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Protoplasma

, Volume 256, Issue 3, pp 839–856 | Cite as

Pest and natural enemy: how the fat bodies of both the southern armyworm Spodoptera eridania and the predator Ceraeochrysa claveri react to azadirachtin exposure

  • Elton Luiz Scudeler
  • Ana Silvia Gimenes Garcia
  • Carlos Roberto Padovani
  • Daniela Carvalho dos SantosEmail author
Original Article

Abstract

The effects of biopesticides on insects can be demonstrated by morphological and ultrastructural tools in ecotoxicological analysis. Azadirachtin-based products are widely used as biopesticides, affecting numerous insect populations. Through morphological biomarkers, this study aimed to characterize the fat bodies of both the southern armyworm Spodoptera eridania and the predator Ceraeochrysa claveri after chronic exposure to azadirachtin. Larvae of S. eridania and C. claveri were fed with fresh purple lettuce leaves (Lactuca sativa) and egg clusters of Diatraea saccharalis treated with azadirachtin solution of 6 mg active ingredient (a.i.)/L and 18 mg a.i./L for 7 days, respectively. The biological data showed a significant reduction in survival and body mass in S. eridania and cytotoxic effects in the parietal and perivisceral fat bodies in both species. Ultrastructural cell damage was observed in the trophocytes of both species such as dilated cisternae of the rough endoplasmic reticulum and swollen mitochondria. Trophocytes of S. eridania and C. claveri of the parietal and perivisceral layers responded to those injuries by different cytoprotective and detoxification means such as an increase in the amount of cytoplasmic granules containing calcium, expression of heat shock protein (HSP)70/HSP90, and development of the smooth endoplasmic reticulum. Despite all the different means of cytoprotection and detoxification, they were not sufficient to recover from all the cellular damages. Azadirachtin exhibited an excellent performance for the control of S. eridania and a moderate selectivity for the predator C. claveri, which presents better biological and cytoprotective responses to chronic exposure to azadirachtin.

Keywords

Biopesticide Cytotoxicity Insect Morphology Ultrastructure 

Introduction

Evaluations using morphological biomarkers of cell and tissue damage have become an important tool in ecotoxicological studies. Through histological, histochemical, immunohistochemical, and ultrastructural techniques, the diagnosis of cellular and subcellular responses may reveal sublethal effects that often go unnoticed in toxicity analyses (Adamski 2007; Fontanetti et al. 2011; Scudeler et al. 2016a).

Due to the multiple metabolic functions performed by the fat body, this organ has been successfully used as a target organ in invertebrate cytotoxicity studies. Due to its important role in detoxification, endocrinology, reproduction, and nutrition, damage to this organ may indicate relevant sublethal effects that may compromise insect development and behavior (Adamski et al. 2005, 2016; Arrese and Soulages 2010; Büyükgüzel et al. 2013; Domingues et al. 2017; Martins et al. 2011), especially in regard to beneficial species such as natural enemies.

We understand that the use of morphological biomarkers in species that share occurrence in similar agroecosystems but occupy different trophic levels in the food chain may contribute data and information on the mechanism of action of biopesticides, since they are exposed to the same substances, which are often understood to be safe and effective for some species and are never evaluated for others that share the same environment (Cloyd 2012).

Azadirachtin-based biopesticides (Azadirachta indica A. Juss) (Meliaceae) are widely used throughout the world in agricultural crops and veterinary field, as they exhibit different activities against insects and others arthropods such as repellency, an antifeeding effect, growth regulation, incomplete ecdysis with malformation of pupae and adults, suppression of reproduction with reduced fertility, and fertility with mortality. These pesticides can act by contact or ingestion (Cloyd 2012; Mordue (Luntz) and Nisbet 2000; Morgan 2009; Schmutterer 1990; Lima de Souza et al. 2017).

The larva of the southern armyworm, Spodoptera eridania (Stoll, 1782) (Lepidoptera: Noctuidae), is a polyphagous pest that has a wide distribution throughout the American continent, affecting plant species from vegetables to those of large agricultural crops (Montezano et al. 2014; Santos et al. 2005; Specht et al. 2016). Because it is an insect pest of great economic importance, S. eridania has become a target to be controlled by the use of biopesticides (Shannag et al. 2015). It is therefore necessary to investigate the existence of mechanisms of resistance exerted by the fat body, which may hinder the management of this insect pest in agroecosystems.

Because they are polyphagous predators found in many cultures of economic interest in the Neotropical region and share the occurrence in agroecosystems with the larvae of S. eridania, the green lacewing Ceraeochrysa claveri (Navás, 1911) (Neuroptera: Chrysopidae) has been shown to be a good biological model to represent natural enemies to be considered predator of eggs and several soft-bodied arthropods. These predators play an important role in biological control, reducing the population density of several arthropods considered as pests (De Freitas and Penny 2001; Pappas et al. 2011). Therefore, the preservation and maintenance of lacewings in agroecosystems should be considered through the search and use of products selective to this natural enemy population (Cloyd 2012; Pappas et al. 2011). The use of neem or azadirachtin products has been shown to have deleterious effects on nontarget organisms such as lacewings, by either direct or indirect exposure through ingestion of contaminated prey (Aggarwal and Brar 2006; Ahmad et al. 2003; Cordeiro et al. 2010; Garcia et al. 2018; Medina et al. 2003; Scudeler and Santos 2013; Scudeler et al. 2013, 2014, 2016a, b).

However, little is known about the fat body of S. eridania and C. claveri. What are the effects of biopesticides in this organ and its possible cellular responses to exposure to a toxic agent? By collecting biological data regarding the morphological and ultrastructural characterization of the fat body of S. eridania and C. claveri after chronic exposure to azadirachtin, we were able to compare the response of these two species to exposure to azadirachtin as a biopesticide and to verify the use of the fat body as a target organ in ecotoxicological studies through the use of morphological biomarkers.

