Phenoloxidase activity among developmental stages and pupal cell types of the ground beetle Carabus (Chaetocarabus) lefebvrei (Coleoptera, Carabidae)

Journal of Insect Physiology 59 (2013) 466–474

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Phenoloxidase activity among developmental stages and pupal cell types of the ground beetle Carabus (Chaetocarabus) lefebvrei (Coleoptera, Carabidae) Anita Giglio a,⇑, Piero Giulio Giulianini b a b

Department of Biology, Ecology and Earth Sciences, University of Calabria, Via P. Bucci, I-87036 Arcavacata di Rende, Italy Department of Life Sciences, Brain Centre, University of Trieste, Via Giorgieri 7-9, I-34127 Trieste, Italy

a r t i c l e

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Article history: Received 3 October 2012 Received in revised form 22 January 2013 Accepted 24 January 2013 Available online 4 February 2013 Keywords: Ecological immunology Hemocytes Life cycle Phagocytosis Microscopy

a b s t r a c t In ecological immunology is of great importance the study of the immune defense plasticity as response to a variable environment. In holometabolous insects the fitness of each developmental stage depends on the capacity to mount a response (i.e. physiological, behavioral) under environmental pressure. The immune response is a highly dynamic trait closely related to the ecology of organism and the variation in the expression of an immune system component may affect another fitness relevant trait of organism (i.e. growth, reproduction). The present research quantified immune function (total and differential number of hemocytes, phagocytosis in vivo and activity of phenoloxidase) in the pupal stage of Carabus (Chaetocarabus) lefebvrei. Moreover, the cellular and humoral immune function was compared across the larval, pupal and adult stages to evaluate the changes in immunocompetence across the developmental stages. Four types of circulating hemocytes were characterized via transmission electron microscopy in the pupal stage: prohemocytes, plasmatocytes, granulocytes and oenocytoids. The artificial non-self-challenge treatments performed in vivo have shown that plasmatocytes and granulocytes are responsible for phagocytosis. The level of active phenoloxidase increases with the degree of pigmentation of the cuticle in each stage. In C. lefebvrei, there are different strategies in term of immune response to enhance the fitness of each life stage. The results have shown that the variation in speed and specificity of immune function across the developmental stages is correlated with differences in infection risk, life expectancy and biological function of the life cycle. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction The invertebrate immune response is based on both cellular and humoral components (Ottaviani, 2005). Cellular immunity refers to responses such as phagocytosis, nodulation and encapsulation which are carried out by hemocytes (Lavine and Strand, 2002; Strand, 2008a,b). The humoral components are antibacterial proteins (i.e. phenoloxidase) and other immune-related molecules secreted in the plasma by hemocytes against bacteria and parasites (Gillespie et al., 1997). Measurements of immune function in animals have shown that it is essential to consider immunocompetence in an ecological and evolutionary context. These aspects of immunity have been reviewed extensively and can be summarized as follows: (i) immune function is specific to the species and varies according to ecological parameters; (ii) immune systems impose evolutionary and mainte⇑ Corresponding author. Tel.: +39 0984 492982; fax: +39 0984 492986. E-mail address: [email protected] (A. Giglio). 0022-1910/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jinsphys.2013.01.011

nance costs on different levels; (iii) trade-offs exist between immune defenses and life-history traits (reproduction, growth and development) in order to maximize an animal’s fitness (Lazzaro and Little, 2009; Rolff and Siva-Jothy, 2003; Sadd and SchmidHempel, 2009; Schmid-Hempel, 2003, 2005; Schmid-Hempel and Ebert, 2003; Schulenburg et al., 2009). Moreover, many organisms increase their fitness against parasites and pathogens by mounting a non-immunological defense pre- or post-infection, such as behavioral mechanisms, symbiont-mediated immunity and fecundity compensation (Cotter et al., 2010; Rolff and Siva-Jothy, 2004; Parker et al., 2011). The relationship between immune response and an organism’s life history has been defined and measured in very different ways in invertebrates, including insects (Boughton et al., 2011; Marmaras and Lampropoulou, 2009; Schmid-Hempel, 2005; Siva-Jothy et al., 2005). However, although it is well known that species with a complex life cycle show changes in their ecology and physiology, the ability to mount an immune response across developmental stages has not been thoroughly investigated. In holometabolous insects, the larval stage (concerned with feed-

