Immune challenges trigger cellular and humoral responses in adults of Pterostichus melas italicus (Coleoptera, Carabidae)

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Immune challenges trigger cellular and humoral responses in adults of Pterostichus melas italicus (Coleoptera, Carabidae) Q3

Anita Giglio a, *, Pietro Brandmayr a, Teresa Pasqua a, Tommaso Angelone a, Silvia Battistella b, Piero G. Giulianini b a

Q1

b


della Calabria, Via P. Bucci, I-87036 Rende, Italy Dipartimento di Biologia, Ecologia e Scienze della Terra, Universita
di Trieste, Via Giorgieri 5, I-34127 Trieste, Italy Dipartimento di Scienze della Vita, Universita

a r t i c l e i n f o

a b s t r a c t

Article history: Received 17 July 2014 Received in revised form 20 January 2015 Accepted 23 January 2015 Available online xxx

The present study focuses on the ability of Pterostichus melas italicus Dejean to mount cellular and humoral immune responses against invading pathogens. Ultrastructural analyses revealed the presence of five morphologically distinct types of hemocytes: prohemocytes, plasmatocytes, granulocytes, oenocytoids and macrophage-like cells. Differential hemocyte counts showed that plasmatocytes and granulocytes were the most abundant circulating cell types and plasmatocytes exhibited phagocytic activity following the latex bead immune challenge. Macrophage-like cells were recruited after the immune challenge to remove exhausted phagocytizing cells, apoptotic cells and melanotic capsules formed to immobilize the latex beads. Total hemocyte counts showed a significant reduction of hemocytes after latex bead treatment. Phenoloxidase (PO) assays revealed an increase of total PO in hemolymph after immune system activation with lipopolysaccharide (LPS). Moreover, the LPS-stimulated hemocytes showed increased protein expression of inducible nitric oxide synthase, indicating that the cytotoxic action of nitric oxide was engaged in this antimicrobial collaborative response. These results provide a knowledge base for further studies on the sensitivity of the P. melas italicus immune system to the environmental perturbation in order to evaluate the effect of chemicals on non target species in agroecosystems. © 2015 Published by Elsevier Ltd.

Keywords: Carabid beetle Cellular immunity Microscopy Nitric oxide synthase Phenoloxidase Phagocytosis

1. Introduction Once pathogens enter the hemocoel of the host, they encounter a complex system of innate defense mechanism involving cellular and humoral responses. These responses are based on a sequence of events including the recognition of invaders and their immobilization (Gillespie et al., 1997; Siva-Jothy et al., 2005; Ottaviani, 2005). This non-self recognition involves a series of membrane receptors of hemocytes which are essential to recognize pathogenassociated molecular patterns (PAMPs) and to trigger an immune response. The cellular immune response involves different hemocyte types which participate in pathogen clearance by phagocytosis, nodule formation, encapsulation and cytotoxic reactions. Morphological, histochemical and functional characteristics and monoclonal antibody and genetic markers have been used to characterize many hemocyte types in insects. The most common

* Corresponding author. Tel.: þ39 0984492982; fax: þ39 0984492986. E-mail address: [email protected] (A. Giglio).

types of hemocytes described in species of diverse orders are prohemocytes, granulocytes, plasmatocytes, spherule cells and oenocytoids (Gillespie et al., 1997; Lavine and Strand, 2002; Giulianini et al., 2003; Jiravanichpaisal et al., 2006; Giglio et al., 2008). Humoral defenses include the production of antimicrobial peptides (AMPs), reactive intermediates of oxygen or nitrogen and the prophenoloxidase enzymatic cascade (proPO) regulating melanization of hemolymph. The inducible isoform of the enzyme nitric oxide synthase (iNOS) is rapidly synthesized by a wide array of cells and tissues and it catalyzes nitric oxide synthesis until the substrate is depleted in response to acute infections in invertebrates (Nappi and Ottaviani, 2000; Nappi et al., 2000; Nappi and Vass, 2001; Rivero, 2006). In insect hemocytes, iNOS is synthesized in response to immune insult and the resulting nitric oxide acts cytotoxically on many kinds of pathogens (Ratcliffe et al., 1985; Davies, 2000; Siva-Jothy et al., 2005; Marmaras and Lampropoulou, 2009). The proPO-activating system comprises a complex cascade of serine proteases allowing the conversion of proPO to phenoloxidase (PO) (Marmaras et al., 1996; Gillespie et al., 1997; Rolff and Siva-Jothy, 2003; Schmid-Hempel, 2005; Siva-Jothy

http://dx.doi.org/10.1016/j.asd.2015.01.002 1467-8039/© 2015 Published by Elsevier Ltd.

