI (1994). Immunoreactivity of
neurosecretory granules in the brain-retrocerebral complex
of Manduca sexta to heterologous antibodies against Bombyx
prothoracicotropic hormone and bombyxin. Invert Reprod
Dev 26: 187–196.
Demir İ, Eryüzlü E, Demirbağ Z (2012). A study on the
characterization and pathogenicity 311 of bacteria from
Lymantria dispar L. (Lepidoptera: Lymantriidae). Turk J Biol
36: 312 459–468.
Barbehenn RV, Jones CP, Karonen M, Salminen JP (2006). Tannin
composition affects the oxidative activities of tree leaves. J
Chem Ecol 32: 2235–2251.
Demir İ, Gürel N, Nalçacioğlu R, Demirbağ Z (2009). Comparative
susceptibilities of six insect cell lines to infection by
Malacosoma neustria nucleopolyhedrovirus (ManeNPV). Turk
J Biol 33: 259–273.
Barbehenn RV, Martin MM (1994). Tannin sensitivity in Malacosoma
disstria: roles of the peritrophic envelope and midgut oxidation.
J Chem Ecol 20: 1985–2001.
Felton GW, Workman J, Duffey SS (1992). Avoidance of antinutritive
plant defense: role of midgut pH in Colorado potato beetle. J
Chem Ecol 18: 571–578.
Barbosa P, Krischik VA (1987). Influence of alkaloids on feeding
preference of eastern deciduous forest trees by gypsy moth
Lymantria dispar L. Am Nat 130: 53–69.
Fridovich I (1995). Superoxide radical and superoxide dismutases.
Annu Rev Biochem 64: 97–112.
Beutler E (1982). Catalase. In: Beutler E, editor. Red Cell Metabolism:
A Manual of Biochemical Methods. New York, NY, USA:
Grune and Straton, pp. 105–106.
Blagojević D, Grubor-Lajšić G (2000). Multifunctionality of
antioxidant system in insects. Arch Biol Sci 52: 185–194.
Bradford M (1976). A rapid and sensitive method for the quantization
of microgram quantities of protein utilizing the principle of
protein-dye bending. Anal Biochem 72: 248–254.
Cai YJ, Dai JQ, Fang JG, Ma LP, Hou LF, Yang L, Liu ZL (2002).
Antioxidative and free radical scavenging effects of ecdysteroids
from Serratula strangulata. Can J Physiol Pharmacol 80: 1187–
1194.
Canada AT, Giannella E, Nguyen TD, Mason RP (1990). The
production of reactive oxygen species by dietary flavonols. Free
Radic Biol Med 9: 441–449.
Gade G, Goldsworthy GJ (2003). Insect peptide hormones: a selective
review of their physiology and potential application for pest
control. Pest Manag Sci 59: 1063–1075.
Gade G, Marco G (2006). Structure, function and mode of action
of select arthropod neuropeptides. In: Ur-Rhaman A, editor.
Studies in Natural Products Chemistry (Bioactive Natural
Products). Amsterdam, the Netherlands: Elsevier Science, pp.
69–139.
Griffith OW (1980). Determination of glutathione and glutathione
disulfide using glutathione reductase and 2 vinyl pyridine.
Anal Biochem 106: 207–212.
Grubor-Lajšić G, Petri ET, Kojić D, Purać J, Popović ZD, Worland
RM, Clark MS, Mojović M, Blagojević D (2013). Hydrogen
peroxide and ecdysone in the cryoprotective dehydration
strategy of Megaphorura arctica (Onychioridae: Collembola).
Arch Insect Biochem Physiol 82: 59–70.
409
PERIĆ-MATARUGA et al. / Turk J Biol
Gruntenko NE, Karpova EK, Adonyeva NV, Chentsova NA,
Faddeeva NV, Alekseev AA, Rauschenbach IY (2005). Juvenile
hormone, 20-hydroxyecdysone and dopamine interaction in
Drosophila virilis reproduction under normal and nutritional
stress conditions. J Insect Physiol 51: 417–425.
Halliwell B (1996). Free radicals, proteins and DNA: oxidative
damage versus redox regulation. Biochem Soc Trans 24: 1023–
1027.
Halliwell B, Gutteridge JMC (2007). Free Radicals in Biology and
Medicine. 4th ed. New York, NY, USA: Oxford University
Press.
Ilijin L, Vlahović M, Mrdaković M, Lazarević J, Matić D, Nenadović V,
Perić-Mataruga V (2012a). Activity of gypsy moth dorsolateral
neurosecretory neurons under increased rearing density. Arch
Biol Sci 64: 1085–1092.
Ilijin L, Vlahović M, Mrdaković M, Mirčić D, Todorović D, Lazarević
J, Perić-Mataruga V (2012b). Responses of PTTH-producing
neurosecretory neurons in Lymantria dispar caterpillars
exposed to cadmium. Environ Toxicol DOI: 10.1002/tox.21804
(in press).
Ilijin L, Vlahović M, Perić-Mataruga V, Kmetič I, Gavrilović A, Matić
D, Mrdaković M (2014). Temperature-induced stress response
in Lymantria dispar neurosecretory neurons. Turk J Biol 38:
157–167.