A. Giglio, P.G. Giulianini / Journal of Insect Physiology 59 (2013) 466–474

ing and growth) is separated ecologically from the adult stage (concerned with reproduction and dispersion) by metamorphosis to avoid resource competition and to increase the species fitness at each stage. As a result, the influence of ecological factors on the life cycle and the immunological outcomes varies both across and within individuals (Gillott, 2005; Gullan and Cranston, 2010) in response to factors such as age, dietary quality and quantity, and reproductive effort (Chambers and Schneider, 2012; De Block and Stoks, 2008; Shi and Sun, 2010; Stoehr, 2007; Thomas and Rudolf, 2010; Wilson-Rich et al., 2008). Studies on insect immunity have focused on larval or adult stages with regard to their economic or ecological value, but few studies have been conducted on pupal stages. The present study investigated the poorly documented immune defenses in pupal stages of ground beetles by comparing the variation in humoral and cellular responses across the developmental stages. Carabid beetles are among the most important groups of terrestrial predators in many ecosystems. They are mostly polyphagous, although some species may be oligophagous and specialized feeders. They are well known as bioindicators and natural enemies of agricultural pests (Avgin and Luff, 2010; Luff, 1983; Rainio and Niemelä, 2003). Because of their bio-indication and economic importance, the role of biotic and abiotic factors in carabid population dynamics has been studied extensively. Abiotic factors and predation are the main causes of mortality for all carabid beetle life-cycle stages, while pathogens and parasites can be important for larvae and pupae which have less chitinization and limited or absent mobility (Lovei and Sunderland, 1996; Luff, 2003; Turin et al., 2003). However, information on immunocompetence is necessary to complete the ecological framework of ground beetle species. The object of this study is Carabus lefebvrei Dejean, 1826, an Italian endemic species that lives in beech, oak, chestnut and pine forests of the Central and Southern Apennines, from lower altitudes to about 1500 m a.s.l. It reproduces in spring, is active from April until September and hibernates as adults. Eggs are laid in humus-rich soils in April–June and larvae are active on the surface of the leaf litter from June to August (Thiele, 1977; Turin et al., 2003). Larvae molt to the pupal stage in a cell dug in the ground by the 3rd instar larva. The pupa is exarate and is able to defend itself using a defensive secretion produced by abdominal glands (Giglio et al., 2009). The habit of adults and larvae is typically that of a snail-eating predator: adults have a prolonged and narrowed head as in Cychrus spp. (cychrization) and larvae have broad tergite projections to protect the abdominal spiracles (Giglio et al., 2012; Turin et al., 2003). Previous studies on both the adult and larval immune system have shown four morphotypes of circulating hemocytes: prohemocytes, granulocytes, oenocytoids and plasmatocytes. Moreover, C. lefebvrei shows a non-specific immune response involving phagocytosis performed by plasmatocytes in both adults and larvae and by oenocytoids in larvae (Giglio et al., 2008). To evaluate the immune function in the pupal stage, we measured a set of immune variables: total and differential number of hemocytes and phagocytosis after in vivo artificial non-self-challenges as a trait of cellular response; the activity of phenoloxidase as a component of the humoral defense. Moreover, based on the ecology and life habit of C. lefebvrei, the results were compared across the larval, pupal and adult stages to evaluate the changes in immunocompetence across the developmental stages.

2. Material and methods 2.1. Sample rearing and hemolymph collection C. lefebvrei males and females were hand-collected from under rotten pine bark in the Catena Costiera Mountains (39°190 N,

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16°70 E, 900–1000 m a.s.l.; southern Italy, Calabria) in early spring 2010. In the laboratory, the beetles were sexed and kept in groups (males and females) in 10 L plastic boxes filled to a depth of 6.0 cm with moistened humus. After copulation, males were removed from the boxes to reduce the disturbance to females, which readily laid eggs. The eggs were transferred singly into 150 ml glass jars filled to a depth of 4.0 cm with moistened humus. Egg production and larval developmental time were recorded every 2 days. Adults and larvae were reared with a light regime of L/D = 15/9 h, 70% RH and a day/night room temperature of 23/18 °C. All specimens were fed with snails. The animals used for this study were last (3rd) instar larvae, 10-day-old pupae and 10day-old adults. The animals were CO2 anesthetized before hemolymph collection. The hemolymph was collected from pupae and larvae by puncturing the soft cuticle between the second and third dorsal abdominal segment at the level of the hemolymphatic vessel with a 26-gauge needle. Adults were punctured at the ventral level of the pro-mesothorax articulation. The first droplet of about 50 lL of hemolymph was collected.