Please cite this article in press as: Giglio, A., et al., Immune challenges trigger cellular and humoral responses in adults of Pterostichus melas italicus (Coleoptera, Carabidae), Arthropod Structure & Development (2015), http://dx.doi.org/10.1016/j.asd.2015.01.002

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et al., 2005). Synthesis of proPO occurs mostly in hemocytes with species-specific variation in relation to hemocyte types. The main role of PO in melanogenesis is to convert phenols to quinones which subsequently polymerize to form melanin. Natural activators of the proPO system are pathogen cell surface molecules such as b1,3 glucans from fungal cell walls and lipopolysaccharides (LPS) and peptidoglycans from microbial cells. The melanin deposited onto the foreign target prevents the pathogen growth and reproduction and thus melanization is an important cell-mediated immune response in tissue repair and in pathogen sequestration (Soderhall € derha €ll, 2004; Gonza lez-Santoyo and et al., 1994; Cerenius and So  rdoba-Aguilar, 2012). Studies on insect immunity have Co measured a number of immune effectors related to both cellular and humoral reactions, focusing on defense variation in both the evolutionary and the ecological context (for a review see SchmidHempel and Ebert, 2003; Schmid-Hempel, 2003, 2005). There is also increasing interest in this field with regard to the economic or ecological value of the investigated species. Carabid beetles are one of the most important groups of predators in most terrestrial ecosystems acting as beneficial arthropods in the agroecosystem food chain in which they are natural enemies of pests such as aphids, lepidopterans, slugs and dipterans (Luff, €, 2003; Avgin and Luff, 2010). 1983, 1996; Rainio and Niemela Because of their use as bioindicators and their economic importance, many species of this coleopteran family have been extensively studied. However, information on immunocompetence is necessary to complete the ecological framework of ground beetles. Pterostichus melas italicus (Dejean, 1828), a eurytopic and thermophilous species inhabiting clay soils, shows generalist predator habits in adulthood and a bimodal activity period. Adults appear in April and September and breed during autumn in the Mediterranean biome. They show activity throughout the winter on the soil surface, while the larval stage hibernates. This species is very common in agroecosystems of Calabria (southern Italy) (i.e. olive groves) and acts as a predator against insect pests (i.e. Bactrocera oleae). A previous study demonstrated that this beneficial species is sensitive to agricultural management practices, especially the use of pesticides. Laboratory tests showed that a sublethal dose of dimethoate affects cell-mediated immune responses, causing a decrease of the total number of circulating hemocytes (Giglio et al., 2011). This prompted our study of the immune competence of P. melas italicus as a baseline for future research to define the modulation of the beetle's immune system in polluted conditions. This parameter could be used as a biomarker to monitor the effect of toxicants on this non-target species integral to the agroecosystem community. To evaluate the immune function of P. melas italicus adults, we measured a set of the most common immune markers used in ecological and evolutionary studies to define the immune defenses of insects. The following key components of insect immune function were scored: 1) total and differential number of hemocytes and their morphological characterization by light and transmission electron microscopy, 2) phagocytosis after in vivo artificial non-self challenge with latex beads, and 3) inducible nitric oxide synthase (iNOS) after in vivo lipopolysaccharide challenge as a means to assess the cellular immune ability, 4) basal and total activities of phenoloxidase (PO) as components of the humoral defense.

specimens were reared with a light regime of L8:D16, 70% r.h. and at a day/night temperature of 16/13  C. They were fed on homogenized meat and fruit ad libitum. The animals were CO2 anesthetized and the hemolymph was collected in two different ways. For the in vivo phagocytosis assay, the last two abdominal segments of each specimen were laterally torn, a 29-gauge needle was inserted in the neck membrane and 10 mM sterile phosphate-buffered saline (PBS, SigmaeAldrich) was slowly injected to bring about hemolymph flow through the tear in the abdomen. The first droplet of about 30 mL of hemolymph was collected directly in the fixative. For all other assays the adults were punctured at the ventral level of the pro-mesothorax articulation with a 29-gauge needle. The first droplet of about 5 mL of hemolymph was collected. 2.2. Light and transmission electron microscopy

