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Insect Science (2015) 22, 748–760, DOI 10.1111/1744-7917.12141

ORIGINAL ARTICLE Modulation of immune responses of Rhynchophorus ferrugineus (Insecta: Coleoptera) induced by the entomopathogenic carpocapsae (Nematoda: )

Maristella Mastore1, Vincenzo Arizza2, Barbara Manachini2 and Maurizio F. Brivio1 1Department of Theoretical and Applied Sciences, University of Insubria, Varese, Italy and 2Department of Sciences and Biological, Chemical and Pharmaceutical Technologies, University of Palermo, Palermo, Italy

Abstract Aim of this study was to investigate relationships between the red palm (RPW) Rhynchophorus ferrugineus (Olivier) and the entomopathogenic nematode Stein- ernema carpocapsae (EPN); particularly, the work was focused on the immune response of the insect host in naive larvae and after infection with the EPN. Two main immunological processes have been addressed: the activity and modulation of host prophenoloxidase- phenoloxidase (proPO) system, involved in melanization of not-self and hemocytes recog- nition processes responsible for not-self encapsulation. Moreover, immune depressive and immune evasive strategies of the parasite have been investigated. Our results suggest that RPW possess an efficient immune system, however in the early phase of infection, S. carpocapsae induces a strong inhibition of the host proPO system. In addition, host cell- mediated mechanisms of encapsulation, are completely avoided by the parasite, the elusive strategies of S. carpocapsae seem to be related to the structure of its body-surface, since induced alterations of the parasite cuticle resulted in the loss of its mimetic properties. S. carpocapsae before the release of its symbiotic bacteria, depress and elude RPW immune defenses, with the aim to arrange a favorable environment for its bacteria responsible of the septicemic death of the insect target. Key words encapsulation; immune depression; immune evasion; proPO system; Rhyn- chophorus ferrugineus; Steinernema carpocapsae

Introduction Japan (Roda et al., 2011; Huang, 2013). As reported, the RPW is one of the most serious pests affecting at least 19 The international trade of plants and goods around the palm species (EPPO, 2008; Dembilio et al., 2009; Wang world may cause the unintentional diffusion of various et al., 2013), including ornamental palms and species of alien insect pests in new areas, this is the case of Rhyn- economic interest such as coconut, date, and oil palms. chophorus ferrugineus (Coleoptera: Curculionidae), also RPWs is wood-boring insect, often attack healthy palms named red palm weevil (RPW). RPW is native to South- and larvae feed on the soft fibers and on apical meristem east Asia but today it is distributed in almost all Mediter- of the palms creating massive damage to palm tissues, ranean areas, China, the United States, Middle East, and weakening the structure of the palm trunk and leading to the death of infested palms. The resulting cutting down Correspondence: Maurizio Francesco Brivio, Department of of infested trees is very significant in countries where Theoretical and Applied Sciences, University of Insubria, via palm trees are ornamental, changing the landscape; also, JH Dunant 3, Varese 21100, Italy. Tel: +390332421403; email: the damage is economically considerable where palms are [email protected] grown as crops.