2.2. In vivo phagocytosis assay To assess the ability of pupal hemocytes to phagocytize, we used a 26-gauge needle to inject 20 lL of carboxylate-modified polystyrene latex beads (0.9 lm in diameter, aqueous suspension, 10% solids content, Sigma) diluted 1:10 in 0.15 M sterile phosphate buffered saline (PBS, Sigma) into the abdomen of five pupae at the level of the dorsal hemolymphatic vessel. Parallel controls were run with five specimens. After 2 h, 20 lL of hemolymph were collected from treated and control pupae, transferred to a 1 mL microcentrifuge tube with a solution containing 2.5% glutaraldehyde, 1% paraformaldehyde and 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 of untreated and latex bead-treated animals were pelleted by 5000 rpm centrifugation for 10 min. The pellets obtained from pooled hemolymph of the five animals were then post-fixed 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, 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. For transmission electron microscopy, images were acquired with a Veleta – 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 deviation.

2.3. Hemocyte counts For total hemocyte counts (THCs), 5 lL of hemolymph were collected and hemocytes were counted in a Bürker’s chamber (Carlo Erba, Italy) without dilution, observed under differential interferential contrast (DIC) in light microscopy (LM) at 40 magnification. THC was expressed as the number of cells per mL. For differential hemocyte counts (DHCs), semi-thin sections from pellets, obtained as described in the previous section, were stained by the Humphrey and Pittman method (Mazzi, 1977) and observed under a light microscope (Zeiss AXIOSKOP). Images were acquired with a Nikon Coolpix 4500 camera. Five DHCs were carried out by two different operators from 2 lm semi-thin sections (from three slides at the beginning, center and end of the pellet).

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2.4. Plasmatic phenoloxidase (PO) assay Phenoloxidase was measured using the procedure of Krishnan et al. (2006). Five microliters of whole hemolymph were diluted with 95 lL of 10 mM sterile phosphate buffered saline (PBS, Sigma) and centrifuged at 5000g at 4 °C for 5 min. Forty microliters of hemolymph-buffer supernatant were taken and mixed with 160 lL of DL-DOPA (3,4-dihydroxy-DL-phenylalanine, Sigma; 3 mg/mL phosphate buffer) in a microtiter plate. PO activity at 25 °C was measured (in duplicate) at 492 nm for 30 min in 5 min intervals using a plate reader (Sirio S, SEAC). Enzyme activity was expressed as absorbance units representing an absorbance for lL of hemolymph. 2.5. Statistical analyses THCs of larvae, pupae and adults were checked for normality with the Shapiro–Wilk test and the homogeneity of variance among stages was checked with the Bartlett test. The differences among THCs were assessed by nonparametric statistics, i.e. Kruskal–Wallis rank sum test followed by post-hoc Wilcoxon rank sum test pairwise comparisons with Bonferroni correction, since the null hypothesis of the Bartlett test could not be rejected. The box and whiskers plots were drawn with the boxplot command. For the comparison of hemolymph plasmatic PO activity, the differences among larvae, pupae and adults were assessed by enzymatic activity with DL-DOPA. 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 enzymatic activity. The absorbance units at different time were plotted for each stage and their regression lines calculated. The differences in slopes and intercepts of regression lines showing the PO activity as absorbance units per min were tested by means of analysis of covariance (ANCOVA). Statistical analyses were performed using R version 2.13.1 software (R Development Core Team 2011). 3. Results 3.1. Hemocyte types and morphology Four morphological types of circulating cells were identified in the hemolymph of C. lefebvrei pupae: prohemocytes (Fig. 1A),