2. Material and methods

To assess the ability of hemocytes to phagocytize, we used a 29gauge needle to inject 20 mL of carboxylate-modified polystyrene latex beads (0.9 mm in diameter, aqueous suspension, 10% solids content, SigmaeAldrich) diluted 1:10 in 0.15 M sterile PBS into the body of five adults at the ventral level of the pro-mesothorax articulation. Parallel controls were run with five untreated animals and with five animals injected with 20 mL of sterile PBS alone. After 2 h, 20 mL of hemolymph were collected from treated and control specimens, transferred to a 1.5 mL eppendorf tube with a fixative solution containing 2.5% glutaraldehyde, 1% paraformaldehyde and a 7.5% saturated aqueous solution of picric acid in 0.1 M cacodylate buffer, pH 7.4, with 1.5% sucrose, and fixed for 2 h at 4  C. The hemocytes were pelleted by 1700 g centrifugation for 10 min at 4  C. The pellets obtained from the pooled hemolymph of five animals for each experimental group were then postfixed in 1% osmium tetroxide in the same buffer, serially dehydrated in acetone and embedded in Embed812/Araldite (Electron Microscopy Sciences, Fort Washington, PA). For transmission electron microscopy (TEM), thin sections were cut with a Leica Ultracut UCT ultratome, stained with uranyl acetate and lead citrate and examined with a Zeiss EM10 electron microscope at 60 kV. Images were acquired with a Veleta e 2k  2k side-mounted TEM CCD Camera (Olympus, Germany) provided with an iTEM imaging platform and saved in JPEG format. Hemocyte measurements were taken with Image-Pro Plus version 4.5 software (Media Cybernetics, Bethesda, MD, USA) on digitized images and processed as means ± standard error. For light microscopy, 2 mm semi-thin transverse sections from pellets, were stained with toluidine blue and observed under a Zeiss AXIOSKOP microscope. Images were acquired with a Nikon Coolpix 4500 camera. Differential hemocyte counts (DHCs) were carried out by three different operators from three slides at the beginning, center and end of the pellet using morphological characteristics (sensu Ribeiro and Brehelin, 2006). The relative percentage of hemocyte types was counted for P. melas italicus adults treated with in latex beads, injected with sterile PBS or left untreated. For total hemocyte counts (THCs), 5 mL of hemolymph were collected from animals treated as described above and the hemocytes were counted in a Bürker's chamber (Carlo Erba, Italy) without dilution and observed under differential interferential contrast (DIC) in light microscopy (LM) at 40 magnification (Zeiss AXIOSKOP). THC was expressed as the number of cells per mL.

2.1. Insect rearing and hemolymph collection

2.3. Western blot analysis

Adults of P. melas italicus were collected by bait traps in an area of the Botanical Garden of the University of Calabria, Italy (39 40 N and 16 20 E) from October to November 2011 and 2013. The

To verify whether lipopolysaccharide (LPS) treatment is able to modify inducible nitric oxide synthase (iNOS) protein expression in hemocytes, we injected adults (n ¼ 6) with 4 mL of LPS (0.5 mg/mL

Please cite this article in press as: Giglio, A., et al., Immune challenges trigger cellular and humoral responses in adults of Pterostichus melas italicus (Coleoptera, Carabidae), Arthropod Structure & Development (2015), http://dx.doi.org/10.1016/j.asd.2015.01.002