748 C 2014 Institute of Zoology, Chinese Academy of Sciences Rhynchophorus ferrugineus immune responses 749

In order to limit the uncontrolled diffusion of this et al., 1997; Bowen et al., 1998, ffrench-Constant et al., insect pest, chemical, biological, and physical methods 2007a,b). Beyond the action of bacteria, physiological are being tested (Gitau et al., 2009). The efficacy of interactions between the parasite and its host must be chemical treatments has been evaluated when applied carefully considered; the life-cycle of an endoparasite to the soil, when injected into palm trunks or sprayed is strongly related to its ability to evade and/or to sup- on the foliage, procedures currently used to control RPW press host immune responses in the early phase of infec- are largely based on dispersal of large quantities of chem- tion. A successful infection is subjected to interactions ical pesticides, although these treatments cause great con- with the host, which can culminate in the undisturbed cern for the environment and health which is not advisable growth and reproduction of the parasite inside the host in the urban areas (Faleiro, 2006). However, the cryptic body (Toubarro et al., 2010, 2013; Castillo et al., 2011). habitat of RPW during its life-cycle makes these treat- Insects are able to maintain self-integrity discriminating ments ineffective unless they are extensive and protracted self from not self by means of a powerful immune sys- over long periods (Ferry & Gomez, 2002). tem (Schmid-Hempel, 2005); consequently, endoparasites An alternative to chemical treatments could be the use have to overcome the host immune defenses to complete of biocontrol agents such as bacteria, fungi, or ento- their life-cycles (Loker, 1994, Loker et al., 2004; Carton mopathogenic . Biological control with Bacil- et al., 2008; Schmid-Hempel, 2012). lus thuringiensis (Bt) is now common in many agricultural Invertebrates (i.e., insects) lack finely tuned im- systems, besides the troubles related to the concealed habi- munorecognition receptors (antibodies and B and T cells tat of the RPW, a major weakness in the use of Bt is the are absent) but they possess a pool of cellular and hu- onset of resistance to the bacterial toxin in the host target moral Pattern-Recognition Receptors (PRRs) able to in- populations (Pardo-Lopez et al., 2013). The use of fungi teract specifically with a broad range of foreign antigenic as biopesticides is now well established (Pedrini et al., surface compounds, commonly named PAMPs (pathogen- 2007), to date, few studies about pathogenic fungi with associated molecular patterns). PAMPs-PRRs interaction specific effects versus RPW have been carried out is a key process of the discriminatory steps of innate im- (Dembilio et al., 2010a; Guerri-Agullo,` 2010). At present, munity that usually lead to effectors mechanisms respon- the most promising biological agents against RPW seem sible for the elimination of not self (Medzhitov, 2001; to be entomopathogenic nematodes (EPNs). Several stud- Janeway & Medzhitov, 2002; Kanost et al., 2004). Insect ies have been performed with EPNs, these parasites spend immune responses involve both humoral and cellular de- most of the life-cycle within insects body and through- fenses mechanisms (Hoffmann et al., 1996; Janeway & out this endoparasitic phase, they kill the host and repro- Medzhitov, 2002; Hoffmann, 2003); humoral defenses in- duce (Llacer´ et al., 2009; Dembilio, 2010b; Triggiani & clude processes such as, antimicrobial peptides synthesis, Tarasco, 2011). EPNs are safe for both the environment lectin-mediated recognition and melanization (also called and vertebrates and the operating cost is now reduced by humoral encapsulation), the latter is consequent to the ac- mass production in liquid media (Ehlers, 2003). With re- tivation of the prophenoloxidase-phenoloxidase (proPO) gard to RPW, the cryptic habitat inside the palm seem system (Cerenius & Soderhall,¨ 2004; Wang & Jiang, 2004; could be favorable to EPNs action, since this dark and wet Xue et al., 2006; Cytrynska´ et al., 2007). Moreover, main environment minimize nematode damage due to ultravio- cellular processes such as phagocytosis and encapsulation let light and dehydration (Mracek, 2003). Temperature is are carried out by immunocompetent cells called hemo- also an important factor sometimes limiting the efficacy cytes (Ribeiro et al., 1999; Ribeiro & Brehelin, 2006; Tojo of the parasite, for instance, warm habitats of Mediter- et al., 2000; Lavine & Strand, 2002). ranean areas suggest the use of EPNs as Steinernema car- Though, both proPO system activity and cellular encap- pocapsae, which seems to be more effective under these sulation are the more effective defenses processes against conditions (Kaya & Stock, 1997; Llacer´ et al., 2009). foreign multicellular organisms, in previous works we The EPNs are mutualistically associated to bacteria, described how the entomoparasite Steinernema feltiae particularly, S. carpocapsae is symbiotic with the bac- is able to escape from immunological detection of the terium nematophila and the infective 3rd host Galleria mellonella (Lepidoptera: Pyralidae), avoid- juvenile stage of the nematocomplex (stage IJ3) reaches ing hemocytes encapsulation and inhibiting the activity the hemocoel cavity of its host in a range from 1 to 2 h. of the host proPO system (Brivio et al., 2002, 2004; Ma- When parasites are inside the hemocoel, symbiotic bac- store & Brivio, 2008). The majority of studies regard- teria are released and the subsequent death of the in- ing the interaction between EPNs and immune host sys- sect is mainly due to bacteria-induced septicaemia in the tem, are focused on lepidopteran and dipteran species (Li late phase of infection (Akhurst & Dunphy, 1993; Ehlers et al., 2007; Castillo et al., 2013), few data are available

