Gypsy moth 4th instar caterpillars were fed for 3 days with an artificial diet supplemented with increasing cadmium (Cd) concentrations (0, 10, 30, 100 and 250 μg⁄g of dry food weight). Changes in the morphometric characteristics of A1' dorso-medial and L2 dorso-lateral neurosecretory neurons (nsn) were analyzed. In the A1' nsn, Cd supplements led to an enhanced nuclear size, except in the group treated with 250 μg Cd⁄g in the form of dry food. The size of L2 type nsn was increased in the groups provided with 30 and 100 μg Cd⁄g, while no differences in the size of nuclei was detected in L2 neurons among the experimental groups.Projekat ministarstva br. 7302

Ilijin, Larisa

Lazarević, Jelica

Matić, Dragana

Mrdaković, Marija

Perić Mataruga, Vesna

Todorović, Dajana

Vlahović, Milena

Digital Repository of Archived Publications - Institute for Biological Research Sinisa Stankovic (RADaR)

Arch. Biol. Sci., Belgrade, 63 (4), 1287-1292, 2011 DOI:10.2298/ABS1104287I
1287
CHANGES IN LYMANTRIA DISPAR PROTOCEREBRAL NEUROSECRETORY NEURONS  
AFTER EXPOSURE TO CADMIUM 
LARISA ILIJIN*,  MILENA VLAHOVIĆ, MARIJA MRDAKOVIĆ, DRAGANA MATIĆ,  
JELICA LAZAREVIĆ, DAJANA TODOROVIĆ and VESNA PERIĆ-MATARUGA
Department of Insect Physiology and Biochemistry, Institute for Biological Research „Siniša Stanković”,  
University of Belgrade, 11060 Belgrade, Serbia
Abstract - Gypsy moth 4th instar caterpillars were fed for 3 days with an artificial diet supplemented with increasing cad-
mium (Cd) concentrations (0, 10, 30, 100 and 250 µg⁄g of dry food weight). Changes in the morphometric characteristics 
of A1’ dorso-medial and L2 dorso-lateral neurosecretory neurons (nsn) were analyzed. In the A1’ nsn, Cd supplements led 
to an enhanced nuclear size, except in the group treated with 250 µg Cd⁄g in the form of dry food. The size of L2 type nsn 
was increased in the groups provided with 30 and 100 µg Cd⁄g, while no differences in the size of nuclei was detected in L2 
neurons among the experimental groups.
Key words: Cadmium, Lymantria dispar, protocerebral neurosecretory neurons
UDC 595.78:591:54
INTRODUCTION
Lymantria dispar is a polyphagous herbivorous insect 
pest with a host range estimated at more than 500 
plant species (Lance, 1983). Cadmium accumulation 
in plants causes changes in the dynamics of animal 
populations, including L. dispar and other phytopha-
gous insects (Williams and Liebhold, 1995).
The amount of Cd in nature has been increasing 
as a consequence of anthropogenic activities. Nu-
merous data indicate the influence of this heavy met-
al on insect development, growth, reproduction and/
or hatching (Gintenreiter et al., 1993; Rayms-Keller 
et al., 1998; Sildanchandra and Crane, 2000), respira-
tion (Ortel and Vogel, 1989) and metabolic processes 
(Bischoff, 1995; Niu et al., 2002). 
The neuroendocrine reorganization in the in-
sect’s protocerebral region (dorsomedial part) of the 
brain during nutritive stress changes protein and li-
pid metabolism as well as digestive enzyme amounts 
and activities (Ivanović and Janković-Hladni, 1991; 
Mrdaković et al., 2008). 
Our previous investigations revealed that acute 
exposure of gypsy moth caterpillars to a Cd sup-
plemented diet induced changes in the activity of 
the neurosecretory neurons (nsn) from the pars 
intercerebralis. We found alterations in bombyxin 
(a small form of PTTH) immunopositive A2 nsn 
(Ilijin et al., 2010; Ilijin et al., 2011) and L2’ nsn, 
suggesting they are the sites of synthesis of the 
large form of PTTH in L. dispar caterpillars (Ili-
jin, 2009). To further elucidate the hormonal re-
organization in response to heavy metal stress, we 
analyzed the morphometric changes in two other 
types of nsn: A1’ and L2. The aim of this work was 
to overview specific and nonspecific responses to 
the acute effects of Cd in gypsy moth caterpillars 
by analyzing two different types of neurosecretory 
neurons.
1288 LARISA ILIJIN   ET AL.
MATERIALS AND METHODS
Insect rearing 
Egg masses were collected in a poplar forest at Opovo 
30 km from Belgrade and kept in a refrigerator at 4ºC 
until they were set to hatch. Larvae were reared on a 
synthetic HWG (high wheat germ) diet (O’Dell et al., 
1985) in transparent plastic containers (V=200ml) at 
23˚C and a 12h light/dark photoperiod. On the first 