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phosphate buffer) from Escherichia coli 0127:B8 (SigmaeAldrich, L3129). Twenty-four hours after injection, the beetles were anesthetized and 5 mL of hemolymph were collected, transferred into 95 mL ice-cold PBS (10 mM) and centrifuged at 1700 g for 5 min at 4  C. Parallel controls were run with a group of untreated insects. The supernatant containing a solution of PBS and hemolymph plasma was stored at 20  C prior to the measurement of phenoloxidase enzyme activity. Hemocytes were resuspended and lysed in ice-cold radioimmunoprecipitation assay buffer (RIPA, R0278, SigmaeAldrich) containing a mixture of protease inhibitors (1 mmol/L aprotinin, 20 mmol/L phenylmethylsulfonyl fluoride and 200 mmol/L sodium orthovanadate). Lysates were then centrifuged at 6000 g for 10 min at 4  C for debris removal. Protein concentration was determined using Bradford reagent according to the manufacturer's instructions (SigmaeAldrich). Equal amounts of proteins (60 mg) were: (1) separated on 8% SDS-PAGE gels at 90 V for about 2 h; (2) transferred to PVDF (polyvinyl difluoride) membranes (RPN303F, GE Healthcare) at 60 V for 2 h using a Mini-PROTEAN Tetra Cell plus Tetra blotting Module (BioRad) equipment; (3) blocked with nonfat dry milk and incubated overnight at 4  C with a polyclonal rabbit anti-iNOS antibody (sc-649, Santa Cruz Biotechnology, Santa Cruz, CA) used as a primary antibody diluted 1:1000 in TBS-T containing 5% non-fat dry milk. The anti-rabbit peroxidase-linked secondary antibody (goat anti-rabbit IgG HRP linked; sc-2004, Santa Cruz Biotechnology, Santa Cruz, CA) was diluted 1:2000 in TBS-T containing 5% non-fat dry milk. Membranes were incubated with the secondary antibody at room temperature for 1 h. A polyclonal rabbit anti-b-actin antibody was used as loading control (Santa Cruz Biotechnology, Santa Cruz, CA). Immunodetection was performed using an enhanced chemiluminescence kit (ECL PLUS, Amersham). Autoradiographs were obtained by exposure to X-ray films (Hyperfilm ECL, Amersham). Immunoblots were digitized and the densitometric analysis of the bands was carried out using NIH IMAGE 1.6 for a Macintosh computer based on 256 grey values (0 ¼ white; 256 ¼ black). 2.4. Measurement of phenoloxidase enzyme activity Phenoloxidase enzyme activity was measured in both untreated animals and those injected (treated) with 4 mL of LPS as described above. Each hemolymph sample (5 mL) was immediately transferred into 95 mL ice-cold PBS in a 1.5 mL eppendorf tube and centrifuged at 1700 g for 5 min at 4  C. The cell-free hemolymph obtained as supernatant was stored at 20  C prior to spectrophotometric measurement of the enzyme activity. For determination of basal phenoloxidase (PO), 20 mL of hemolymph-buffer solution were taken and mixed with 180 mL of L-DOPA (3,4-dihydroxy-L-phenylalanine, SigmaeAldrich; 3 mg/mL in PBS) in a microtiter plate. For determination of total phenoloxidase enzyme activity, 30 mL of hemolymph-buffer solution were added to 30 mL of methanol which chemically activates PO from its inactive zymogen, prophenoloxidase (proPO). The hemolymph-methanol mixture was incubated for 5 min at room temperature and 20 mL were mixed with 180 mL of L-DOPA (3 mg/mL in PBS) in a microtiter plate. The basal and total phenoloxidase enzyme activity at 20  C was measured (in duplicate) at 492 nm for 30 min in 5 min intervals using a plate reader (Sirio S, SEAC) and was expressed as absorbance units representing an absorbance per mL of hemolymph. The plasmatic PO activity was compared between control and treated animals. The increase of absorbance units per min is linear within 30 min and the slope of the calculated linear regression represents the Vmax of the enzyme activity. The absorbance units at different times were plotted and their regression lines calculated.

3

2.5. Statistical analysis Statistical analyses were performed using R version 3.0.1 software (R Development Core Team 2013). The THC and DHC data and the optical density of iNOS immunoreactive bands in the western blot analysis were compared by both parametric statistics, i.e. Welch two sample t-test, and nonparametric statistics, i.e. Wilcoxon rank sum test, due to the small numbers of data. The box and whiskers plots were drawn with the boxplot command. The differences in slopes and intercepts of regression lines showing the basal and total PO activity as absorbance units per min were tested by analysis of covariance (ANCOVA). 3. Results 3.1. Morphological characterization of hemocytes Four types of circulating hemocytes were identified in the hemolymph of control P. melas italicus adults (Figs. 1 and 2): prohemocytes (Fig. 1A), plasmatocytes (Fig. 1B and C), granulocytes (Fig. 1D and E) and oenocytoids (Fig. 1F). Prohemocytes are the smallest circulating hemocyte types with a maximum diameter of ca. 5.5 mm and a round profile (Fig. 1A). In section, the ratio nucleus/cell surface ratio is 0.7. The cytoplasm contains a well-developed rough endoplasmic reticulum (RER) and small mitochondria. Plasmatocytes are irregularly shaped cells with a maximum diameter up to 16 mm  7.7 mm (Fig. 1B). The nucleus is large, lobed and euchromatic with a diameter ca. of 9.4 mm  2.5 mm and a prominent large nucleolus. They are characterized by numerous electron-dense granules with a mean diameter of 0.34 ± 0.1 mm (n ¼ 56) (Fig. 1B and C). The cytoplasm contains a well developed rough endoplasmic reticulum, free ribosomes, Golgi complexes and numerous elongated mitochondria. The plasma membrane exhibits irregular pseudopodia (Fig. 1B). Occasionally plasmatocytes exhibit pinocytotic vesicles (Fig. 1C). Granulocytes are oval cells with a maximum diameter up to 13 mm characterized by a number of electron-dense dishomogeneous granules with a round irregular to elliptical profile (Fig. 1D and E). The granules have a mean diameter of 0.5 ± 0.1 mm (n ¼ 36). The cytoplasm contains rough endoplasmic reticulum, Golgi complexes and elongated mitochondria. The plasma membrane is irregular, displaying filopodia on its surface (Fig. 1D). Oenocytoids are round cells, about 12 mm in diameter, characterized by an eccentric nucleus (Fig. 1F). The cytoplasm has few organelles, although small oval mitochondria, free ribosomes, numerous polysomes and a rough endoplasmic reticulum are sometimes present. The round profile of the cell shows several short filopodia. 3.2. In vivo phagocytosis assay Plasmatocytes are the only hemocyte type involved in phagocytosis in P. melas italicus adults and, in section, they present up to 40 phagocytized beads (Fig. 2AeD). Granules fusing with a phagosome are evident in the cytoplasm, demonstrating their role as primary lysosomes (Fig. 2B). Aggregated phagocytizing cells around clusters of localized latex beads (Fig. 2C and D) and beads entrapped in melanotic brown bodies (Fig. 3D) are found in response to non-self-challenge. Moreover, 2 h after the latex bead injections, macrophage-like cells were identified in the hemolymph (Fig. 3A and B), involved in the phagocytosis of melanotic brown bodies (Fig. 3D and E). This cell type is spherical, ca. 30 mm in diameter and characterized by an eccentric nucleus (ca. 14 mm in diameter). The cytoplasm has numerous, large mitochondria, rough and smooth