C 2014 Institute of Zoology, Chinese Academy of Sciences, 22, 748–760 750 M. Mastore et al. regarding the huge order of Coleoptera. Although studies a formula based on apple slices and only healthy lar- on the distribution of RPWs (Rugman-Jones et al., 2013) vae were selected for the experiments. Entomopathogenic and on the damage caused to palms are available in litera- nematodes, S. carpocapsae were provided by Koppert Bi- ture (Gitau et al., 2009), data about the immune defenses ological System (Koppert BV, AD Berkel en Rodenrijs, of this species are scarce (Manachini et al., 2011, 2013), the Netherlands); parasites are available as commercial thus, we believe that it is important to study this aspect of preparation (CapsanemR ) at the infective juvenile stage the physiology of R. ferrugineus with the aim to improve L3, in cryptobiosis. To maintain the parasites in infective control strategies against this insect pest. stage the preparation was kept at 4 °C. Before assays, S. In this paper, we have examined some aspects of the carpocapsae were purified from inert material and about immune response of R. ferrugineus larvae, considering 2–3 g of the formulation (nematode-clay) was dissolved the effects induced by the presence of S. carpocapsae. in dechlorinated tap water. The suspension was layered on The potential interference of the EPNs in host cellular and a sucrose gradient (75 %–50 %–25 %) and centrifuged at humoral immunological processes has been assessed; par- 100× g, for 10 min at room temperature; nematodes were ticularly, we have evaluated the activity of the host proPO recovered at the 25 %–50 % interface then, washed several system and the efficiency of hemocytes to recognize and times with sterile tap water to remove contaminants. encapsulate parasites. From the obtained data, we have as- certained the ability of S. carpocapsae nematocomplexes Isolation and purification of S. carpocapsae cuticles to modulate proPO system activity of RPW, thus interfer- ing with insect melanization processes. Moreover, we have Cuticles were obtained from previously washed nema- also confirmed the mimetic role of the nematode body- todes (as describe above). Processed parasites were sus- surface to circumvent host cell recognition and encapsu- pended in 20 volumes of CEB (cuticle extraction buffer: lation processes, as a result of immune-depressive/elusive 10 mmol/L Tris-HCl, 10 mmol/L EDTA, 1 mmol/L PMSF, strategies; the parasite seems to be able to overcome suc- pH 7.2) and subjected to 2 cycles of sonication (150 cessfully the immune responses also in this insect species. watts for 30 sec), in a Labsonic-L Ultrasonic processor (B. Braun Biotech Inc., Allentown, PA, USA). Parasites Materials and methods body fragments were homogenized using a Potter Dounce (B. Braun, pestle B) to remove tissue and body flu- Reagents and instruments ids, finally the cuticles were washed several times with 10 mmol/L of Tris-HCl pH 7.2, to remove tissue debris Instruments and reagents were purchased from Bio- and contaminants. To verify the purification degree, cuti- Rad Laboratories (Detroit, MI, USA), Sigma Chemicals cles fragments were checked by light microscopy. (St. Louis, MO, USA), Millipore Corporation (Billerica, MA, USA), Merck (Darmstadt, Germany) and Celbio Microorganisms SpA (Milan, Italy). Centrifugations were performed by an Eppendorf MiniSpinR and Eppendorf 5804R (Eppendorf Gram-negative bacteria (Escherichia coli C1a), Gram- AG, Hamburg, Germany). Spectrophotometric measure- positive bacteria (Bacillus subtilis, ATCC 6051) and yeast ments were carried out using a Jasco V-560 ( Easton, (Saccharomyces cerevisiae) were used to verify the ef- MD, USA). All materials and buffers were autoclaved. fects on the host proPO system in vitro. Bacterial cultures All assays were performed at 4 °C under sterile condi- were grown overnight at 37 °C in Luria-Bertani broth (1 % tions when required. tryptone, 0.5 % yeast extract, 1.0 % NaCl) and the bac- teria concentration was estimated by spectrophotomet- Insects and parasites ric reading of absorbance (λ = 600 nm). Subsequently, the cultures were centrifuged at 1700 × g for 15 min, R. ferrugineus later instars larvae were used to study the bacterial pellet was washed several times with ster- relationships between insect hosts and the parasites. ile PBS (138 mmol/L NaCl, 2.7 mmol/L KCl, 10 mmol/L Larvae were collected from Canary palm trees (Phoenix Na2HPO4/KH2PO4, pH 7.4), cells were killed (20 min at canariensis), in Palermo area (Italy) and transferred (au- 95 °C), then washed with sterile phosphate buffer. The thorization MIPAF prot. 0025254) to our laboratory for bacteria strains were used at a final concentration of experimental tests. RPW at various stages were main- 103 cfu/mL. S. cerevisiae (0.025 g/L) was inoculated in tained in a climatic chamber at 30 °C, in dark condition, sterile media (1 % yeast extract, 2 % D-glucose monohy- with a relative humidity of 75 %; insects were fed with drate, 0.05 % peptone) and incubated overnight at 37 °C.

