ADJUSTMENT OF L1 NEUROSECRETORY NEURON ACTIVITY IN RESPONSE TO DIF- FERENT STRESSORS IN GYPSY MOTh CATERPILLARS

Gypsy moth caterpillars were exposed to an increased rearing temperature of 35 ̊C and diet, supplemented with Cd, a heavy metal pollutant, and tannic acid, a plant secondary metabolite. After 3 days’ exposure to stressors, changes in the number, morphometric parameters of L1 neurosecretory neurons (nsn) (sizes of the nsn and their nuclei), and the quantity of neurosecretory material in the cytoplasm of the neurons were estimated. Acute exposure to the high temperature of 35 ̊C induced increases in the number of L1 nsn, their size and the size of their nuclei with prolonged exposure time. After acute exposure to different Cd concentrations, the number of L1 nsn was reduced, their size increased and the size of their nuclei decreased. Together with the enhanced relative density of the cytoplasm, our results point to an intensive synthesis and retention of neurosecretory material in the neurons. The relative density of the neurosecretory material in the cytoplasm increased at the thermal treatment, suggesting intensive synthesis and secretory activity in L1 nsn. Caterpillars reared on an artificial substrate with the addition of high concentrations of tannic acid (TA) showed a decreased number of nsn, increased cell size and decreased size of their nuclei. The reduction in the relative density of the cytoplasm led us to conclude that this treatment induced a high synthetic activity of L1 nsn.


INTRODUCTION
Insect hormones, such as Lepidopteran brain neurohormones and allatostatins have dominant roles in stress response mechanisms.(Homberg et al., 1991;Stay and Tobe, 2007).Allatostatins are neuropeptides that rapidly and reversibly inhibit the synthesis of juvenile hormones in the corpora allata (secretory and neurochaemal structure).Juvenile hormones (JH) play an important role in all insect life processesdevelopment, physiology, behavior, etc. Allatostatins largely determine the rate at which the corpora allata synthesizes JH (Hoffmann et al., 1999).Cadmium (Cd) is an endocrine disrupting chemical (EDC).Acute and chronic exposure of gypsy moth caterpillars to Cd produces toxic effects on their life processes (Ilijin et al., 2010;2011;2012;Vlahović et al., 2012;2013).In addition, Cd appears to inhibit ecdysone secretion and interfere with other insect hormones that regulate stress response, development, reproduction, etc. (Rodrigez et al., 2007).
Expected global climate changes will raise the mean temperature by 1.5-4.5˚C in this century (Houghton et al., 1996).Augmented temperature leads to an increase in insect growth rate (Levesque et al., 2002), changes in developmental time (Petersen et al., 2000), as well as duration of each larval instar (Levesque et al., 2002).Previously, we detected changes in the activity of gypsy moth protocerebral nsn upon exposure to increased rearing temperature.Analyzed medial (A1') and lateral (L2 and L2') nsn showed changes in morphometric parameters and a low level of secretion (Ilijin et al., 2013a;2013b).
Tannins, as plant secondary metabolites, have various effects on herbivorous insects.They prolong the duration of the larval stage (Ruuhola et al., 2007) and induce negative effects on development, relative growth rate and food assimilation efficiency (Kopper et al., 2002), as a consequence of complex formation between tannins and proteins and carbohydrates from leaves.These complexes can prevent or reduce ingestion and absorption of macromolecules from leaves (Kumar and Singh, 1984).Upon chronic exposure to tannic acid (TA), larval mass was reduced (Mrdaković et al., 2011) and the activity of total proteases and lipases was increased (Mrdaković et al., 2013).After 3-day feeding of L. dispar caterpillars with an artificial diet supplemented with TA, Ilijin (2009) observed enhanced synthesis in lateral nsn.
Our aim in this work was to analyze the changes of lateral L1 type of nsn (a potential source of alla-  tostatins) upon acute exposure of caterpillars to Cd (a heavy metal pollutant), high temperature (global warming effects) and TA (plant secondary metabolite).

Insect rearing
Gypsy moth larvae were reared on an artificial diet (O'Dell et al., 1985) in plastic containers (V=200 ml) at 23˚C and a 16 h light:8 h dark photoperiod.On the first day of 4 th instar, larvae were exposed to three different acute stressors (Cd, high temperature of 35°C, and tannic acid) for 3 days.The first group of caterpillars was fed on a diet with 10, 100, 250 µg of added Cd/g of dry food (group designations: 10 µg, 100 µg and 250 µg) or without Cd (control group or C).The second group of caterpillars was reared at 23˚C until entry into the 4 th instar, when they were acutely exposed to a stressful high temperature for 1 h, 12 h, 12 h followed by transfer to 23°C for 12 h recovery, and 24 h (group designations: 1h, 12h, 12/12h, 24h), in a rearing chamber, with constant humidity.For this acute stressor, the control group was reared at 23°C (C).The third acute stressor was tannic acid (Sigma) added to the artificial diet.The experimental groups of caterpillars were fed a diet containing 1%, 2.5% or 5% TA (group designations: 1% T, 2.5% T, 5% T).For this acute stressor, the control group (C) was provided with a basic artificial diet.For all treatments, caterpillars (n = 15) were randomly assigned to the experimental groups for histochemistry.