Materials and methods

Insects

Larvae of S. eridania (7 days old) and C. claveri (3 days old) used in the bioassays were obtained from a colony maintained at the Laboratory of Insects (22° 53′ 39.2″ S, 48° 29′ 42.4″ W) in the Department of Morphology at the Biosciences Institute at UNESP, Botucatu, São Paulo, Brazil. Both insect species were maintained in an environmental chamber with controlled conditions (25 °C ± 1 °C; 70% ± 10% RH, and photoperiod of 12 h). The S. eridania larvae were fed with fresh purple lettuce leaves (Lactuca sativa) and the adults with an artificial diet (1:10 honey/water). C. claveri was reared with eggs of Diatraea saccharalis (Lepidoptera: Crambidae) in the larval stage and with an artificial diet (1:1 honey/yeast solution) for the adults. Eggs of D. saccharalis were provided by the Cetma Comércio de Agentes para Controle Biológico, Lençóis Paulista, São Paulo State, Brazil.

Bioassays

Azadirachtin was used in its commercial formulation (AzaMax, 12 g (a.i.)/L, emulsifiable concentrate, UPL do Brasil Indústria e Comércio de Insumos Agropecuários S.A., Ituverava, SP, Brazil), chronically exposing larvae via ingestion. Two concentrations (6 mg a.i./L and 18 mg a.i./L) were administered, which correspond to 50% of the minimum and maximum concentrations recommended and registered for use in the field in agricultural crops in which the lacewings occur (MAPA 2018).

Azadirachtin solutions (6 mg a.i./L and 18 mg a.i./L) were diluted in distilled water and prepared starting from the commercial formulation and made daily for use. Fresh purple lettuce leaves and egg clusters of D. saccharalis were collected and dipped once in the azadirachtin solution for 5 s and air-dried at room temperature for 1 h. For the control groups of both species, purple lettuce leaves and egg clusters were dipped into distilled water.

Larvae of S. eridania (7 days old) were selected randomly and divided into three experimental groups (n = 15 per group), where they were maintained in polyethylene pots (9 cm height × 18 cm diameter). Nonetheless, larvae of C. claveri (3 days old) were selected and placed individually in polyethylene pots (2 cm height × 6 cm diameter). The larvae of C. claveri were divided similarly into three experimental groups (n = 15 per group).

Each experimental group of both species comprised six replicates. The chronic oral exposure ad libitum occurred during 7 days. Due to the sensitivity of the azadirachtin to ultraviolet light, purple lettuce leaves and egg clusters not consumed or not preyed upon by S. eridania and C. claveri larvae, respectively, were replaced every 2 days. The entire experiment was conducted under the same environmental conditions as described for insect rearing. The mortality data from each species/experimental group were recorded daily until the last day of the bioassay. After 7 days of exposure, larvae were weighed using a precision scale. The larval body mass (mg) was recorded for six individuals selected randomly from each experimental group/replicate (n = 36 individuals).

Light microscopy

Morphological and histochemical analyses

Larvae of S. eridania and C. claveri obtained from each experimental group were quickly cryoanesthetized at − 4 °C. Their fat bodies (parietal and perivisceral layers) were dissected in saline solution for insects (0.1 M NaCl, 0.1 M Na2HPO4, and 0.1 M KH2PO4). The insects were cut dorsally under a stereomicroscope for removal of the fat body layers of the abdominal region. The fat body was isolated and fixed in a 2.5% glutaraldehyde and 4% paraformaldehyde solution in 0.1 M phosphate buffer (pH 7.3) for a period of 24 h for the morphological and histochemical analyses. Five insects of each species/group were used for morphological analysis. For the morphological and the same histochemical analyses, the fat bodies were washed in phosphate buffer (0.1 M, pH 7.3) and dehydrated in graded ethanol solutions (70–95%) and then embedded in glycol methacrylate historesin (Leica Historesin Embedding Kit; Leica Biosystems, Wetzlar, Germany) according to the manufacturer’s instructions. Sections with a thickness of 3 μm were cut with a Leica RM 2045 microtome and stained with hematoxylin and eosin (HE) (Pearse 1972) for morphological analysis, bromophenol blue for the detection of total proteins (Junqueira and Junqueira 1983), and periodic acid-Schiff (PAS) for the detection of neutral polysaccharides (Pearse 1972).

For other histochemical tests, the samples were dehydrated in graded ethanol solutions (70–100%), cleared in xylene, and embedded in Paraplast® (Leica Biosystems, Richmond, IL, USA). The blocks were sectioned at 5 μm thickness, and the slides were submitted to histochemical tests for the detection of calcium (von Kossa technique) (Junqueira and Junqueira 1983) and Sudan Black B for the detection of total lipids (Pearse 1972). The slides were analyzed and documented using a Leica DM500 photomicroscope. The qualitative analysis of the histochemical tests was based on the intensity of reaction/staining in the fat body cells: (−) absence of reaction/staining, (+) reaction/staining with weak intensity, (++) reaction/staining with median intensity, and (+++) reaction/staining with strong intensity.

In the morphological analysis, the percentage of oenocytes in the parietal layer of C. claveri was estimated under a Leica DM500 photomicroscope at × 400 magnification. The total number of cells was counted in five fields of three longitudinal sections from five specimens of each experimental group. The percentage was calculated dividing the number of oenocytes by the total number of fat body cells analyzed, and the final value was multiplied by 100.