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Cultures were centrifuged at 1700 × g for 10 min at Effects of parasites on host proPO system 20 °C, pellets were harvested and washed several times in PBS, yeast cells were counted in a Burker¨ chamber The relative activity of host phenoloxidase was recorded and suspended to a final concentration of 105 cells/mL. by spectrophotometric assays carried out after parasitiza- Finally, yeast cells were heat-killed (at 95 °C for 30 min), tion (in vivo), or by coincubation of S. carpocapsae with centrifuged and washed several times in sterile buffer, host CFF (in vitro). To investigate the effects of para- aliquots of all microorganisms were immediately used or sites in vivo on host proPO system, 30–50 μL of a sus- stored at −20 °C. pension containing about 20–30 (living or cold-killed) nematodes in PBS were injected into the hemocoelic cav- ity of R. ferrugineus. The parasitization was carried out Propagation of S. carpocapsae in R. ferrugineus by microinjection into host larvae in order to establish a “time zero,” required for a correct kinetic of activation. To evaluate the ability of penetration of S. carpocapsae Injections were performed using microsyringes Hamilton inside R. ferrugineus larvae, the method of Dutky et al. (mod. Gas-tight), equipped with thin needles (diameter (1964) was used. Ten to fifteen milliliters of an aqueous 0.13 mm internal, 0.26 mm external). solution of S. carpocapsae (approximately 2 × 103 nema- Thirty minutes after parasites injection, larval todes/mL) was layered on a sterile filter paper placed in a hemolymph was collected and the CFF was used for spec- Petri dish (90 mm diameter), larvae were incubated up to trophotometric measurements. For in vitro assays, about 48 h at 20 °C. After incubation, dead or living larvae were 30–50 nematodes or cuticles fragments, were added to dissected to assess the presence of parasites in the hemo- host CFF aliquots (200 μL), after 30 min of incubation un- coelic cavity. Host body cavity was washed with Mead der a gentle nitrogen flow at 25–26 °C, parasites (or cuti- buffer (98 mmol/L NaOH, 145 mmol/L NaCl, 17 mmol/L cles) were pelleted by centrifugation (200 × g for 10 min) EDTA, 41mmol/L citric acid, pH 4.5), the buffer was har- and the relative activity of phenoloxidase was recorded in vested and the number of nematodes evaluated under a supernatants as describe above. All kinetics assays were stereomicroscope. carried out as described in “proPO system activity of R. ferrugineus” section. As controls in all in vivo assays, proPO system activity of R. ferrugineus larvae were injected with sterile PBS.

proPO system relative activity of RPW larvae was mon- itored by spectrophotometric analysis in hemolymph cell- RPW hemocytes primary cultures free samples. The activity was examined in vitro, both in the presence of microorganisms (Bacillus subtilis, Es- To investigate in vitro the process of cellular encap- cherichia coli, Saccharomyces cerevisiae) and in the pres- sulation we established primary cells isolated from the ence of isolated PAMPs compounds (Lipopolysaccha- hemolymph of R. ferrugineus. Briefly, healthy larvae rides, β-Glucan, Peptidoglycans). Cell-free fraction (CFF) were sterilized in 70 % ethanol, anesthetized on ice and supernatants were obtained by 2 low-speed centrifuga- bled by puncturing the dorsal vessel by a sterile nee- tions (200 × g, for 10 min, at 4 °C) of whole hemolymph, dle. Hemolymph was flushed out in a refrigerated ster- previously flushed out from healthy larvae. Bacteria (2 × ile Eppendorf tube with anticoagulant buffer (98 mmol/L 102), yeast cells (2 × 102), lipopolysaccharides (10 μg), NaOH, 145 mmol/L NaCl, 17 mmol/L EDTA, 41 mmol/L 10 μL of a saturated solution of β-Glucans, or peptido- citric acid, pH 4.5), to avoid undesired cells degranulation. glycans (10 μg), were added to 200 μL aliquots of CFF; Cells were separated by low speed centrifugation (200 × 30 min after incubation (under nitrogen flow at 25–26 °C) g for 10 min at 4 °C), humoral fraction was discarded and time courses of phenoloxidase activity were recorded. All collected hemocytes washed with sterile PBS, the proce- kinetics were done with 5 μL of hemolymph added to dure was repeated a few times to avoid any contamination 1 mL of L-Dopa (8 mmol/L L-Dopa in 10 mmol/L Tris- by tissues or cells debris. Hemocytes were suspended in a HCl, pH 7.2) as substrate; time courses of absorbance complete culture medium (10 % fetal bovine serum, 1 % changes were recorded (A 490 nm 5 min−1,at20°C), antibiotic antimycotic, 1 % glutamine in Grace’s insect by evaluating the dopachrome formation from L-Dopa medium), and 2 × 105 cells were cultured in 96 microw- substrate. As control, basal activity of proPO system ells plates (Cell cultures cluster, flat bottom, Iwaki) and was analyzed in extracted hemolymph samples without kept at 25–26 °C, in a humidified incubator (Cellstar) activators. without CO2.