histological techniques
Caterpillars were sacrificed on the 3 rd day of the 4 th instar and head capsules were fixed in Bouin's solution (Merck, Darmstadt, Germany).Brain complexes were dissected and embedded in paraffin wax (59°C, Merck, Darmstadt, Germany).Serial sections were cut at 3.5 µm and stained by a paraldehyde fuchsin technique (Ewen, 1962) modified by Panov (1980).Based on their size and morphological characteristics, we divided the lateral protocerebral nsn of L. dispar into L1, L2, L2' groups.The activity of protocerebral L1 type nsn was determined by monitoring the number of nsn, the size of nsn, and their nuclei (expressed as mean values of the smallest and largest diameters in µm).Measurements were made using a DMLB light microscope (Leica).The data were analyzed by one-way analysis of variance (ANOVA) and Fisher's least significant difference (LSD).The relative density of neurosecretory material in L1 nsn cytoplasm was estimated using NIH software Image J 1.42q.

The effects of acute exposure to Cd on L1 nsn
In Fig. 1, L1 nsn in 4 th instar gypsy moth caterpillars after a 3-day feeding with various Cd concentrations are presented.The presence of Cd in the artificial diet reduced the number of these neurons (Fig. 2A), especially in lower Cd concentrations (10 µg).Enlargement in size of L1 nsn was observed for all three Cd concentrations used in this experiment in comparison to control group (Fig. 2B).The trend of reduced size of L1 nuclei in larvae reared on 250 µg of Cd is obvious in Fig. 2C.The relative density of neurosecretory material present in L1 nsn cytoplasm was enhanced in all Cd-treated groups (Fig. 2D).We can conclude that intensive synthesis and retention of neurosecretory material is characteristic of L1 nsn upon acute exposure to different Cd concentrations.
The effects of acute exposure to high temperature on L1 nsn L1 nsn in 4 th instar caterpillars, after a 3-day exposure to 35°C for 1 h (1h), 12 h (12h), 12 h followed by transfer to 23°C, (12/12h) and 24 h (24h) are presented in Fig. 3.With prolonged exposure and after re-covery (12/12h), the number of L1 nsn rose (Fig. 4A).Only caterpillars from the 24h group reduced the number of these nsn.Neurons decreased their size after 1 h exposure to 35°C in comparison to control group (Fig. 4B).The size of nuclei increased significantly (Fig. 4C) in all groups exposed to high temperature in comparison to control group (one-way ANOVA, F 4, 111 = 2.6551, p<0.05).After 12 and 24 h of exposure, the nuclei size was increased in comparison to control group (Fig. 4C).Relative density of the neurosecretory material present in cytoplasm increased after all thermal treatments (Fig. 4D).In Fig. 3, we can see the presence of axonal endings of L1 nsn filled with neurosecretory material upon the caterpillars' exposure to high temperature.We can conclude that acute exposure to high temperature has induced intensive activity in L1 nsn (synthesis and secretion).

The effects of acute exposure to TA on L1 nsn
Changes in L1 nsn in caterpillars exposed to different concentrations of tannic acid added to artificial diet (1%, 2.5% or 5%) are presented in Fig. 5.A reduced number of these cells was observed after the addition of 2.5 and 5% of TA (Fig. 6A).Exposure to 2.5% of TA was correlated with enlargement of L1 nsn in comparison to the 1% T group (Fig. 6B).In groups fed with 2.5 and 5% TA, the nuclei size of L1 nsn dropped in comparison to the control group (one-way ANOVA, F 3, 141 = 3.6025, p<0.05).Also, a significant decline in the size of nuclei was detected in the group fed 1% of TA in comparison to all the other experimental groups (Fig. 6C).The relative density of the neurosecretory product of L1 nsn waned in caterpillars fed with 1 and 5% of TA, while feeding with 2.5% of this allelochemical led to enhancement (Fig. 6D).We can say that the addition of 2.5% of TA induced high synthetic activity of L1 nsn in comparison to the control as well as the groups with added 1 and 5% of TA.