Immunohistochemical analyses

Immunohistochemical detection of cellular stress

For immunohistochemical analyses, fat body layers from five insects of each species/group were removed and fixed in a 4% paraformaldehyde solution in 0.1 M phosphate buffer (pH 7.3) for 12 h. The samples were dehydrated in graded ethanol solutions (70–100%), cleared in xylene, and embedded in Paraplast® (Leica Biosystems, Richmond, IL, USA). Fat body section slides (5 μm) were deparaffinized, rehydrated, and treated for antigen retrieval in 0.01 M citrate buffer (pH 6.0) in a 500-W microwave for 15 min. Thereafter, the slides were left for 30 min to cool to room temperature. After washing in phosphate-buffered saline (PBS; 0.01 M, pH 7.3), the sections were subjected to the blocking of endogenous peroxidase by treatment with 0.3% H2O2 in PBS for 15 min at room temperature. The sections were then rinsed with PBS and covered with Protein Block (SPD-125, Reveal Polyvalent HRP-DAB Detection System; Spring Bioscience, Pleasanton, CA, USA) for 10 min at room temperature to block nonspecific background staining as described previously by Scudeler et al. (2016a). Afterwards, the sections were incubated for 2 h at room temperature with the primary antibodies mouse anti-heat shock protein 70 (HSP70; 1:100 dilution) (clone 5A5, ab2787; Abcam®, Inc., Cambridge, MA, USA) and mouse anti-heat shock protein 90 (HSP90; dilution 1:250) (clone AC-16, H-1775; Sigma-Aldrich®, St. Louis, MO, USA) for immunolocalization of cellular stress. After incubating the primary antibodies, the sections were submitted to the immunoenzymatic antigen detection system (SPD-125, Reveal Polyvalent HRP-DAB Detection System; Spring Bioscience, Pleasanton, CA, USA). The methodology was performed according to the manufacturer’s instructions for the kit. Diaminobenzidine (DAB) chromogen developed a brown reaction product for positive signals. Finally, the slides were lightly counterstained with Harris hematoxylin. The slides were examined and photographed with a Leica DM500 photomicroscope.

Immunohistochemical detection of cell death by TUNEL assay

Fat body sections (5 μm) were deparaffinized, rehydrated, and washed in tris-buffered saline (TBS; 20 mM Tris, pH 7.6, 140 mM NaCl). Next, they were submitted to the TUNEL assay, which was used to label the 3′ ends of fragmented DNA, a technique widely used to identify cell death by microscopy. Sections were permeabilized by incubation in proteinase K (20 μg/ml in 10 mM Tris, pH 8) at room temperature for 30 min, and further procedures were performed according to the manufacturer’s instructions (TdT FragEL™ DNA Fragmentation Detection Kit; Calbiochem®, Merck KGaA, Darmstadt, Germany) as reported previously by Scudeler et al. (2016a). All TUNEL-positive nuclei from five fields (× 400 magnification) were counted for the three longitudinal sections from five samples in each species/experimental group, and the positivity percentages were calculated for the total nuclei counted.

Electron microscopy

Conventional transmission electron microscopy

After 7 days of oral exposure, five larvae of each species/experimental group were selected and dissected. Fragments of fat body (parietal and perivisceral layers) were collected and fixed in 2.5% glutaraldehyde and 4% paraformaldehyde solution in 0.1 M phosphate buffer (pH 7.3) for 24 h at room temperature and then postfixed in 1% osmium tetroxide in the same buffer for 2 h. After washing in distilled water, the samples were contrasted with an aqueous solution of 0.5% uranyl acetate for 2 h at room temperature, dehydrated in a graded acetone series (50%, 70%, 90%, and 100%), and embedded in Araldite resin. Ultrathin sections were contrasted with uranyl acetate and lead citrate and then analyzed in a Tecnai Spirit transmission electron microscope (FEI Company, Eindhoven, Netherlands) at the Electron Microscopy Center of the Institute of Biosciences of Botucatu.

Ultrastructural cytochemical analysis

To evaluate subcellular compartments as endomembrane systems that can react using the zinc iodide-osmium tetroxide (ZIO) method, fat body fragments were fixed for 12 h with 3% glutaraldehyde in 0.1 M phosphate buffer (pH 6.8) with 8.5% sucrose and incubated for 18 h at 4 °C in the ZIO incubation medium (zinc powder, resublimated iodide, osmium tetroxide, and tris(hydroxymethyl)-aminomethane buffer) (Reinecke and Walther 1978). The specimens were then washed in the same buffer, dehydrated, and embedded using routine procedures.

The presence of acid phosphatase was demonstrated using a standard technique as reported by Scudeler et al. (2016a), where the samples of fat bodies were fixed in 2.5% glutaraldehyde and 2% paraformaldehyde solution in 0.1 M sodium cacodylate buffer (pH 7.2) with 5% sucrose for 20 min. Immediately thereafter, the tissue samples were washed several times in sodium cacodylate buffer with 5% sucrose and washed twice in 0.05 M acetate buffer (pH 5.0) with 5% sucrose. Afterwards, the samples were incubated in a solution of cytidine-5′-monophosphate and 1% lead nitrate in acetate buffer (pH 5.0) for 1 h at 37 °C (Pino et al. 1981). Postfixation was performed in 2.5% glutaraldehyde and 2% paraformaldehyde solution in 0.1 M sodium cacodylate buffer (pH 7.2) with 5% sucrose for 45 min and with 1% osmium tetroxide in cacodylate buffer for 1 h. Finally, the samples were dehydrated and embedded following the procedures for conventional analysis. For all cytochemical analyses, the ultrathin sections unstained with uranyl acetate and lead citrate were examined in a Tecnai Spirit transmission electron microscope.

Statistical analysis

The data for accumulated mortality of both species were analyzed using the Goodman test, which involves contrasts among and within multinomial populations (Goodman 1964, 1965). Other quantitative variables were compared using an analysis of variance, which was complemented with multiple comparison tests. To assess the occurrence of oenocytes and cell death, the procedure was parametric, which was complemented with the Tukey and Bonferroni tests, respectively (Zar 2009; Johnson and Wichern 2007). Data on body mass were submitted to nonparametric testing complemented with Dunn’s test of multiple comparisons (Zar 2009). All tests were performed at a significance level of 5%.