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Encapsulation assays Table 1 Propagation test, white trap infestation of R. ferrug- ineus (RPW) with S. carpocapsae (EPN). Data refer to the num- To examine the ability of host hemocytes to encapsu- ber of parasites inside host hemocoel cavity and to mortality of late foreigners such as nematodes (live, heat-killed, and RPW (means ± SEM, n = 20). cold-killed), parasites isolated cuticles and synthetic mi- Time (h) No. EPNs (means ± SEM) RPW mortality(%) crobeads and we carried out in vitro coincubations of host primary cells with potential targets. 10 0 Before assays, all the targets samples, were washed in 62± 0.55 0 sterile buffer and resuspended in Grace’s insect medium, 12 3 ± 0.70 0 after 30 min from the hemocytes adhesion to the sub- 24 5 ± 0.91 10 strate, targets were added to cells cultures at a concentra- 48 5 ± 0.74 80 tion of about 10–15 units/well. The encapsulation process was examined at 2 and 8 h after the start of incubation, observations were made under an inverted microscope E. coli), peptidoglycans (from B. subtilis), or β-glucans (Olympus I×51) and images were acquired by a digital (from S. cerevisiae), resulted in a strong increase of ac- system (Nikon digital camera DXM1200F). For in vivo tivity (Fig. 1A, LPS, PGN, GLU); absorbance recorded assays a slight modification of the procedure described values were, on average, more than twice as compared to above was used (cold-killed and heat-killed nematodes the control (Figs. 1A and B). were suspended in sterile PBS, before injection into lar- The proPO system modulation induced by isolated vae); 30 min, or 2 h, after hosts infection, larvae were bled PAMPs was comparable to that obtained in presence of and extracted nematodes and hemocytes were observed microorganisms (Fig. 1B); graphs Ec, Bs, and Sc show by microscopy. an evident increase of activity in hemolymph samples incubated with E. coli, B. subtilis and S. cerevisiae,re- Data processing and statistical analysis spectively. The host proPO system was, as expected, very responsive, since the extraction procedures induced a Statistical analysis were performed using the Student’s basal activity of the phenoloxidase; the basal activity was unpaired t-test, differences between mean values were considered as control (Fig. 1). analyzed and considered significant when P < 0.05 or < considered extremely significant when P 0.0001 with Host proPO modulation induced by parasites respect to control values. All experiments were replicated at least 10 times. Data were processed with GraphPad The effects of S. carpocapsae on the activity of host Prism 4 and GraphPad InStat 4 (GraphPad Software, Inc., proPO system were assessed by in vitro and in vivo as- CA, USA). says (Fig. 2). Fig. 2A shows the phenoloxidase relative activity when live parasites, or isolated parasites cuticles, Results were coincubated with CFF in vitro; the presence of whole parasites (Nem) resulted in a marked decrease of the en- RPW infestation zyme relative activity, instead, isolated parasite cuticles (Cut) lacked inhibiting properties. When parasites were The capability of S. carpocapsae to penetrate and kill injected into the host hemocoel cavity in the early phase RPW larvae was assessed, data outlined in Table 1 show of infection (Fig. 2B, Nem30), a level of inhibition com- the number of penetrated parasites at various times within parable to that recorded in the in vitro assay (Fig. 2A, 48 h and the R. ferrugineus mortality. Nem) was observed. In the late phases after injection (60 and 120 min) a marked activation of proPO system was observed (Fig. 2B, Nem60,Nem120). The proPO system of RPW We also performed in vivo assays with cold-killed ne- matodes, Fig. 2C shows the proPO activity after injection Relative activity of host phenoloxidase was evaluated of cold-killed parasites; the strong inhibition effects ob- by means of spectrophotometric analyses either in naive served at 30 min with live nematodes (Fig. 2B) seem to be CFF samples or in pretreated hemolymph samples, in the lost; for all the analyzed time periods (Fig. 2C, dNem30, presence of PAMPs or whole microorganisms. As shown dNem60,dNem120) a weak inhibition was evident when in Fig. 1A, the presence of lipopolysaccharides (from compared to the control (Fig. 2C).

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Fig. 1 (A) In vitro host proPO system modulation in the presence of various PAMPs. C, control; LPS, lipopolysaccharides; PGN, peptidoglycans; GLU, β-glucans. (B) In vitro host proPO system modulation in the presence of various microorganisms. C, control; Ec, Escherichia coli; Bs, Bacillus subtilis;Sc,Saccharomyces cerevisiae. Mean ± SEM, n = 5, **P < 0.0001.