day of the 4th instar, larvae were fed on diets with 10, 
30, 100, 250 µg of added Cd per g of dry food (10 
µg, 30 µg, 100 µg and 250 µg groups) or without Cd 
(control group) for 3 days and then sacrificed by de-
capitation. The caterpillars were randomly assigned 
to five groups for histochemistry (n=15). 
Histological techniques
After decapitation, the head capsules were fixed in 
Bouin’s solution for 24 h (Merck). The brain com-
plexes were dissected, dehydrated in a graded series 
of ethanol (from 80% to 100%) (Hemos, Belgrade), 
and then embedded in paraffin wax (59ºC, Merck). 
Serial sections (3.5 µm) of the brain complexes were 
cut for histochemistry (microtome “820”Spencer). 
After drying, the sections were deparaffinized in xy-
lene (Hemos), and stained by the modified (Panov, 
1980) Ewen paraldehyde fuchsin technique (Ewen 
1962). The neurosecretory neurons were stained 
dark purple - paraldehyde fuchsin positive. The size 
of the A1’and L2 nsn and their nuclei (expressed as 
means of the shortest and longest diameters in µm) 
were analyzed. Measurements were made using the 
image processing and analysis system (QWin image 
analysis tool kit) linked to a Leica DMLB light mi-
croscope (Leica). Data were evaluated by one-way 
ANOVA and a post-hoc multiple range test (Fisher’s 
least significant difference (LSD)).
RESULTS
A1’nsn are located in the dorsomedial part of the 
L. dispar protocerebrum, and their average size was 
25.96 µm (Fig. 1). After exposure of the caterpillars 
to different Cd concentrations we observed a ten-
dency in the A1’nsn to increase in size (Fig. 2A). The 
average nuclear size was significantly greater in the 
groups of caterpillars fed with diets containing 10, 30 
and 100 µg of added Cd per g dry weight (one-way 
ANOVA, P<0.001) (Fig. 2B). The majority of A1’nsn 
in all groups had a large amount of neurosecretory 
material in the cytoplasm (Fig. 2C). In the A1’ nsn 
from the caterpillars receiving the diets with 10 and 
100 µg of added Cd, a fine-grained neurosecretory 
material was present, while in the other groups the 
neurosecretory material was large-grained (Fig. 2D). 
All these results point to elevated synthetic and se-
cretory activity in A1’nsn in response to Cd.
The L2 nsn are located in the dorsolateral part 
of the caterpillar protocerebrum, and their average 
size is 16.15 µm (Fig. 3). Statistically significant in-
creases in nsn size were detected in the groups re-
Fig. 1. – Brain transverse cross-sections of Lymantria dispar 4th 
instar caterpillars after feeding for 3 days with an artificial diet 
supplemented with various cadmium concentrations (0 µg; 10 
µg; 30 µg; 100 µg; 250 µg Cd/g of dry food weight). Arrows in-
dicate the protocerebral A1’ neurosecretory neurons. The bar 
represents 10 µm.
CHANGES IN LYMANTRIA DISPAR PROTOCEREBRAL NEUROSECRETORY NEURONS AFTER EXPOSURE TO CADMIUM 1289
ceiving 30 and 100 µg of added Cd per g of dry food 
weight in comparison to the control group (Fig. 4A). 
There were no significant changes in nuclei size in 
any of the experimental groups (Fig. 4B). The cyto-
plasm of most of the L2 nsn was filled with a large 
amount of large-grained neurosecretory material 
(Figs. 4C and 4D). These cytological characteristics 
indicate a low level of secretion and the retention 
of neurosecretory material in the cytoplasm of L2 
neurons.
DISCUSSION
Neurosecretory cells from the pars intercerebralis in 
the brain of insects release various hormonal factors 
that control the essential physiological and devel-
opmental functions and these cells are therefore of 
considerable biological significance. These neuro-
hormones are included in all phases of the stress re-
sponse mechanism, providing the energy necessary 
for the mechanisms of stress tolerance (Gäde and 
Goldsworthy, 2003).
The insect’s gut is an accumulative organ for 
Cd. The gut epithelium and peritrophic matrix of 
insects is an important target organ for heavy met-
als (Lauverijat et al., 1989). Clearly, cadmium in 
food destroys the midgut structure and this im-
pacts fundamental processes (digestion and ab-
sorption of food) and could cause starvation and 
have a dramatic impact on an insect’s life processes 
and survival (Rayms-Keller et al., 1998, 2000). High 
dose Cd toxicity causes effects similar to starvation. 