DISCUSSION
Having in mind their role and the fact that insect neuropeptides and peptide hormones are produced in less than 1% of the brain neurosecretory neurons, it is essential to understand the way that nervous system regulates life processes through the synthesis and release of neurohormones.In addition, the nervous system has a tremendous plasticity, achieved mainly by the regulation of intracellular Ca 2+ in presynaptic nerve terminals, i.e. by modulation of synaptic transmission.Neuron discharge is regulated by up-and downmodulation of Ca 2+ -activated K + currents (Wicher et al., 1994), i.e. the Ca 2+ influx in nsn influences the level of synthesis and release of secretory products.
Insect larvae respond to Cd intoxication by changing Ca 2+ concentration (Craig et al., 1998).Terrestrial insects accumulate Cd in the same region of the gut where essential elements such as Ca 2+ are taken up (Taylor, 1986), probably due the similar ionic radii of both Ca 2+ (0.97 Å) and Cd 2+ (0.99 Å) ions (Pauling, 1960), they compete for the same antry sites.In their central nervous system, Cd acts as a dose-dependent Ca 2+ channel blocker preventing Ca 2+ permeation of the membrane and activating K + currents in nsn (Bickmeyer et al., 1994).After supplementing Cd in different concentrations to gypsy moth caterpillars' artificial diet, the morphological parameters of medial A1 (Ilijin, 2009), A1' (Ilijin et al., 2011) and A2 (Ilijin et al., 2010), as well as lateral L2 (Ilijin et al., 2011) and L2' (Ilijin et al., 2012) protocerebral nsn were increased.At the same time, we detected an augmented level of synthesis and retention of neurosecretory material in the cytoplasm of nsn.Probably as the consequence of Cd inhibiting Ca influx in nsn, upon acute exposure to different Cd concentrations gypsy moth L1 nsn also increased in size, with enhanced relative density of cytoplasm, indicating an intensive synthesis and retention of neurosecretory material.
Inorganic Ca 2+ has many important roles as an intracellular messenger that controls development and differentiation, and coordinates cellular functions by passing on information within cells (Berridge et al., 2000).Large amounts of free Ca 2+ trigger a secretion by secretory cells.Membrane calcium ATPase pumps found in the membranes are the main regulators of free Ca 2+ concentration in the cytosol (Vasser and Lytton, 2007).During hyperthermia, this regulatory pump works in reverse mode, pumping the extracellular Ca 2+ into the cytosol (Klose et al., 2008).We have observed that prolonged exposure of gypsy moth caterpillars to high temperature induced increased size of L1 nsn as well as increased relative density of neurosecretory material in the cytoplasm.The detected presence of a large amount of neurosecretory material in the axonal endings of L1 nsn is probably due to the spontaneous release of neurosecretory material at high temperatures.In Drosophila melanogaster, increased temperature induced a lack of ATP and the accumulation of Ca 2+ in the nerve terminal cytosol (Klose et al., 2009).Finally, a spontaneous release of neurotransmitter occurs and normal synaptic transmission is absent.
Tannins are plant secondar y metabolites (Khanababaee and van Ree, 2001), and they have an important role in protection from herbivorous attacks (Strauss and Zangerl, 2002).The effects of tannins, like the effects of other environmental factors, are expressed through specific changes in neurotransmitters and neuromodulators at the level of the neuroendocrine system.In gypsy moth caterpillars fed with locust leaves, which are rich in tannins, changes in the number and activity of medial nsn was observed (Perić Mataruga and Lazarević, 2004).Acute feeding of gypsy moth caterpillars with tannic acid showed less influence on the activity of nsn (Ilijin, 2009).Bombyxin and PTTH immunopositive nsn have modulated their activity after tannic acid treatment.Medial A1 nsn activity was reduced in response to high concentrations of tannic acid.In this paper, we present results showing the increased number and size of L1 nsn as well as enhanced synthetic activity after the addition of 2.5% of tannic acid.Lymantria dispar is a herbivorous insect that feeds on 500 different host plant species (Lance, 1983).Acute exposure to tannic acid is not a strong environmental stressor for caterpillars.
Neurohormonal controllability of the stress response in insects requires adjustment of many components, including neurosecretory neurons of the lateral part of the protocerebral brain as a region of allatostatin synthesis, (Veelaert et al., 1995).Depending on the duration and type of stressors, neurosecretory neuron activity and their requirement for allatostatin are different due to their roles in the regulation of JH release, food intake, digestive function, energy reserves and metabolism in several insect species (Hergarden et al., 2012).

Fig. 2 .
Fig. 2. The number of L1 nsn (A), size of L1 nsn (B), size of L1 nsn nuclei (C), and relative density of cytoplasm, expressed in % (D), in Lymantria dispar 4 th instar caterpillars exposed to different Cd concentrations.All abbreviations are as in Fig. 1.Different letters (a, b) indicate significant differences between groups (LSD test, P<0.01).

Fig. 1 .
Fig. 1.Brain transverse cross-sections of Lymantria dispar 4 th instar caterpillars after feeding for 3 days with an artificial diet supplemented with 10, 100, 250 mg of Cd/g of dry food weight.Arrows indicate the protocerebral lateral L1 nsn.The bar represents 10 µm.

Fig. 4 .
Fig. 4. The number of L1 nsn (A), size of L1 nsn (B), size of L1 nsn nuclei (C), and relative cytoplasmic density, expressed in % (D) in Lymantria dispar 4 th instar caterpillars exposed to 35˚C.All abbreviations are as in Fig. 3. Different letters (a, b) indicate significant differences between groups (LSD test, P<0.01).

Fig. 6 .
Fig. 6.The number of L1 nsn (A), size of L1 nsn (B), size of L1 nsn nuclei (C), and relative cytoplasmic density, expressed in % (D) in Lymantria dispar 4 th instar caterpillars exposed to different concentrations of tannic acid.All abbreviations are as in Fig. 5. Different letters (a, b) indicate significant differences between groups (LSD test, P<0.01).