plasmatocytes (Fig. 1B), granulocytes (Fig. 1C) and oenocytoids (Fig. 1D). Prohemocytes are the smallest cells found in the hemolymph and display a spherical profile with a maximum diameter of about 5 lm (Fig. 2A). The nucleus almost fills the cell and the nucleus/cell surface ratio is 0.6 in section. The cytoplasm is basophilic (blue color) in LM and contains a well developed rough endoplasmic reticulum and small mitochondria. The plasmatocytes are irregularly shaped cells with a maximum diameter up to 16 lm (Fig. 2B–D). They are characterized by numerous electron dense granules, slightly acidophilic in LM, with a mean diameter of 0.23 ± 0.078 lm (N = 95). The nucleus is large (5.31 ± 1.10  2 ± 0.90 lm, N = 11), lobed and euchromatic with a prominent large nucleolus. The rough endoplasmic reticulum and the Golgi complexes are well developed. Numerous elongated mitochondria with tabular cristae are observed. The plasma membrane exhibits irregular pseudopodia. Occasionally plasmatocytes exhibit pinocytotic vesicles, multilamellar bodies, multivesicular bodies and electron-lucent vesicles 0.8 ± 0.4 lm (N = 51) in diameter (Fig. 2B and E). Granulocytes are oval cells with a maximum diameter up to 13 lm (Fig. 2F and G). They are characterized by a number of electron-dense dishomogeneous granules with a round irregular to elliptical profile. The granules are acidophilic in LM and have a mean diameter of 0.83 ± 0.48 lm (n = 36). The cytoplasm contains rough endoplasmic reticulum, Golgi complexes and elongated mitochondria. The plasma membrane is irregular, displaying filopodia on its surface. Oenocytoids are rare compared to the other cell types encountered in the hemolymph (Fig. 3A and B). They are round cells, about 12 lm in diameter, characterized by an eccentric nucleus. The cytoplasm has few organelles and is acidophilic in LM, although small oval mitochondria, free ribosomes, numerous polysomes and a rough endoplasmic reticulum are sometimes present. Other peculiar circulating cells are observed in the pupal stage. The first type is about 8 lm in diameter with a lobate nucleus (Fig. 3C). The cytoplasm contains rough endoplasmic reticulum, Golgi complexes, a large number of elongated mitochondria and electron-transparent granules. The plasma membrane is regular with filopodia on its surface. The second type is about 14 lm in diameter with large lipid droplets in the cytoplasm (Fig. 3D). The last type is an apoptotic hemocyte showing chromatin condensation and nuclear fragmentation (nuclear material surrounded by an intact nuclear membrane) (Fig. 3E). The granulocytes recognize and phagocytize these apoptotic cells (Fig. 3E). 3.2. Hemocyte phagocytic responses Plasmatocytes and granulocytes of pupae were able to phagocytize latex beads 2 h after the injection (Fig. 4A–D). We observed both hemocyte types with up to 30 phagocytized beads within the cytoplasm. In plasmatocytes, granules fusing with a phagosome are evident, demonstrating their role as primary lysosomes (Fig. 4C). Oenocytoids with internalized latex beads were not found, suggesting that this hemocyte population does not perform phagocytosis in the pupal stage. 3.3. Total and differential hemocyte counts

Fig. 1. Light microscope semithin sections of prohemocytes (A), granulocytes (B), plasmatocytes (C) and oenocytoid (D) of C. lefebvrei pupa. Scale bar: 5 lm.

The circulating hemocyte numbers in larvae, pupae and adults are shown in Fig. 5. THCs of pupae (n = 7, mean ± SE = 36.378.571 ± 4.873.622 hemocytes/mL) are significantly higher than the THCs of both larvae (n = 5, mean ± SE = 4.852.000 ± 1.760.691; ⁄⁄p = 0.0076) and adults (n = 5, mean ± SE = 12.774.000 ± 2.022.767; ⁄p = 0.0152). No significant difference was found between larval and adult THCs.

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Fig. 2. Transmission electron microscopy of hemocytes from C. lefebvrei pupa. (A) prohemocyte showing a high nucleus/cell surface ratio. (B and C) longitudinal section of plasmatocyte. (D and E) transversal section of plasmatocytes. (F and G) granulocytes. arrowheads: Golgi complexes, asterisks: granules, v: electron lucent vesicles, ly: lysosomes, m: mitochondria, mlb: multilamelar body, mv: multivasicular body, n: nucleus, nu: nucleolus, p: pseudopodia, pv: pinocytotic vesicles, rer: rough endoplasmic reticulum. Scale bars: 1 lm (A), 5 lm (C and D), 2 lm (B and E–G).