Results

After 5 days of chronic exposure to azadirachtin, a significant mortality was observed in larvae of S. eridania exposed to both azadirachtin groups (Table 1). Meanwhile, the predator C. claveri remained stable throughout the exposure period. Although there was no significant mortality for C. claveri, both species had a reduction in larval body mass at the end of the bioassay, highlighting the reduction in body mass in S. eridania for both azadirachtin-treated groups (Table 2).
Table 1

Accumulated mortality (%) of C. claveri and S. eridania larvae subjected to bioassays of chronic exposure to azadirachtin by ingestion

Species

Treatment

Accumulated mortality (%) per day

1

2

3

4

5

6

7

C. claveri

Control

0.0 aA

0.0 aA

0.0 aA

0.0 aA

0.0 aA

0.0 aA

0.0 aA

6 mg

0.0 aA

0.0 aA

0.0 aA

0.0 aA

0.0 aA

0.0 aA

2.2 aA

18 mg

0.0 aA

2.2 aA

2.2 aA

6.7 aA

6.7 aA

6.7 aA

6.7 aA

S. eridania

Control

0.0 aA

0.0 aA

0.0 aA

2.2 aA

4.4 aA

4.4 aA

4.4 aA

6 mg

0.0 aA

6.7 aA

8.9 aA

13.3 aB

24.4 bB

40.0 bB

40.0 bB

18 mg

0.0 aA

6.7 aA

6.7 aA

11.1 aA

24.4 bA

42.2 bB

42.2 bB

p value

 

> 0.05

> 0.05

> 0.05

< 0.05

< 0.05

< 0.05

< 0.05

The data followed by different letters are significantly different. Lowercase letters indicate the comparison of treatments within the species. Uppercase letters indicate the comparison of species fixed to treatments (p < 0.05, Goodman test)

Table 2

Larval body mass (mg) [median (minimum; maximum value)] of C. claveri and S. eridania after treatment with azadirachtin for 7 days

Species

Treatment

Control

6 mg

18 mg

C. claveri

7.10 (2.10; 10.50)#

3.85 (1.00; 8.70)

4.35 (0.60; 8.70)

S. eridania

504.00 (255.00; 700.00)#*

54.50 (26.00; 570.00)*

69.50 (23.00; 360.00)*

#p < 0.05, control vs. 6 mg and 18 mg

*p < 0.001, C. claveri vs. S. eridania (Dunn’s test, n = 36 larvae per concentration)

Through morphological analysis, the abdominal fat body in larvae of S. eridania and C. claveri is divided into the parietal layer, which is characterized by the accumulation of small cellular masses adhering to the epidermis and associated with muscle fibers (Figs. 1a and 2a) and the perivisceral layer, which is much more developed, consisting of elongated and convoluted cell leaflets (Figs. 1d and 2d) that contact the digestive and reproductive organs.
Fig. 1

Photomicrography of the morphological analysis of the parietal (PA) and perivisceral (PV) fat bodies of the larvae of S. eridania after azadirachtin treatment. af Hematoxylin-eosin stain. gl Periodic acid-Schiff (PAS) stain. a, d In the control group, trophocytes (T) showed a basophilic cytoplasm with lipid droplets (Li) of different sizes and regular nucleus (N). b, c Note the vesiculation (Ve) of the cytoplasm and the occurrence of intercellular spaces (arrow). e, f Trophocytes have lost their morphology, giving an irregular shape to the convoluted leaflets of the perivisceral fat body. Condensed and fragmented nuclei (arrowhead) were observed. g, j PAS staining showed trophocytes with strong positive reaction for neutral polysaccharides and glycoconjugates (asterisk) mainly in the parietal layer. h, i, k, l Azadirachtin treatments showed reduced staining for PAS in trophocytes for both fat body layers. Cu cuticle, Ep epidermis, Mf muscle fiber. Bars = 20 μm

Fig. 2

Morphological changes in the parietal (PA) and perivisceral (PV) fat bodies of the larvae of C. claveri after azadirachtin treatment. af Hematoxylin-eosin stain. gl Periodic acid-Schiff (PAS) stain. a, d In the control group, trophocytes (T) showed the cytoplasm rich in lipid droplets (Li) with a regular shape, many acidophilic cytoplasmic granules (Gr), and irregular nuclei (N). b, c, e, f Note the decrease of the acidophilic cytoplasmic granules and the occurrence of irregular lipid droplets in the trophocytes. g, j PAS staining showed trophocytes with positive reaction for neutral polysaccharides and glycoconjugates (asterisk), mainly in the parietal region. h, i, k, l Treatment groups with azadirachtin showing the reduced stain of PAS-positive regions of the trophocyte cytoplasm. Cu cuticle, Ep epidermis, Mf muscle fiber, O oenocyte, Mt Malpighian tubule. Bars = 20 μm

In S. eridania, the parietal and perivisceral fat bodies are composed of only trophocytes with a basophilic cytoplasm that contain lipid droplets of varying sizes, distributed throughout the cell (Fig. 1a, d). Neutral polysaccharides were identified by PAS staining mainly in the parietal layer (Fig. 1g, j). Table 3 lists the results obtained in the histochemical analyses of the fat body. We highlight prominent changes in both layers for the groups exposed to azadirachtin, such as vesiculation of the cytoplasm, occurrence of intercellular spaces in the parietal layer, condensed and fragmented nuclei, and irregular shapes of trophocytes in the perivisceral leaflets (Fig. 1b, c, e, f). The azadirachtin-treated groups presented a strong reduction in glycogen deposits (Fig. 1h, i, k, l) and lipid droplets (Table 3).
Table 3

Histochemical analyses of the parietal and perivisceral fat bodies from S. eridania after exposure to azadirachtin

Fat body region

Treatment

Bromophenol blue

Periodic acid-Schiff

Sudan Black

Trophocyte

Trophocyte

C

GR

N

GR

GR

Parietal

Control

++

+

+++

++

6 mg

+

+

+

+

+

18 mg

+

+

+

+

+

Perivisceral

Control

++

+

++

+++

6 mg

+

+

+

+

+

18 mg

+

+

+

+

+

−, absence of reaction/staining; +, reaction/staining with weak intensity; ++, reaction/staining with median intensity; +++, reaction/staining with strong intensity