Ishizaki H, Suzuki A (1994). The brain secretory peptides that
control moulting and metamorphosis of the silkmoth, Bombyx
mori. Int J Dev Biol 38: 301–310.
Janković-Hladni MI (1991). Hormones and metabolism in insect
stress (historical survey). In: Ivanović J, Janković-Hladni M,
editors. Hormones and Metabolism in Insect Stress. Boca
Raton, FL, USA: CRC Press, pp. 5–26.
Jellinck PH, Bradlow HL (1990). Peroxidase-catalyzed displacement
of tritium from regiospecifically labeled estradiol and
2-hydroxyestradiol. J Steroid Biochem 35: 705–710.
Karp DR, Shimooku K, Lipsky PE (2001). Expression of gammaglutamyl transpeptidase protects ramos B cells from oxidationinduced cell death. J Biol Chem 276: 3798–3804.
Kawakami A, Kataoka H, Mizoguchy A, Kimura-Kavakami M,
Adachi T, Iwami M, Magasawa H, Suzuki A, Ishizaki H (1990).
Molecular cloning of the Bombyx mori prothoracicotropic
hormone. Science 247: 1333–1335.
Kelly JT, Masler EBP, Bell RBA, Thyagaraje BS, Davis RE, Fescemyer
HW, Borkovec AB (1991). Gypsy moth prothoracicotrophic
hormone. In: Menn JJ, Kelly TJ, Masler PE, editors. Insect
Neuropeptides: Chemistry, Biology and Action. Washington,
DC, USA: American Chemical Society, pp. 27–37.
Krishnan N, Kodrik D (2012). Endocrine control of oxidative
stress. In: Farooqui T, Farooqui A, editors. Oxidative Stress
in Vertebrates and Invertebrates – Molecular Aspects of Cell
Signaling. New York, NY, USA: Wiley-Blackwell, pp. 261–271.
Krishnan N, Sehnal F (2006). Compartmentalization of oxidative
stress and antioxidant defense in the karval gut of Spodoptera
littoralis. Arch Insect Biochem Physiol 63: 1–10.
410
Krishnan N, Večera J, Kodrik F, Sehnal F (2007). 20-Hydroxyecdysone
prevents oxidative stress damage in adult Pyrrhocoris apterus.
Arch Insect Biochem Physiol 65: 114–124.
Kulfan M (2012). Lepidoptera on the introduced Robinia pseudoacacia
in Slovakia, Central Europe. Check List 8: 709–711.
Lazarević J, Perić-Mataruga V, Stojković B, Tucić N (2002).
Adaptation of the gypsy moth to an unsuitable host plant.
Entomol Exp Appl 102: 75–86.
Liebhold AM, Gottschalk KW, Muzika RM, Montgomery ME, Young
R, O’Day K, Kelly B (1995). Suitability of North American Tree
Species to Gypsy Moth: A Summary of Field and Laboratory
Tests. General Technical Report NE-211. Randor, PA, USA:
USDA Forest Service.
Liehr JG, Roy D (1998). Pro-oxidant and antioxidant effects
of estrogens In: Armstrong D, editor. Free Radicals and
Antioxidant Protocols (Methods in Molecular Biology). New
York, NY, USA: Humana Press, pp. 425–435.
Maksimović M (1987). Preventive monitoring of Lymantria dispar.
Forestry 50: 5–66.
Martemyanov VV, Dubovskiy IM, Rantala MJ, Salminen JP,
Belousova IA, Pavlushin SV, Bakhvalov SA, Glupov VV (2012).
The association between leaf phenolics of the Silver Birch,
Betula pendula and the gypsy moth’s performance, immune
defense and its resistance to nucleopolyhedrovirus. Arthropod
Plant Interact 6: 507–518.
McGraw JB, Gottschalk KW, Vovrek MC, Chester AL (1990).
Interactive effects of resource availabilities and defoliation on
photosynthesis, growth and mortality of red oak seedlings.
Tree Physiol 7: 247–254.
McHugh NA, Merrill GF, Powell SR (1998). Estrogen diminishes
postischemic hydroxyl radical formation. Am J Physiol Heart
Circ Physiol 274: 1950–1954.
Mirčić D, Blagojević D, Perić-Mataruga V, Ilijin L, Mrdaković M,
Vlahović M, Lazarević J (2013). Cadmium effects on the
fitness-related traits and antioxidative defense of Lymantria
dispar L. larvae. Environ Sci Pollut Res 20: 209–218.
Misra HP, Fridovich I (1972). The role of superoxide anion in the
autoxidation of epinephrine and sample assay for superoxide
dismutase. J Biol Chem 247: 3170–3175.
Mizoguchi A, Tadanori O, Kataoka H, Nagasawa H, Suzuki A,
Ishizaki H (1990). Immunohistochemical localization of
prothoracicotropic hormone–producing neurosecretory cells
in the brain of Bombyx mori. Dev Growth Differ 32: 591–598.