Host hemocytes and encapsulation of abiotic not self Encapsulation properties of RPW larvae hemocytes were assessed in vitro in the presence of inert materi- Main hemocytes populations from late stage larvae of als, the process was tested versus synthetic microbeads, R. ferrugineus are showed in Fig. 3A (left and right), as observable in Fig. 3B, RPW cells are able to re- granulocytes (Gr), plasmatocytes (Pl), and larger oenocy- act against not self. Agarose beads were coincubated toids (Oe) are clearly observable. Granulocytes (Gr) are with hemocytes, after 2 h (left), both plasmatocytes and rounded cells with a small nucleus; in the cytoplasm sev- granulocytes move toward and surround the bead, in eral granules (detected by focusing through the cells) are panel right (8 h), beads are enclosed by several cellu- present. Plasmatocytes (Pl) are also evident, they are eas- lar layers and melanin formation within the capsule is ily recognized by their spreading behavior and however, evident. they can be clearly distinguished by their spindle-shaped appearance. Membrane extensions, which originate from the cell surface can be seen in culture (after 30 min), Host cells reaction vs. parasites (in vitro) which later develop into motile pseudopodia that move and spread on the surface of culture wells (not shown). We tested the host cellular response against live and Oenocytoids (Oe) are larger round cells, they are gener- dead parasites. Fig. 4A shows in vitro incubations of ally refractive, and occasionally the nucleus in peripheral host hemocytes with live and dead S. carpocapsae (left position can be seen. and middle). After 8 h of incubation, both living and

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Fig. 2 (A) In vitro host proPO system modulation in the presence of parasites and isolated cuticles. C, control; Nem, S. carpocapsae; Cut, S. carpocapsae isolated cuticles. Mean ± SEM, n = 5, **P < 0.0001. (B) In vivo host proPO system modulation in the presence of live parasites at various times. C, control, Nem30, Nem60, Nem120, S. carpocapsae-injected larvae, 30, 60, and 120 min after infection. Mean ± SEM, n = 5, **P < 0.0001; *P < 0.05. (C) In vivo proPO system modulation in the presence of cold-killed parasites. C, control; dNem30, dNem 60, dNem 120, dead S. carpocapsae-injected larvae, 30, 60, and 120 min after infection. Mean ± SEM, n = 5, *P < 0.05. cold-killed parasites were not recognized and not encap- ascertained by encapsulation assays against isolated cu- sulated, hemocytes seem to be healthy, no esocytosis pro- ticles, as shown in Fig. 4B, cuticles were not detected cess was observable and melanin formation was absent. by host cells, no migration and encapsulation were ob- In order to verify if the presence of parasites affected cell served (panels left and middle, 2 and 8 h postincubation, health and functions, nematodes and microbeads were respectively). However, when parasite body-surfaces were incubated concurrently with host hemocytes (Fig. 4A, damaged by heat-treatment, S. carpocapsae seems to lose right); the presence of cold-killed parasites did not inter- its mimetic features; as shown in Fig. 4B (right), cuticle- fere with hemocytes encapsulation properties since host damaged parasites are recognized and surrounded by cel- cells were able to encapsulate beads (arrow head). Given lular multilayered capsules. Cell migration processes to- that hemocytes did not recognize S. carpocapsae as not ward parasites were usually preceded by the formation self, the parasite itself seems to possess mimetic prop- of several stretched motile pseudopodia (Fig. 4B, right, erties. The elusive role of the parasite body-surface was arrowheads).