C cytoplasm, N nucleus, GR cytoplasmic granules

For C. claveri, the fat body is composed of trophocytes and oenocytes in the parietal layer and only trophocytes in the perivisceral region. The trophocytes showed an acidophilic cytoplasm that contains an abundant number of lipid droplets and acidophilic cytoplasmic granules throughout the cytoplasm and around the irregular nucleus (Fig. 2a, d). Oenocytes have a homogeneously stained acidophilic cytoplasm (Fig. 2a). Similar to that of S. eridania, the parietal layer has more glycogen deposits than the perivisceral fat body (Fig. 2g, j) (Table 4). In the azadirachtin exposition, it was possible to note lipid droplets with irregular shapes and a decrease in the amount of acidophilic granules, possibly protein granules stained by bromophenol blue, as well as the reduced staining of PAS and lipid droplets (Fig. 2b, c, e, f, h, i, k, l) (Table 4). For the highest azadirachtin concentration, a significant increase in the number of oenocytes in the parietal layer of larvae of C. claveri (Table 5) was observed.
Table 4

Histochemical analyses of the parietal and perivisceral fat bodies from C. claveri after exposure to azadirachtin

Fat body region

Treatment

Bromophenol blue

Periodic acid-Schiff

Sudan Black

Oenocyte

Trophocyte

Cytoplasm

Cytoplasm

C

N

C

GR

N

GO

GT

GO

GT

Parietal

Control

+

+

+++

++

+

+++

++

6 mg

+

+

++

+

+

+

++

18 mg

+

+

++

+

+

+

++

Perivisceral

Control

++

++

+

++

+++

6 mg

+

+

+

+

++

18 mg

+

+

+

+

++

−, absence of reaction/staining; +, reaction/staining with weak intensity; ++, reaction/staining with median intensity; +++, reaction/staining with strong intensity

C cytoplasm, N nucleus, GR cytoplasmic granules, GO cytoplasmic granules in the oenocytes, GT cytoplasmic granules in the trophocytes

Table 5

Percentage of oenocytes (mean (SD)) in the parietal fat body of the larvae of C. claveri after azadirachtin treatment

Treatment

 

Control

4.561 (2.036)a

6 mg

8.092 (1.943)a, b

18 mg

10.025 (3.041)b

The means followed by different letters are significantly different (p < 0.05, Tukey test)

The histochemical analysis for calcium detection showed a positive reaction to cytoplasmic granules in the trophocytes of both layers of the fat bodies of S. eridania and C. claveri. When the azadirachtin-treated groups were analyzed, we observed an increase in these positive signals in all azadirachtin dosages in both layers and species (Fig. 3a–l).
Fig. 3

Histological sections of the parietal (PA) and perivisceral (PV) fat bodies of C. claveri and S. eridania larvae were analyzed for calcium localization by the von Kossa technique. afC. claveri. glS. eridania. In the control group, trophocytes (T) showed few cytoplasmic calcium granules (arrow). Differently, in the azadirachtin treatments, it is possible to observe the increase in calcium granules in both concentrations. Mf muscle fiber, Tr trachea, Cu cuticle, Ep epidermis, O oenocyte, Mt Malpighian tubule. Bars = 20 μm

The immunohistochemical analyses for HSP70/HSP90 revealed the existence of the expression of these proteins in both layers of the fat body of S. eridania and C. claveri. In the larvae of S. eridania, HSP70 had a medium intensity of expression in the cytoplasm and nucleus in both layers of the fat body, while HSP90 had a low level of positive reaction in the control group (Fig. 4a, d, g, j). The opposite was observed in the C. claveri fat body, with major expression of HSP90 occurring in the control group. The cytoplasm of the oenocytes had a strong positive reaction for HSP70/HSP90 (Fig. 5a, d, g, j). All tested concentrations of azadirachtin in the larvae of S. eridania and C. claveri induced an increased level of staining for HSP70/HSP90 in trophocytes of the parietal and perivisceral fat bodies (Figs. 4 and 5) (Table 6).
Fig. 4

Photomicrography of the immunohistochemical analysis of HSP70 and HSP90 expression in the parietal (PA) and perivisceral (PV) fat bodies of larvae of S. eridania subjected to azadirachtin treatments. af HSP70. gl HSP90. The brown stain indicates the in situ expression of HSP70 or HSP90. Note the upregulation of HSP70 and HSP90 expression in the cytoplasm (asterisk) and nucleus (N) of trophocytes (T) in both fat body regions subjected to azadirachtin. Cu cuticle, Ep epidermis, Mf muscle fiber, Tr trachea. Bars = 20 μm

Fig. 5

Histological sections of the parietal (PA) and perivisceral (PV) fat bodies of larvae of C. claveri were analyzed by HSP70 and HSP90 immunodetection. af HSP70. gl HSP90. The brown stain indicates a positive signal for HSP70 or HSP90. Note the increase in staining for HSP70 and HSP90 in the cytoplasm (asterisk) and nucleus (N) of the trophocytes (T) in the treatments for both fat body regions and azadirachtin concentration. A positive signal in the cytoplasm of the oenocyte (O) was observed in the control and all treatment groups. Cu cuticle, Ep epidermis, Mf muscle fiber, Tr trachea, Mt Malpighian tubule. Bars = 20 μm

Table 6

Immunohistochemical analyses of HSP70/HSP90 in the parietal and perivisceral fat bodies from C. claveri and S. eridania after exposure to azadirachtin

Species

Treatment

HSP70

HSP90

Parietal

Perivisceral

Parietal

Perivisceral

C. claveri

Control

+

+

++

++

6 mg

++

++

+++

+++

18 mg

++

++

+++

+++

S. eridania

Control

++

++

+

+

6 mg

+++

+++

++

++

18 mg

+++

+++

++

++

+, reaction/staining with weak intensity; ++, reaction/staining with median intensity; +++, reaction/staining with strong intensity

Complementing the cytotoxicity analyses of azadirachtin performed in the fat body using the TUNEL method, cell death was induced in both dosages in S. eridania, being more intense in the perivisceral layer. For C. claveri, significant cell death can be observed only at the highest dose, being more pronounced in the perivisceral layer. Despite the occurrence of cell death in both species, the incidence was more intense in S. eridania (Table 7).
Table 7