Please cite this article in press as: Giglio, A., et al., Immune challenges trigger cellular and humoral responses in adults of Pterostichus melas italicus (Coleoptera, Carabidae), Arthropod Structure & Development (2015), http://dx.doi.org/10.1016/j.asd.2015.01.002

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Fig. 2. Transmission electron micrographs of plasmatocytes after in vivo artificial non-self-challenge, longitudinal sections. A and B: Higher magnifications show a large number of latex beads (asterisks) in the cytoplasm included into the phagosomes (B) containing cluster or single latex beads. Many granules appear fusing with phagosomes (arrowheads). C: The longitudinal section shows the latex bead phagocytosis at the membrane level of plasmatocytes. D: Aggregate phagocytizing cells around clusters of localized latex beads.

endoplasmic reticulum, Golgi complexes, peroxisomes and a large number of electron-lucent granules. 3.3. Total and differential hemocyte counts The total circulating hemocyte counts (THCs) in treated and control adults of P. melas italicus are shown in Table 1. THCs of latex bead-challenged animals (n ¼ 5, mean ± SE: 0.65 ± 0.07  106 cells/ mL) are significantly lower than the THCs of control animals

(n ¼ 10, mean ± SE: 1.47 ± 0.21  106 cells/mL; Welch two sample ttest: p ¼ 0.0039; Wilcoxon rank sum test: p ¼ 0.0126) while there is no significant difference between PBS sham-injected (n ¼ 5, mean ± SE: 1.41 ± 0.44  106 cells/mL) and control animals (Welch two sample t-test: p ¼ 0.9139; Wilcoxon rank sum test: p ¼ 0.7679). The differential hemocyte counts (DHCs) shows that plasmatocytes are the main hemocyte type in hemolymph (76.13 ± 7.01%) of the untreated P. melas italicus adults (Table 2, n ¼ 3). Their number increases after latex beads injection (89.13 ± 3.85%) and

Fig. 1. Transmission electron micrographs of circulating hemocytes from P. melas italicus control adults. A: Prohemocyte, longitudinal section. B: Plasmatocyte, longitudinal section. C: Detail of cytoplasmic organelles of plasmatocytes, longitudinal section. D and E: Granulocytes: longitudinal (D) and transversal (E) section. Note the bundle of microtubules running just beneath the plasma membrane (D) (mt). F: Oenocytoid, longitudinal section. Abbreviations: arrowhead: electron dense granules, f: filopodia, g: Golgi complexes, mi: mitochondria, mt: microtubules, n: nucleus, nu: nucleolus, p: pseudopodia, RER: rough endoplasmic reticulum.

Please cite this article in press as: Giglio, A., et al., Immune challenges trigger cellular and humoral responses in adults of Pterostichus melas italicus (Coleoptera, Carabidae), Arthropod Structure & Development (2015), http://dx.doi.org/10.1016/j.asd.2015.01.002

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Fig. 3. Macrophage-like cells and melanized brown bodies after immune challenge with latex beads, longitudinal sections. A, B and C: Transmission electron micrographs of cells macrophage-like cells. B: detail of A showing mitochondria and cytoplasmatic vescicle. C: Melanized brown bodies embedding latex beads are identified in the hemolymph after the immune challenge (detail of D). D and E: Light micrographs of melanized brown bodies absorbed in the cytoplasm of the macrophage-like cell (C and D). Abbreviations: asterisk: latex beads, g: Golgi complexes, mi: mitochondria, n: nucleus.