Table 1 shows the relative percentages (DHC) of hemocyte types (counted in 2 lm semi-thin sections of pellets under LM) in latexbead treated and untreated C. lefebvrei pupae. Plasmatocytes are

the main hemocyte type; the percentage in control animals is higher than in treated animals, although there is no difference if the percentage of phagocytizing plasmatocytes is added. The same

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Fig. 3. Transmission electron microscopy of hemocytes from C. lefebvrei pupa. (A and B) oenocytoid. (C and D) activated hemocytes, (E): apoptotic hemocyte. asterisks: granules, v: electron-transparent vesicles, l: lipid droplets, m: mitochondria, n: nucleus, p: philopodia, rer: rough endoplasmic reticulum. Scale bars: 2 lm (A and C), 1 lm (B and D), 4 lm (E).

behavior was recorded for granulocytes: the control animals show a higher percentage than latex bead-treated animals, but not if phagocytizing granulocytes are added. 3.4. Phenoloxidase (PO) activity The PO activity of the animals varies significantly at different stages (ANCOVA: F5,386 = 22.19, p < 0.0001). The pupae presented a highly significant lower PO activity than the adults (ANCOVA: p = 0.000342; nadults = 21, nlarvae = 19, npupae = 16) (Fig. 6), whilst the enzymatic activity does not differ significantly between larvae and pupae and between larvae and adults. 4. Discussion This paper provides a first description of the pupal immunocompetence in C. lefebvrei. The experimental results support our hypothesis that cellular responses and PO activity levels vary among the three life stages of this holometabolous ground beetle.

In an ultrastructural analysis, we characterized four types of circulating hemocytes in the pupal stage and classified them as prohemocytes, plasmatocytes, granulocytes and oenocytoids as in previous studies on adult and larval immunity (Giglio et al., 2008). Morphological criteria have been set up to describe hemocytes in studies supporting the ‘‘multiple-cell theory’’ (Brehelin et al., 1978; Ribeiro and Brehelin, 2006; Strand, 2008a,b). This theory suggests that hemocytes are populations of separate immutable cell lines differentiating from a single stem cell forming the first population of hemocytes produced during embryogenesis. In C. lefebvrei, the number of cellular morphotypes is rather low compared with other insect species described thus far. Our results are in agreement with the data on insect immunology supporting a limited number of hemocytes involved in cellular defense, and the various hemocyte types are considered stages with separate functions of a single cell line derived from prohemocytes (Chain et al., 1992; Manfredini et al., 2008). This so-called ‘‘single-cell theory’’ suggests that the high number of indeterminate cells (Figs. C– E; Table 1) found in the differential hemocyte count of C. lefebvrei pupae are transitional stages of hemocyte types. Moreover, they

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Fig. 4. Transmission electron microscopy of hemocytes from C. lefebvrei pupa after in vivo artificial non-self-challenge. (A and B) Plasmatocytes showing phagosomes containing clusters or single latex beads. (C) Detail of the plasmatocyte cytoplasm showing some electron-dense granules (head arrow) fusing with a phagosome. (D) granulocyte showing latex beands in the cytoplasm. arrow: latex beads, asterisks: granules, head arrows: lysosomes, n: nucleus, nu: nucleolus. Scale bars: 2 lm (A, B, D), 1 lm (C).

could be activated hemocytes involved in the first stages of nodule formation. The differential hemocyte counts after in vivo artificial non-selfchallenge treatments show that plasmatocytes and granulocytes are the most abundant circulating cell types and are responsible for phagocytosis. As described for adults and larvae, the pupal plasmatocytes are large, flattened, elongated circulating cells with many small lysosomes in the cytoplasm, while the granulocytes have numerous granules in the cytoplasm that are twice the diameter of those in adults and larvae (0.46 ± 0.12 lm, n = 10; Giglio et al., 2008). Many studies have shown that granulocytes and plasmatocytes are the primary cells responsible for phagocytosis (Amaral et al., 2010; Huang et al., 2010; Firlej et al., 2012; Lavine and Strand, 2002; Ling et al., 2005; Ling and Yu, 2006a,b; Manachini et al., 2011; Tojo et al., 2000). In some cases, other types of hemocytes can also perform this function, such as prohemocytes (Ling et al., 2005) and oenocytoids (Giglio et al., 2008; Giulianini et al., 2003). From an ecological prospective, phagocytosis is a non-specific cellular response that plays a key role against parasites and parasitoids (Lavine and Strand, 2002; Marmaras and Lampropoulou, 2009; Ottaviani, 2005; Strand, 2008b). In C. lefebvrei, microorganisms entering the hemocoel must contend with a high