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trol the insect pest. Some works on RPW have been performed in field (Dembilio et al., 2011; Triggiani & Tarasco, 2011), but only a few papers address both host immunological responses and evasive strategies of the S. carpocapsae (Manachini et al., 2011, 2013). From the data previously obtained with S. feltiae and G. mellonella, we ascertained the primary role of the par- asite cuticle, the epicuticular zone seems to be the key element responsible of molecular disguise strategies and at the same time, able to cause host immune suppression. The dual role of the cuticle was evident when we assayed the biochemical properties of its lipidic moiety; lipids of S. feltiae epicuticle interacted and removed 3 main humoral components from the host hemolymph (named HIPs). The subtraction consequent to cuticle adhesion of specific host proteins was the main mechanism responsi- ble of the observed disguise, resulting from the covering of the parasite with host antigens, (Mastore & Brivio, 2008). The removal of hemolymph host factors downregulated antimicrobial peptide synthesis (Brivio et al., 2006), in- Fig. 3 In vitro assay with Rhynchophorus ferrugineus hemo- hibited proPO system activation (Brivio et al., 2004) and cytes. (A) micrographs show main cell populations from last finally affected encapsulation and phagocytosis capability instar larvae. Gr, granulocytes; Pl, plasmatocytes; Oe, oenocy- toids (400×). (B) Agarose beads were coincubated in vitro with of host hemocytes (Brivio et al., 2010). larvae hemocytes. Left (2 h), hemocytes are close to the bead Interactions between S. carpocapsae and R. ferrug- and the encapsulation process begins; right, (8 h), various beads ineus seem to be only partly similar to that described are enclosed by the cells and melanin formation is evident inside for S. feltiae–G. mellonella (for a review see: Brivio the capsule (200×). et al., 2005); according to literature (Balasubramanian et al., 2009; Hao et al., 2009; Toubarro et al., 2009, 2010) we observed immune depressive effects of living S. car- Host immune reaction versus parasites (in vivo) pocapsae culminating in the fast inhibition of host proPO system. The decrease of phenoloxidase activity was evi- The host–parasite interaction assays were carried out in dent only in the early phase of infection (30 min), since vivo by microinjection of nematodes into the host hemo- after 1 h the parasite did not induce activation of proPO coel (Fig. 4C). When injected into the host, cold-killed system. As described by Shi et al. (2012), the delayed parasites show mimetic properties (Fig. 4C, left); the lack activation of phenoloxidase could be due to the release of of cell responses was similar to that observed when the Xenorhabdus toxins into the hemocoelic cavity, though, assays were carried out in vitro (Fig. 4A, middle). Instead, inhibitory effects of symbionts bacteria have been ascer- when heat-killed parasites were injected, cellular and hu- tained in other insect species (de Silva et al., 2000; Song moral reactions were faster and stronger with respect to et al., 2011). Even if the efficacy of EPNs is based on in vitro assays. Fig. 4C (middle) shows the formation of the lethal action of symbiotic bacteria in the late phase dark melanin aggregates around a parasite after 30 min of infection, results confirmed that, immediately after the and after 2 h (Fig. 4C, right) melanin masses fully engulf entry, parasites must neutralize a complex series of ad- parasites (arrowheads) and hemocytes are barely visible verse immune reactions performed by the host (Dunphy since they were enclosed inside the dark capsule. & Webster, 1987; Brivio et al., 2005; Brivio et al., 2010). The early inhibitory effects did not seem be mediated Discussion by processes comparable to that observed with S. feltiae, because only living S. carpocapsae possess inhibitor ef- In this work, we investigated the interaction of S. car- fects, probably induced by their secretions, for example, pocapsae with R. ferrugineus; in particular, some im- serine proteases (Toubarro et al., 2013) and not to cuti- munological features of the host and elusive/depressive cle properties. Moreover, when EPNs are used against a strategies of the parasite have been elucidated, the ob- range of insect targets, the behavior of parasites and host tained data could be of value to improve actions to con- responses are often inhomogeneous (Gaugler & Kaya,

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Fig. 4 (A) S. carpocapsae living (left) and cold-killed (middle) were incubated in vitro with host hemocytes (8 h). In both cases parasites were not recognized and encapsulation process was not observed (100×). Right, nematodes and microbeads were coincubated, even if in the presence of parasites cells are able to encapsulate agarose beads (arrowhead) (200×). (B) Immune evasion properties of the parasite body surface. Isolated cuticles were incubated with host hemocytes for 2 h (left) and 8 h (middle), either at short or long in vitro incubations, cuticles seem to avoid cell recognition. Right, heat-induced cuticle damage resulted in the loss of the mimetic properties of the parasite (n), thus a strong encapsulation was observed (200×). (C) In vivo humoral and cellular reaction against S. carpocapsae: cold-killed (left) and heat-killed (middle and right) nematodes were injected into RPW larvae hemocoelic cavity, then larvae were bleeded, hemocytes and parasites were placed in microwells and observed under a light microscope. Left, cold-killed parasites after 2 h from injection were not encapsulated. Panels middle and right, show assays carried out with heat-killed nematodes 30 min and 2 h after injection, respectively, conspicuous formation of melanin was evident; in right panel, parasites (arrowheads) are completely surrounded by dark melanin masses (100×).

1990), this could explain the variety of effects observed larvae showed a basal activity, which is strongly elicited when S. feltiae or S. carpocapsae infected different hosts. in the presence of foreign bodies (E. coli, B. subtilis, S. In addition, our data show that cold-killed parasites do cerevisiae) or their cell wall compounds (LPS, PGNs, and not inhibit the RPW proPO system, confirming that this β-glucans). These data seem to confirm that, although the mechanism implies active secretion processes and it is RPW proPO system is very responsive to not self, the par- achieved early by living parasites. asite is able to modulate temporarily the phenoloxidase After we verified the ability of parasites to depress host activity, ensuring its survival and creating a favorable en- proPO system, we analyzed the system activity either in vironment for its symbionts. physiological conditions or in the presence of not ento- A further interesting aspect of the immune response mopathogenic microorganisms; we observed that na¨ıve of RPW is related to recognizing processes carried out