Intoxication with a starvation effect obviously in-
fluences both the quality and quantity of midgut 
proteins, including digestive enzymes and brain 
Fig. 2. - The size of A1’ neurosecretory neurons (nsn) (A) and their nuclei (B), the amount (C) and the type of neurosecretory material 
(D) in protocerebrum of L. dispar 4th instar caterpillars after receiving artificial diets supplemented with various cadmium concentra-
tions (0 µg; 10 µg; 30 µg; 100 µg; 250 µg Cd/g of dry food weight) for 3 days. Error bars indicate the standard error of the mean (SEM) 
for (n = 15). Different letters (a,b,c) indicate significant differences between groups (LSD test, P<0.01).
1290 LARISA ILIJIN   ET AL.
proteins. These changes may indicate the role of the 
neuroendocrine system in environmental changes 
(Ivanović et al., 1991). Neurosecretory neurons, like 
A1’nsn, located in the medial part of the pars in-
tercerebralis, synthesize neuropeptides that have a 
regulatory role in the synthesis and release of the 
adipokinetic neurohormone, secretion of midgut 
digestive enzymes (proteases and amylases), diu-
resis, water balance, etc. (Gäde and Goldsworthy, 
2003), (Raabe, 1989). Activation of these neurons 
could be necessary for a digestive and metabolic re-
organization which compensates for the disturbed 
homeostasis under stressful conditions. Prolonged 
larval development (Ilijin et al., 2010), reduction 
of larval mass (Ilijin et al., 2010) and inhibition of 
alkaline phosphatase, trypsin, leucine aminopepti-
dase, α and β glucosidase and esterase (Vlahović et 
al., 2009, 2011), changes in protein, lipid and car-
bohydrate levels in the hemolymph and whole body 
(Ortel 1995 a, 1995 b, 1996) were detected under 
the influence of cadmium. 
In this work we have revealed an elevation of the 
synthetic and secretory activity of A1’ medial nsn. 
The results presented here led us to assume that the 
defense strategy of L. dispar caterpillars in the pres-
ence of cadmium includes digestive reorganization 
allocation of resources towards the activation of 
energy metabolism, regulated by neurohormones, 
synthesized in pars intercerebralis nsn. All defense 
mechanisms, such as increasing excretion, detoxifi-
cation or repair of the damage are energetically ex-
pensive.
In the L2 nsn from the lateral part of the pro-
tocerebrum the retention of neurosecretory mate-
rial in their cytoplasm was obvious and a low level 
of secretion was detected. One of the responses 
of insect larvae to Cd intoxication is the chang-
ing of cell Ca2+ concentration (Craig et al., 1998). 
These two ions compete for passage into the cell 
through the same site of entry. Bickmeyer et al., 
(1994) showed a dose-dependent block of Ca 2+ 
channel currents in Locusta migratoria neurons 
treated with Cd. Calcium is a universal intracellu-
lar messenger that controls secretion, metabolism, 
or gene expression. Also, Cd can effect transmitter 
release (Soliakov and Wonnacott, 1996). It is plau-
sible that the retention of neurosecretory material 
in L2 nsn is the consequence of an inhibited Ca2+ 
influx caused by cadmium. The results also show 
the different sensitivity to certain types of nsn to 
Cd intoxication. Research into the neuroendo-
crine response in stress could provide useful in-
formation about the regulation of the endocrine 
system in Lymantria dispar caterpillars, and could 
contribute to the development of pest-controlling 
substances.
Acknowledgement - This study was supported by the Serbian 
Ministry of Education and Science (grant No.73027).
Fig. 3. – Brain transverse cross-sections of Lymantria dispar 4th 
instar caterpillars after receiving artificial diets supplemented 
with various cadmium concentrations (0 µg; 10 µg; 30 µg; 100 
µg; 250 µg Cd/g of dry food weight) for 3 days. Arrows indicate 
the protocerebral L2 neurosecretory neurons. The bar represents 
10 µm.
CHANGES IN LYMANTRIA DISPAR PROTOCEREBRAL NEUROSECRETORY NEURONS AFTER EXPOSURE TO CADMIUM 1291
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Changes in Lymantria dispar protocerebral neurosecretory neurons after exposure to cadmium

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Changes in Lymantria dispar protocerebral neurosecretory neurons after exposure to cadmium

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