Table 1 THCs in control (CTRL) and challenged adults with sterile buffer (PBS) and latex beads (LATEX). Values are shown as mean ± SE. Asterisks indicate statistically significant differences at Welch two sample t-test (p < 0.01). CTRL

PBS

Latex

*1.47 ± 0.21  106 cell/mL

1.41 ± 0.44  106 cell/mL

*0.65 ± 0.07  106 cell/mL

42.52 ± 3.36% of cells are involved in the phagocytosis 2 h after this in vivo artificial non-self challenge. Granulocytes make up 13.40 ± 5.84% of circulating cells in hemolymph of untreated adults whereas their number is much lower (6.15 ± 3.37%) after latex bead injections. The number of prohemocytes and oenocytoids is very low and does not vary after immune challenge. Nevertheless, the DHCs of untreated animals do not show significant differences from those of treated ones.

Please cite this article in press as: Giglio, A., et al., Immune challenges trigger cellular and humoral responses in adults of Pterostichus melas italicus (Coleoptera, Carabidae), Arthropod Structure & Development (2015), http://dx.doi.org/10.1016/j.asd.2015.01.002

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Table 2 Percentage value of hemocyte types after latex bead injections (latex) compared to the untreated (CTRL) specimens and injected with sterile buffer (PBS). Values are expressed Q2 as mean percentage ± SE (n ¼ 3) (Welch two sample t-test, p < 0.01).

CTRL PBS LATEX

Granulocytes

Plasmatocytes

13.40 ± 5.84 16.63 ± 5.26 6.15 ± 3.37

76.13 ± 7.01 71.03 ± 2.84 89.13 ± 3.85

Phagocytizing

Oenocytoids

Prohemocytes

Not determined

42.52 ± 3.36

1.93 ± 0.96 2.00 ± 0.36 1.49 ± 0.33

1.10 ± 0.35 1.83 ± 0.06 0.80 ± 0.41

7.40 ± 1.83 8.5 ± 2.32 2.43 ± 0.20

3.4. Hemocyte nitric oxide synthase assay Western blot analysis revealed significantly higher iNOS protein expression in the hemocytes of animals treated with LPS compared with untreated ones (Welch two sample t-test: p ¼ 0.0012; Wilcoxon rank sum test: p ¼ 0.0238) (Fig. 4). 3.5. Activity of proPO and PO The total proPO activities of both untreated and treated animals do not show significant differences in comparison to basal PO activities. The plasmatic basal PO activity of the LPS-treated animals (n ¼ 6) tends to be higher than that of untreated animals (n ¼ 13) (ANCOVA: F3,129 ¼ 8.17, p ¼ 0.056) while the plasmatic total proPO activity of treated animals is significantly higher than that of untreated ones (n ¼ 18) (ANCOVA: F3,164 ¼ 6.86, p ¼ 0.034) (Fig. 5). 4. Discussion In this study, we identified five hemocyte types in the immune system of P. melas italicus adults. According to their shape, size and ultrastructure, they were classified as prohemocytes, plasmatocytes, granulocytes, oenocytoids and macrophage-like cells. The differential hemocyte counts show that plasmatocytes and

granulocytes are the most abundant circulating types. The morphotypes of P. melas italicus are rather few compared with other insect orders, i.e. coleopteran, dipteran and lepidopteran species. In those orders, adipohemocytes, coagulocytes crystal cells, lamellocytes, spherule cells and thrombocytoids were also observed (Hillyer and Christensen, 2002; Lavine and Strand, 2002; Giulianini et al., 2003; Ribeiro and Brehelin, 2006; Araújo et al., 2008; Manfredini et al., 2008; Strand, 2008a; Manachini et al., 2011; Fors et al., 2014), However, morpho-functional studies have shown that some morphotypes are different stages of hemocyte maturation and thus data suggest that problems still remain for an exhaustive comparison between species (Gillespie et al., 1997; Ottaviani, 2005; Jiravanichpaisal et al., 2006; Strand, 2008a). Macrophage-like cells are absent in untreated adults of P. melas italicus but they appear in the hemolymph after immune challenge with latex beads. In insects, circulating macrophage-like cells are well studied mainly in Drosophila and they are involved in the engulfment of apoptotic cells during embryogenesis (Abrams et al., 1993; Honti et al., 2014; Ribeiro and Brehelin, 2006). In P. melas italicus adults, they are recruited to remove exhausted phagocytizing cells, apoptotic cells and melanotic capsules. THC and DHC values recorded in P. melas italicus only 2 h after latex bead treatment indicate that the number and type of hemocytes in circulation can rapidly change in response to an infection. Data on insect