C 2014 Institute of Zoology, Chinese Academy of Sciences, 22, 748–760 Rhynchophorus ferrugineus immune responses 757 by circulating hemocytes; usually, insect immunocompe- point, however, we have to consider that the complete tent cells, such as granulocytes and plasmatocytes are both picture of the immune response of R. ferrugineus and its involved in the recognition often followed by cellular cov- interactions with S. carpocapsae nematocomplexes is not ering surrounding not self bodies (Strand, 2008; Castillo yet exhaustive, further studies are needed to increase the et al., 2011). Considering our previous observations of im- knowledge of the physiology of this insect pest. mune evasion strategies of S. feltiae (Mastore & Brivio, 2008), we investigated the ability of S. carpocapsae to Acknowledgments elude the RPW cell surveillance and avoid encapsulation processes. RPW hemocytes show a typical morphology We are grateful to Dr. Michela Curradi and Dr. Simona and they actively participate in capsule formation also Binda Rossetti for the critical reading of the manuscript. against abiotic targets; when agarose beads were coincu- We express gratitude to Prof. Santi Longo for providing bated with hemocytes, cellular encapsulation was evident, the RPW larvae. This work was supported by funds from beads were surrounded by several cell layers and melanin University of Insubria, Italy. enclosed targets inside the capsule. Parasites may have sharp differences in molecular or- ganization and surface properties, this is particularly true Disclosures for EPNs, which once entered, must interact with a po- tentially harmful insects host environment. Even though Authors declare that there are no interest conflicts and no the body surface of S. carpocapsae did not seem to be in- financial interests concerning the subject presented in the volved in depression of R. ferrugineus humoral responses, manuscript. a key role of the cuticle has been ascertained to avoid host cell surveillance. Recognition and cell encapsulation are References completely lacking against S. carpocapsae, either alive or cold-killed nematodes were not recognized by host cells; Akhurst, R.J. and Dunphy, G.B. (1993) Tripartite interactions also, isolated cuticles are identified as self by RPW hemo- between symbiotically associated entomopathogenic bacteria, cytes confirming the involvement of the parasite body nematodes, and their insect hosts. Parasites and Pathogens of surface in elusive mimetic strategies. Lacking of cellular Insects (eds. N.E. Beckage, S.N. Thompson & B. Federici). recognition against parasites was not due to damage of Academy Press, New York. hemocytes, because when coincubated with EPNs, syn- Balasubramanian, N., Hao, Y.J., Toubarro, D., Nascimento, G. thetic beads were normally encapsulated. and Simoes,˜ N. (2009) Purification, biochemical and molecu- According to the data obtained with S. feltiae (Brivio lar analysis of a chymotrypsin protease with prophenoloxidase et al., 2002, 2004), the mimetic role of S. carpocapsae suppression activity from the entomopathogenic nematode cuticle was lost when the structure was damaged by heat Steinernema carpocapsae. International Journal for Para- treatments, any structural and chemical modifications sitology, 39, 975–984. caused the disappearance of the parasite elusive strate- Blaxter, M.L., Page, A.P., Rudin, W. and Maizels, R.M. (1992) gies culminating in the stimulation of cellular processes. Nematode surface coats: actively evading immunity. Para- Effects of damages of parasite body surface were partic- sitology Today, 8, 243–247. ularly evident when treated S. carpocapsae was injected Brivio, M.F., Pagani, M. and Restelli, S. (2002) Immune sup- into larvae for in vivo assays, immune responses were pression of Galleria mellonella (Insecta, Lepidoptera) hu- strongly enhanced and in particular, melanin formation moral defenses induced by Steinernema feltiae (Nematoda, was very quick and intense. Rhabditida): involvement of the parasite cuticle. Experimen- The hypothesis of a central function of the body surface tal Parasitology, 101(2–3), 149–156. of parasites was proposed early by Vinson (1977, 1990), Brivio, M.F., Mastore, M. and Moro, M. (2004) The role of and further works confirmed the involvement of the cuti- Steinernema feltiae body-surface lipids in host–parasite im- cle in immune evasion and suppression of host defenses, munological interactions. Molecular and Biochemical Para- (Blaxter et al., 1992; Politz & Philipp, 1992; Akhurst & sitology, 135, 111–121. Dunphy, 1993). Brivio, M.F., Mastore, M. and Pagani, M. (2005) Parasite–host Although somewhat different, S. carpocapsae and relationship: a lesson from a professional killer. Invertebrate S. feltiae share an overall strategy aimed to arrange a favor- Survival Journal, 2, 41–53. able environment for their symbiotic bacteria, by means Brivio, M.F.,Moro, M. and Mastore, M. (2006) Down-regulation of the elusion from cell recognition and depression of host of antibacterial peptide synthesis in an insect model in- humoral defenses. Our data can provide a useful starting duced by the body-surface of an entomoparasite (Steinernema

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