Percentage of cell death (mean (SD)) in the fat body regions of C. claveri and S. eridania after azadirachtin treatment for 7 days

Species

Treatment

Fat body region

Parietal

Perivisceral

C. claveri

Control

1.26 (0.72) aAα

3.48 (1.22) aAβ

6 mg

3.34 (1.13) aAα

6.48 (2.12) aAβ

18 mg

8.63 (3.43) bAα

17.38 (3.88) bAβ

S. eridania

Control

1.35 (0.74) aAα

4.58 (0.99) aAβ

6 mg

9.84 (5.40) bBα

17.22 (6.85) bBβ

18 mg

14.16 (3.78) bBα

24.32 (6.99) cBβ

Data followed by different letters are significantly different. Lowercase letters indicate the comparison of treatments fixed to the species and fat body region. Uppercase letters indicate the comparison of species fixed to the treatment and fat body region. Greek letters indicate the comparison of fat body regions of the fixed species and treatment (p < 0.05, Bonferroni test)

The ultrastructural analysis of the parietal and perivisceral fat bodies of S. eridania demonstrated trophocytes with well-developed cisternae of the rough endoplasmic reticulum and mitochondria with varying morphologies, lipid droplets, and glycogen deposits (Figs. 6a, d and 7a, d). The basal lamina surrounds and protects the masses of trophocytes from direct contact with the hemolymph (Fig. 6b). Small lysosomes can be detected by their acid phosphatase activity (Figs. 6g and 7g). However, in the larvae resulting from azadirachtin-treated groups, trophocytes showed severe cell damage, as shown by the dilated cisternae of the rough endoplasmic reticulum (Figs. 6b, c and 7b, c) and swollen mitochondria (Fig. 7b, c). Through the ZIO technique, we noted that the morphology of the Golgi complex changed to a vesicular appearance. In addition, the smooth endoplasmic reticulum presented the development of branched tubules (Figs. 6e, f and 7e, f). The activity of acid phosphatase was observed in vacuoles with membranous structures such as myelin structure, indicating the occurrence of autolysosomes in both experimental groups exposed to azadirachtin (Figs. 6h, i and 7h, i).
Fig. 6

TEM micrographs of trophocytes of the parietal fat body of larvae of S. eridania after azadirachtin treatment. a, d The control group (trophocytes) showed well-developed cisternae of the rough endoplasmic reticulum (Rer), lipid droplets (Li) of different sizes, glycogen (Gly) deposits, and mitochondria (Mi). b, c In the azadirachtin treatments, note the dilated cisternae of the rough endoplasmic reticulum. The myelin structures (Ms) can also be observed in the cytoplasm with vacuolated appearance df The ZIO method revealed changes in the Golgi complex (Gc), where it acquired a vesicular appearance, and there was the development of the smooth endoplasmic reticulum (Ser). gi In the control group, the detection of acid phosphatase indicated small lysosomes (Ly) (arrow). However, in the azadirachtin groups, acid phosphatase occurred in the membranous structures (arrow), indicating the occurrence of autolysosomes. B basal lamina, N nucleus

Fig. 7

Ultrastructure of trophocytes of the perivisceral fat body of larvae of S. eridania after azadirachtin treatment. a, d Cells of the control group showed regular cisternae of the rough endoplasmic reticulum (Rer), electron lucent mitochondria (Mi), and polarized Golgi complex (Gc) with small cisternae and adjacent vesicles. b, c Note swollen mitochondria in the cristolysis process as well as dilated cisternae of the rough endoplasmic reticulum. Autophagosome (Au) was observed with rough endoplasmic reticulum membrane seen inside it. df ZIO technique. Note the morphology of the Golgi complex, with vesiculated appearance, and the large branched tubules of the smooth endoplasmic reticulum (Ser). gi Positive reaction for acid phosphatase (arrow) in lysosomes and myelin structure (Ms). N nucleus, Li lipid droplet, Gly glycogen, V vacuole

In C. claveri, trophocytes of the parietal and perivisceral fat bodies contain large lipid droplets and numerous urate granules around the nuclei. The mitochondria and rough endoplasmic reticulum are visible throughout the cytoplasm (Figs. 8a and 9a, d). In the oenocytes present only in the parietal layer, the cytoplasm is rich in the smooth endoplasmic reticulum and mitochondria (Fig. 8d). Prominent changes in the ultrastructure of trophocytes and oenocytes were observed after azadirachtin exposure at both dosages. Similar to S. eridania, there was a dilatation of the cisternae of the rough endoplasmic reticulum and swollen mitochondria in the cristolysis process, such as vacuoles with membranous content (Figs. 8b, c, and 9b, c, e). Lipid droplets began to fuse with the vacuoles (Fig. 9b). The perinuclear space presented points of dilatation (Figs. 8b and 9f). The ZIO method showed the fragmented appearance of the rough endoplasmic reticulum (Fig. 9g–i). A positive reaction for acid phosphatase was observed in urate granules in the trophocytes for the control for both fat body layers, just decreasing the labeling in the azadirachtin-treated groups (Figs. 8g–i and 9j–l). In these insects, the ultrastructure of the oenocytes was mildly altered. Cytoplasm showed just more tubules of the smooth endoplasmic reticulum (Fig. 8e, f).
Fig. 8

Ultrastructure of parietal fat body cells of larvae of C. claveri subjected to azadirachtin treatments. a In the control group, trophocytes showed a cytoplasm with many urate granules (Ug) presenting near of them glycogen deposits (Gly). Note lipid droplets (Li), cisternae of the rough endoplasmic reticulum (Rer), and elongated mitochondria (Mi) in these cells. d However, oenocytes showed a predominance of cisternae of the smooth endoplasmic reticulum (Ser) and mitochondria. b, c In the azadirachtin groups, trophocytes showed an electron lucent cytoplasm, rich in dilated and fragmented cisterns of the rough endoplasmic reticulum, and the occurrence of swollen mitochondria. Perinuclear space can be observed to be dilated (arrowhead). e, f Note in the oenocyte cytoplasm the development of cisternae of the smooth endoplasmic reticulum. gi In the acid phosphatase method, a positive reaction (arrow) was observed in the urate granules, with decreased staining in the azadirachtin treatments. N nucleus