PO and proPO activities 0.7

iNOS expression 100

*

a− untreated PO b− treated PO c− untreated proPO d− treated proPO

0.6

*

0.2

40

0.4

60

0.3

A/μl hemolymph

0.5

80

densitometric units

0.1

20

0.0

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C groups

Fig. 4. The iNOS protein expression in the hemocytes of P. melas italicus adults. The box represents the interquartile range (IQR ¼ Q3eQ1) and bars represent first (Q1, top) and third quartiles (Q3, bottom) of iNOS expression values from untreated (C) and LPStreated (T) hemocytes. The central horizontal black line indicates the median. The ends of dashed lines (ends of the whiskers) represent the lowest (minimum) datum and the highest (maximum) datum. The enzyme expression was significant higher in LPS-treated (T) hemocytes than untreated (C) ones (Wilcoxon rank sum test, *p < 0.05).

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Fig. 5. The basal (PO, a and b regression lines) and the total phenoloxidase activity (proPO, c and d regression lines) in the hemolymph of untreated and LPS-injected adults (treated) recorded as absorbance units (A) for mL of hemolymph per min. In the LPS-injected adults, the total (proPO, d) phenoloxidase activity was significantly higher than untreated ones (c) (ANCOVA, *p < 0.05). The basal PO enzymatic activity observed in LPS-injected (b) adults tends to be higher than that of untreated animals (a) (ANCOVA, p ¼ 0.056).

Please cite this article in press as: Giglio, A., et al., Immune challenges trigger cellular and humoral responses in adults of Pterostichus melas italicus (Coleoptera, Carabidae), Arthropod Structure & Development (2015), http://dx.doi.org/10.1016/j.asd.2015.01.002

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immunology show that some of these changes recorded as increases of the hemocyte number are due mainly to proliferation events from hematopoietic organs containing prohemocytes or mitotic division of circulating hemocytes (Ottaviani, 2005; Strand, 2008a). Moreover, the THC decrease after latex bead injections could be related to hemocyte involvement in cellular aggregates around clusters of localized latex beads to form melanotic capsules as well as to innate defense mechanisms such as degranulation. Phagocytosis is the primary response of hemocytes for the elimination of pathogenic microbes, removal of apoptotic cells, tissue remodeling and induction of innate and adaptive immune responses (Lavine and Strand, 2002; Ottaviani, 2005; Strand, 2008a, b; Marmaras and Lampropoulou, 2009). The hemocyte types responsible for phagocytosis differ among species of diverse insect orders. Many studies have shown that granulocytes and plasmatocytes are the primary cells responsible for phagocytosis (Tojo et al., 2000; Lavine and Strand, 2002; Ling et al., 2005; Ling and Yu, 2006a,b; Amaral et al., 2010; Manachini et al., 2011; Firlej et al., 2012). In some cases, other types of hemocytes can also perform this function, such as prohemocytes (Ling et al., 2005) and oenocytoids (Giulianini et al., 2003; Giglio et al., 2008). In this study, we used an inert target to record a cellular defense reaction (phagocytosis) not involving the action of pathogen cell surface molecules referred to as PAMPs (pathogen-associated molecular patterns) which elicit the synthesis of hemolymph proteins associated with antimicrobial responses. After the artificial non-self challenge with latex beads, it was clear that plasmatocytes are responsible for phagocytosis. Plasmatocytes of P. melas italicus exhibit a high degree of latex bead immobilization and entrapment. They become activated and latex beads are phagocytized or entrapped by melanotic capsules formed only of acellular material. We have no direct proof that hemocytes adhere to the necrotic core forming the nodule. However, our ultrastructural analyses suggest that melanotic capsules are produced in response to immune challenge and occur in conjunction with phagocytosis as a highly efficient mechanism capable of dealing with massive microbial/test particle insults. Several studies have demonstrated that NO functions as a cell signaling and immune effector molecule displaying high toxicity due to its concentration and its interaction with reactive oxygen intermediates. In the insects, nitric oxide is produced in response to microbial infection in several species of lepidopterans, hemipterans and dipterans (Nappi et al., 2000; Faraldo et al., 2005; Krishnan vez-Lao, 2010). On the basis of the et al., 2006; Hillyer and Este immune response recorded in our study of P. melas italicus adults, non-self recognition of PAMPs such as LPS triggers the proPO system and induces iNOS protein expression in hemocytes. The increase of total PO recorded in hemolymph indicates activation of the proPO system. Moreover, the LPS-stimulated hemocytes show an increased protein expression of inducible NO synthase (iNOS), indicating that the cytotoxic action of nitric oxide (NO) is engaged in this antimicrobial collaborative response. Hence we hypothesize that NO plays an important role as a cytotoxic effector molecule in the P. melas italicus immune system. Recent discussions in the field of evolutionary ecology have focused on the idea that the variability and range of immune response strategies are highly adaptive to maximize the fitness of species (Schmid-Hempel, 2003; Schmid-Hempel and Ebert, 2003; Schulenburg et al., 2009) and that there is a trade-off between immune system activation and other life-history traits. In addition, the activation and use of the immune system is costly from a physiological and evolutionary point of view (Siva-Jothy et al., 2005; Sadd and Schmid-Hempel, 2009). Moreover, each species follows a strategy under environmental selective pressure modulating the cellular and molecular mechanisms involved as first lines