Mrdaković M, Perić-Mataruga V, Ilijin L, Vlahović M, Janković
Tomanić M, Mirčić D, Lazarević J (2013). Response of
Lymantria dispar (Lepidoptera: Lymantriidae) larvae from
differently adapted populations to allelochemical stress: effects
of tannic acid. Eur J Entomol 110: 55–63.
Mrdaković M, Perić-Mataruga V, Ilijin L, Vlahović M, Todorović D,
Nenadović V, Lazarević J (2011). The effects of tannic acid on
the fitness-related traits of Lymantria dispar L. larvae. Arch
Biol Sci 63: 1037–1045.
PERIĆ-MATARUGA et al. / Turk J Biol
Nasir H, Iqbal Z, Hiradate S, Fuji Y (2005). Allelopathic potential of
Robinia pseudoacacia. J Chem Ecol 31: 2179–2192.
Rees HH (1995). Ecdysteroid biosynthesis and inactivation in
relation to function. Eur J Entomol 92: 9–39.
Panov AA (1980). Demonstration of neurosecretory cells in the insect
central nervous system. In: Strausfeld NJ, Miller TA, editors.
Neuroanatomical Techniques – Insect Nervous System. New
York, NY, USA: Springer, pp. 26–49.
Roesijadi G, Rezvankhar S, Binninger DM (2007). Ecdysone
induction of MerA protects against oxidative stress. Biochem
Biophys Res Commun 354: 511–511.
Pecci CD (2011). Oxidation of ingested phenolic compounds creates
oxidative stress in the midgut tissues of Lymantria dispar
caterpillars. BSc, University of Michigan, Ann Arbor, MI, USA.
Perić-Mataruga V, Blagojević D, Lazarević J, Spasić M (2000). Host
plant effects on the presence of superoxide dismutase isoforms
in gypsy moth midgut. Biologia 55: 525–531.
Perić-Mataruga V, Blagojević D, Spasić MB, Ivanović J, JankovićHladni M (1997). Effect of the host plant on the antioxidative
defence in the midgut of Lymantria dispar L. caterpillars of
different population origins. J Insect Physiol 43: 101–106.
Perić-Mataruga V, Janković Hladni M, Ivanović J (1988). The effect of
different feeding substrates on the development of Lymantria
dispar L. Plant Protection 39: 133–138.
Perić-Mataruga V, Lazarević J, Nenadović V (2001a). The effect of the
allelochemical quercetin on the survival of Lymantria dispar L.
Arch Biol Sci 53: 33–38.
Perić-Mataruga V, Lazarević J, Nenadović V (2001b). A possible
role for the dorsolateral protocerebral neurosecretory neurons
in the trophic adaptations of Lymantria dispar (Lepidoptera:
Lymantriidae). Eur J Entomol 98: 257–264.
Perić-Mataruga V, Lazarević J, Vlahović M, Mrdaković M, Ilijin L
(2006a). Histology of the midgut and peritrophic membrane in
Lymantria dispar caterpillars fed on leaves of Quercus cerris or
Robinia pseudoacacia. Phytoparasitica 34: 49–53.
Rossi MA, Cecchini GM, Dianzani M (1983). Glutathione
peroxidase, glutathione reductase and glutathione transferase
in two different hepatomas and in normal liver. IRCS Med Sci
Biochem 11: 805.
Salminen JP, Roslin T, Karonen M, Sinkkonen J, Pihlaja K, Pulkkinen
P (2004). Seasonal variation in the content of hydrolysable
tannins, flavonoid glycosides, and proanthocyanidins in oak
leaves. J Chem Ecol 30: 1693–1711.
Sies H (1999). Glutathione and its role in cellular functions. Free
Radical Biol Med 27: 916–921.
Takeda Y, Noguchi T, Kayiama M (1982). Superoxide dismutase in
various tissues from rabbits bearing the Vx-2 carcinoma in the
maxillary sinus. Cancer Res 42: 4233–4235.
Thiboldeaux RL, Lindroth RL, Tracy JW (1998). Effects of juglone
(5-hydroxy-1,4-naphthoquinone) on midgut morphology and
glutathione status in Saturniid moth larvae. Comp Biochem
Physiol 1: 481–487.
Weissbach H, Resnick L, Brot N (2005). Methionine sulfoxide
reductases: history and cellular role in protecting against
oxidative damage. Biochem Biophys Acta 1703: 203–312.
Zheng J, Cho M, Jones AD, Hammock BD (1997). Evidence of
quinone metabolites of naphthalene covalently bound to sulfur
nucleophiles of proteins of murine Clara cells after exposure to
naphthalene. Chem Res Toxicol 10: 1008–1014.
Perić-Mataruga V, Nenadović V, Lazarević J (2006b). Neurohormones
in insect’s stress. Arch Biol Sci 58: 1–12.
Rauchenbach IY (1991). Changes in juvenile hormone and
ecdysteroid content during insect development under heat
stress. In: Ivanović J, Janković-Hladni M, editors. Hormones
and Metabolism in Insect Stress. Boca Raton, FL, USA: CRC
Press, pp. 116–141.
411