Fig. 9

Transmission electron micrographs of perivisceral fat body cells of the larvae of C. claveri after azadirachtin treatments. a, d, g In the control group, trophocytes showed a cytoplasm with large lipid droplets (Li) around the nucleus (N), urate granules (Ug), cisternae of the rough endoplasmic reticulum (Rer), and elongated mitochondria (Mi). b, c, e, f In the azadirachtin groups, trophocytes showed the fusion (asterisk) of lipid droplets and vacuoles with membranous content (V), besides the dilated and fragmented cisterns of the rough endoplasmic reticulum and the occurrence of mitochondria in the cristolysis process. Perinuclear space can be observed to be dilated (arrowhead). gi The ZIO method revealed changes in the rough endoplasmic reticulum, where we found fragmented appearance. jl Similar to the parietal fat body, trophocytes showed a positive reaction for acid phosphatase (arrow) in the urate granules, and this staining decreased in the azadirachtin groups. B basal lamina

Discussion

Insects vary in their susceptibility to insecticides. In the present report, we showed that dosages of 6 mg and 18 mg of azadirachtin were relatively safe for the larval stage of C. claveri but led to a considerable level of mortality for S. eridania larvae. Azadirachtin- or neem-based products have been shown to be active against Lepidoptera species, an extremely sensitive insect order. These compounds show an effective antifeedant effect (Campos et al. 2016; Mordue (Luntz) and Nisbet 2000; Roel et al. 2010). In addition to affecting survival, antifeedant activity compromises weight gain (Shannag et al. 2015), and mortality may be dominated mainly by starvation (Mordue (Luntz) et al. 1998).

Although we observed the compatibility between the doses of azadirachtin with C. claveri larvae survival, many studies refute this information. According to Cordeiro et al. (2010), there is a delayed mortality that may equivocally suggest higher safety for lacewing larvae. Harmful effects were detected during the formation of pupae leading to inhibition of the adult stage (Medina et al. 2003; Scudeler et al. 2016b). If we think in terms of a short-term effect, we would have a satisfactory form of control of S. eridania with azadirachtin, without great damage to the larval stage of C. claveri. However, in the long term, we would be compromising the lifecycle of this predator and its maintenance in the agroecosystem by affecting reproduction, as indicated in Cloyd (2012), Morgan (2009), and Scudeler et al. (2016a, b). The reduction in body mass may be considered an indirect effect and was accompanied in both species by the loss of glycogen, lipid, and protein stored in the trophocytes of the parietal and perivisceral fat bodies. This mobilization of reserves was possibly used to maintain their survival in response to and in an attempt to minimize the harmful effects of azadirachtin intake.

To obtain more accurate data on the response of the fat body to an exposure of azadirachtin, we opted for a chronic exposure. According to Adamski et al. (2005), a shorter period might result in starvation as a behavioral method of avoiding stress, and the ultrastructural response can be seen more clearly after a few days. These effects can be confirmed by the beginning of the occurrence of significant mortality after 5 days of exposure to S. eridania and by morphological and ultrastructural changes in the fat body after 7 days for both species.

Alterations of histochemical patterns were pronounced in the trophocytes from both larvae exposed to azadirachtin with a non-dose–dependent response. We found a pattern of reduction in mainly lipid droplets and PAS-positive granules, important energy reserves to animal cells. Other experiments with pesticides also reported the occurrence of a decrease in these energy reserves in trophocytes (Alves et al. 2010; Domingues et al. 2017). Arrese and Soulages (2010) relate the mobilization of glycogen in trophocytes under stress conditions (temperature, drought) and lipid (triglycerides) during starvation and immunological responses, respectively. The data obtained indicate that due to the reduction in the body mass of the larvae, energy reserves were mobilized to supply the metabolic activity of other tissues, including the fat body in response to chemical stress.

An important point to highlight was the presence of oenocytes only in the fat body of the predator C. claveri. In the azadirachtin-treated groups, we observed the increase in the number of oenocytes in the parietal fat body, likely as a physiological response of the predator to the stress generated by exposure to the biopesticide. Based on its observed ultrastructure, such as extensive areas of the smooth endoplasmic reticulum, numerous mitochondria, and the occurrence of an increase in the smooth endoplasmic reticulum in the oenocytes of insects exposed to azadirachtin, it is evidenced that these cells play an important role in detoxification (Domingues et al. 2017; Martins and Ramalho-Ortigão 2012; Roma et al. 2010). Other evidence corroborating the detoxification function exerted by oenocytes is the expression of heat shock proteins or HSP70/HSP90 (also referred to as stress proteins) in the control group and in the azadirachtin treatments. The HSP expression suggests a protective role during stressful conditions (Chowanski et al. 2017). According to Lycett et al. (2006), the oenocyte is the major cell type of the fat body-expressing cytochrome P450, an enzyme involved in the pathways of detoxification of a wide range of xenobiotic and endogenous compounds.

In the trophocytes of the control group of both species, we demonstrated immunodetection for HSP70/HSP90 in the cytoplasm and nucleus. Shu et al. (2011) reported a high level of HSP70/HSP90 expression in the fat body of Spodoptera litura. Exposure to azadirachtin caused injury and, therefore, cellular stress, producing the upregulated expression of HSP70/HSP90. This increased expression of HSPs may be a cytoprotective action in the fat body of both species. HSPs are a part of the cellular protective response. Proteins used as cellular markers of cell stress or aggression and their induction were correlated with resistance to induced stress such as heat, pesticides, heavy metals, drugs, and other pollutants (Bierkens 2000; Feder and Hofmann 1999; Ferreira et al. 2013; Lin et al. 2016; Malaspina and Silva-Zacarin 2006; Scudeler et al. 2016a; Shu et al. 2011). Despite the cytoprotective effects made by HSP70/HSP90 as an antiapoptotic response (Lanneau et al. 2008; Scudeler et al. 2016a; Silva-Zacarin et al. 2006; Wang et al. 2014), cell death was observed mainly in the perivisceral layer and with greater intensity in S. eridania. Due to the perivisceral layer being located around the digestive tube, mainly in the abdomen near the midgut, it is possible that trophocytes of this layer interact more intensely with the hemolymph and the absorbed azadirachtin, causing a high percentage of cell death. Using the histological analysis in S. eridania, it was possible to confirm these cell deaths through an observation of condensed and fragmented nuclei in the perivisceral fat body. The lower occurrence of cell death in the predator C. claveri may have occurred due to the presence and increase in oenocyte abundance in the azadirachtin-treated groups. However, the perivisceral layer that does not present oenocytes always shows a higher incidence of cell death than the parietal layer.