of defense against pathogens. In regard to their ecological role, P. melas italicus adults are generalist predators and may encounter pathogens and parasites in their environment. This requires a rapid non-specific response to prevent entry of the pathogen through the most probable paths of the infection; the cuticle and the digestive tract offer most invasion opportunities for external injury. Our findings provide information about two aspect of immune mechanisms and their role in an ecological context. First, the framework of immune effector traits of this species, involving phagocytosis, the PO system and the iNOS action, shows that there is a synergic overlap between cellular and humoral immune responses. Hemocytes are the primary line of defenses against bacteria, engaging in phagocytosis and the production of the rate-limiting enzymes of the melanization pathway. The data presented herein on iNOS protein expression also show, for the first time, that nitric oxide is an essential component of the immune response of this ground beetle. Second, carabid beetles are well-known ecological indicators of changes in terrestrial ecosystems as a result of the human activities. Several species have been used as biological indicators of toxicant effects, as revealed by bioassays such as food consumption, reproductive rate, survivorship and growth (Koivula, 2011; Kotze et al., 2011). Even though pollutants such as pesticides are known to affect insect humoral and cellular immune responses, making the non-target insect more susceptible to disease, very little is known about this aspect in carabid species (Galloway and Depledge, 2001; James and Xu, 2012). In addition, an understanding of the baseline immune response is needed for the more common species in order to reduce mortality of beneficial insects. The limited information on the immune system of carabids shows that the immune response can be used as a highly sensitive early warning parameter to assess the sublethal toxic effects of the pollution when these effects are not evident in conventional bioassays (Giglio et al., 2011; Talarico et al., 2014). Therefore, the results of this study on P. melas italicus adults provide a spring-board for further investigation of topics such as the pathogen resistance and plasticity of the immune system in order to characterize immune responses as biomarkers in an ecotoxicological context. Acknowledgments The authors tank Sig. Enrico Perrotta for the technical assistance in electron microscopy. We thank the anonymous reviewer for helpful comments and suggestions, which significantly contributed to improve the paper quality. This work was supported by the grant (n A.001.2014.EX60) assigned to A. Giglio from the Ministery of Education, University and Research (MIUR). References Abrams, J.M., White, K., Fessler, L.I., Steller, H., 1993. Programmed cell death during Drosophila embryogenesis. Development 117, 29e43. Amaral, I.M.R., Neto, J.F.M., Pereira, G.B., Franco, M.B., Beletti, M.E., Kerr, W.E., Bonetti, A.M., Ueira-Vieira, C., 2010. Circulating hemocytes from larvae of Melipona scutellaris (Hymenoptera, Apidae, Meliponini): cell types and their role in phagocytosis. Micron 41 (2), 123e129. Araújo, H.C., Cavalcanti, M.G., Santos, S.S., Alves, L.C., Brayner, F.A., 2008. Hemocytes ultrastructure of Aedes aegypti (Diptera: Culicidae). Micron 39, 184e189. Avgin, S.S., Luff, M., 2010. Ground beetles (Coleoptera: Carabidae) as bioindicators of human impact. Munis Entomol. Zool. 5 (1), 209e215. €derha €ll, K., 2004. The prophenoloxidase-activating system in inCerenius, L., So vertebrates. Immunol. Rev. 198 (1), 116e126. Davies, S.-A., 2000. Nitric oxide signalling in insects. Insect Biochem. Mol. Biol. 30 (12), 1123e1138. Faraldo, A.C., Sa-Nunes, A., Del Bel, E.A., Faccioli, L.H., Lello, E., 2005. Nitric oxide production in blowfly hemolymph after yeast inoculation. Nitric Oxide 13 (4), 240e246. Firlej, A., Girard, P.-A., Brehlin, M., Coderre, D., Boivin, G., 2012. Immune response of Harmonia axyridis (Coleoptera: Coccinellidae) supports the enemy release hypothesis in North America. Ann. Entomol. Soc. Am. 105 (2), 328e338.

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