percentage of phagocytizing hemocytes in all life stages, although the phagocytic capacity of hemocytes varies among the stages. Plasmatocytes are the main phagocytic hemocytes in each stage and they cooperate with oenocytoids in the larval stage and granulocytes in the pupal stage. The humoral defense components of hemolymph are a variety of effectors either constitutive or inducible with a wide spectrum of biological activity involved against various pathogens (Gramnegative and -positive bacteria, fungi, yeasts, parasites, virus) (Nappi and Ottaviani, 2000; Li et al., 2012). We have focused on prophenoloxidase (proPO)-activating system that is, as well as phagocytosis, one important element of the constitutive immune response involved in an efficient non-self recognition system that recognize and respond to lipopolysaccharides or peptidoglycans from bacteria and b-1,3-glucans from fungi. An effector of the proPO cascade is the phenoloxidase (PO) enzyme (Cerenius and Söderhäll, 2004; González-Santoyo and Córdoba-Aguilar, 2012; Nappi and Christensen, 2005; Siva-Jothy et al., 2005), that is expressed as inactive zymogens and converted to active PO when required (Ling and Yu, 2005). It is present in plasma or hemocytes (or in both) as well as in midgut epithelial cells and cuticle. The total PO activity can be considered an indicator of immunocompetence

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midgut (Barnes and Siva-Jothy, 2000; Wilson et al., 2001). We spectrophotometrically quantified only the active PO inside the cell-free hemolymph, measuring the conversion of the substrate DL-DOPA into dopachrome, and compared the enzymatic activity among the life stages. The level of active PO seems to be correlated with the degree of pigmentation of the cuticle in all stages. Adults and larvae have a high degree of exoskeleton pigmentation and show high PO activity, decreasing the ability of fungal and bacterial proteases to hydrolyze cuticular proteins. Instead, very low PO activity levels occur in the pupal stage in contrast to the high number of hemocytes. The exarate pupa is a vulnerable, non-feeding instar that activates the production and maintenance of dietdependent metabolic systems such as PO only if required, e.g. to repair a wound. Moreover, it lacks physical protection such as a thick, tanned cuticle to block pathogens occurring in the soil of its habitat. Therefore, to increase individual fitness, the larval-pupal molt occurs in a protective cell dug in the ground by the 3rd instar larva, and the pupa obtains additional protection by mounting a non-immunological defense involving chemical protection via abdominal gland secretions. Monoterpenes, especially linalool, are the major volatile chemical compounds of this secretion with antimicrobial and antifungal activity. Hence, monoterpenes saturate the air of the subterranean pupal cell, providing a prophylactic function against pathogens (Giglio et al., 2009). From an evolutionary ecology perspective, the variability and range of immune response strategies are highly adaptive in order to maximize the fitness of species (Schmid-Hempel, 2003; Schulenburg et al., 2009). The costs and trade-offs of immunity can be mapped according to the tactics of use (constitutive versus induced defenses) and the degree of specificity of response (Schmid-Hempel and Ebert, 2003). Besides, in holometabolous insects the mechanisms of immunity are adapted to the specific needs of each life stages. The findings illustrated recently in feeding larvae and immobilized pupae of Galleria mellonella (Meylaers et al., 2007), Manduca sexta (Beetz et al., 2008), Apis mellifera (Wilson-Rich et al., 2008) and Dendroctonus valens (Shi and Sun, 2010) show that

THC

5e+07

hemocytes/mL

4e+07

3e+07

2e+07

1e+07

0e+00 adult

larva

pupa

stage

Fig. 5. Box and whiskers plots of THCs in C. lefebvrei adult, larva and pupa.

only if the variation in enzymatic activity is related to gender, life stage, seasonality and host condition of the species (González-Santoyo and Córdoba-Aguilar, 2012; Laughton et al., 2011) because the PO may be used for other functions such as melanogenesis, a series of diet-dependent chemical reactions involved in cuticle pigmentation, molting, tissue repair and defense against pathogens (Gillespie et al., 1997; Rolff and Siva-Jothy, 2003; Schmid-Hempel, 2005; Siva-Jothy et al., 2005). In many species the degree of cuticular melanization is a strong indicator of resistance to pathogens and is correlated with PO activity in the cuticle, hemolymph and