Another cytoprotective response observed in the trophocytes of both species was the increase in cytoplasmic granules containing calcium. In invertebrates, these cytoplasmic granules indicate the activation of the detoxification mechanisms (Nogarol and Fontanetti 2010; Souza and Fontanetti 2011). Scudeler et al. (2016a) observed this similar response in the columnar cells of the midgut of C. claveri exposed to neem oil intake. Stress conditions may induce the elevation of cytoplasmic calcium levels, and the subsequently, sustained calcium signaling in the cytosol will prevent damage to the endoplasmic reticulum, vesiculation, and the loss of their function (Cerella et al. 2010) as well as stimulate the activation of autophagy (Hoyer-Hansen et al. 2007; Tong and Song 2015).

By ultrastructural analysis, the features of the trophocytes of S. eridania and C. claveri are similar to those described by Paes de Oliveira and Cruz-Landim (2003), Arrese and Soulages (2010), and Roma et al. (2010) in a review on the morphology, ultrastructure, and function of trophocytes. The presence of well-developed rough endoplasmic reticulum, Golgi complexes, mitochondria, and lipid and glycogen deposits suggest the capacity of synthesis, absorption, and storage of energy reserves (Arrese and Soulages 2010; Carvalho et al. 2013; Cunha et al. 2016; Roma et al. 2010). Deposits of urate granules were also observed in C. claveri trophocytes. According to Buckner et al. (1990), these granules contain mainly uric acid and proteins. Uric acid is a product of nitrogen metabolism, and it is sequestered and stored in granules or vacuoles (Buckner et al. 1990; Haunerland and Shirk 1995; Park et al. 2013). This uric acid can be mobilized from urate vacuoles for the synthesis of amino acids under starvation conditions (Park et al. 2013).

The location of a positive reaction to acid phosphatase inside urate granules showed that this enzyme might play a role in the mobilization of uric acid reserves. Poiani and Cruz-Landim (2012) observed that the presence of acid phosphatase in the stored products might be responsible for their mobilization of fat body reserves accumulated in trophocytes. The positive reaction decrease in the azadirachtin-treated groups may indicate needless for this catabolic process in attempting to supply amino acid requirements, since protein synthesis was compromised due to the cell injuries observed in the trophocytes. For S. eridania, the lytic activity of acid phosphatase was observed in vacuoles containing myelin structures through activation of an autophagic process. This increase in acid phosphatase expression is related to cytotoxicity processes, since their activity contributes to detoxification processes (Amirmohammadi et al. 2012; Scudeler et al. 2016a; Valizadeh et al. 2013). Another attempt at detoxification observed in the trophocytes of S. eridania was the development of the smooth endoplasmic reticulum (Catae et al. 2014; Cheville 1994, 2009; Scudeler and Santos 2013; Scudeler et al. 2014, 2016a).

Even though the trophocytes have shown different cytoprotective responses opposing the injuries caused by exposure to azadirachtin in both species, ultrastructural analysis showed that these responses have not proved totally effective, considering a high accumulated mortality and cell death of the trophocytes of S. eridania larvae. In general, the main ultrastructural lesions in the trophocytes of parietal and perivisceral layers that we have identified in the two species involved swollen mitochondria in the cristolysis process, dilated cisternae of the rough endoplasmic reticulum, fusion of lipid droplets, and Golgi complexes with vesiculated appearance and dilatation of the perinuclear space. These aforementioned changes are similar to those described in the cells of the fat body or other organs of arthropods in which the effects of azadirachtin, neem oil, plant derived, and insecticides are evaluated (Adamski et al. 2016, 2005; Adamski 2007; Büyükgüzel et al. 2013; Catae et al. 2014; Nasiruddin and Mordue (Luntz) 1993; Scudeler and Santos 2013; Scudeler et al. 2014, 2016a; Remedio et al. 2016).

In conclusion, the data indicate that azadirachtin intake during the larval stage of S. eridania and C. claveri impairs the fat body by inducing cellular injuries and can impact its functions. Although we observed different cytoprotective and detoxification means, they were not sufficient to recover from all the cellular damages. Azadirachtin exhibited an excellent performance for the control of S. eridania. The predator C. claveri, which presented better biological and cytoprotective responses to this chronic exposure, was indicated to be more selective to possible toxic agents used in the control of insect pests such as azadirachtin. Furthermore, despite the fact that the fat body was not directly exposed to azadirachtin by intake, it is shown to be a good target organ in ecotoxicological studies by providing the details of injuries and cellular responses.

Notes

Acknowledgments

We are grateful to the Electron Microscopy Center of the Institute of Biosciences of Botucatu, UNESP.

Funding

This study was supported and funded by the São Paulo Research Foundation (Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)) (2014/15016-2).

Compliance with ethical standards

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Conflict of interest

The authors declare that they have no conflict of interest.

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Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Laboratory of Insects, Department of Morphology, Institute of Biosciences of BotucatuSão Paulo State University (UNESP)BotucatuBrazil
  2. 2.Department of Biostatistics, Institute of Biosciences of BotucatuSão Paulo State University (UNESP)BotucatuBrazil
  3. 3.Electron Microscopy Center, Institute of Biosciences of BotucatuSão Paulo State University (UNESP)BotucatuBrazil

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