Table 1 Percentage variation of hemocyte types in C. lefebvrei pupae after carboxylated bead injections (latex) compared to the controls. Values are expressed as mean ± SE. Granulocytes CTRL LATEX

16.54 ± 4.37 3.20 ± 1.17

Phagocyting

Plasmatocytes

7.64 ± 2.14

70.54 ± 3.57 48.30 ± 7.46

larva III instar PO activity

Phagocyting

Oenocytoids

Prohemocytes

Not determined

30.60 ± 4.32

1.92 ± 1.08 1.04 ± 0.64

1.40 ± 0.23 1.06 ± 0.36

9.62 ± 2.19 8.12 ± 2.66

pupa PO activity

adult PO activity ● ●

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0.25

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0.20

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0.15

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Fig. 6. Changes of phenoloxidase activity recorded as absorbance units per min in relation to life stages, the regression lines are shown (nadult = 21, nlarva = 19, npupa = 16).

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the costs to mounting an immune response implicate a trade-off between immunity and physiological traits of development. Therefore, each species adopts diverse strategies in immunocompetence during different developmental stages and the variation of one or more cellular and humoral components is both closely related to lifespan of each stage and is an integral aspect of life-history of species. Contrary to larvae and pupae, adults need to find a balance for resource allocation between survival and reproduction. Moreover, the cost of mounting an immune response against pathogens may be sex-specific having males and females different life-histories. The sexual dimorphism has been shown in the components of insect immune system, such as PO and lysozyme-like activity, hemocyte number, and encapsulation assay (Adamo et al., 2001; Schwarzenbach et al., 2005; Meylaers et al., 2007; Beetz et al., 2008; Shi and Sun, 2010). In C. lefebvrei, further investigations are needed to test trade-off between immunity and reproduction. The results of this study show that differences recorded in adults, larvae and pupae for the total hemocyte number, the cells involved in phagocytosis and the PO activity are not necessarily interrelated because each life stage has a different strategy to enhance its fitness in relation to the life history traits of the species. The data reflect a programmed change in immune function across the developmental stages (from cellular-based to PO-based immunity) which varies in speed and specificity and is correlated with differences in infection risk, life expectancy and biological function of the life cycle. The non-specific, constitutive, fast immune response of C. lefebvrei is related both to differences among the pupal, larval and adult stages in physiological flexibility and functional versatility in the use of habitat resources and to the prevailing selection pressures exerted by pathogens in the natural habitat of each stage. The cuticle is an efficient barrier against pathogens in adults and larvae, even though the adult needs to find a balance between the application of resources for survival and reproduction while the larva use its resources only for survival, mounting and maintaining an efficient immune response. The pupa occupies a radically different ecological niche with respect to the adult and larval stages. The pupa is quiescent but metabolically active and more sensitive to biotic factors such as predators and pathogens. It compensates for the lack of behavioral avoidance both with a greater hemocyte load, which plays a crucial role in the dramatic tissue remodeling during metamorphosis (Thomas and Rudolf, 2010), and with a chemical barrier. Acknowledgements The authors are grateful to Prof. P. Brandmayr for his stimulating suggestions concerning the topic of carabid beetles and to Mr. Enrico Perrotta for technical support. This research was supported by grants assigned to A. Giglio from the Ministry of Education, University and Research (MIUR). References Adamo, S.A., Jensen, M., Younger, M., 2001. Changes in lifetime immunocompetence in male and female Gryllus texensis (formerly G. integer): trade-offs between immunity and reproduction. Animal Behaviour 62, 417–425. 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, 123–129. Avgin, S.S., Luff, M., 2010. Ground beetles (Coleoptera: Carabidae) as bioindicators of human impact. Munis Entomology and Zoology 5, 209–215. Barnes, A.I., Siva-Jothy, M.T., 2000. Density-dependent prophylaxis in the mealworm beetle Tenebrio molitor L. (Coleoptera: Tenebrionidae): cuticular melanization is an indicator of investment in immunity. Proceedings of the Royal Society B: Biological Sciences 267, 177–182. Beetz, S., Holthusen, T.K., Koolman, J., Trenczek, T., 2008. Correlation of hemocyte counts with different developmental parameters during the last larval instar of

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