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Hemocytes of Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) and their response to Saccharomyces cerevisiae and Bacillus thuringiensis
Journal of Invertebrate Pathology 106 (2011) 360–365
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Hemocytes of Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) and their response to Saccharomyces cerevisiae and Bacillus thuringiensis Barbara Manachini ⇑, Vincenzo Arizza, Daniela Parrinello, Nicolò Parrinello Dipartimento di Biologia Animale ‘‘G. Reverberi’’, Università di Palermo, Palermo, Italy
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Article history: Received 15 September 2010 Accepted 4 December 2010 Available online 13 December 2010 Keywords: Red Palm Weevil Infection Insect immunity Hemolymph Biological control
a b s t r a c t Originally from tropical Asia, the Red Palm Weevil (RPW Rhynchophorus ferrugineus (Olivier)) is the most dangerous and deadly pest of many palm trees, and there have been reports of its recent detection in France, Greece and Italy. At present, emphasis is on the development of integrated pest management based on biological control rather than on chemical insecticides, however the success of both systems is often insufficient. In this regard, RPW appears to be one pest that is very difficult to control. Thus investigations into the natural defences of this curculionid are advisable. RPW hemocytes, the main immunocompetent cells in the insect, are described for the first time. We identified five hemocyte cell types from the hemolymph of R. ferrugineus: plasmatocytes (50%), granulocytes (35%), prohemocytes (8%), oenocytes (4%) and spherulocytes (3%). SEM observations were also carried out. Some aspects of RPW interaction with non-self organisms, such as Saccharomyces cerevisiae and the entomopathogen bacterium, Bacillus thuringiensis (Bt), are discussed. Plasmatocytes and granulocytes were involved in nodules and capsule formation as well as in the phagocytosis of yeast. The hemocyte response of RPW larvae to sub-lethal doses of commercial products containing Bt was examined. In vivo assays were carried out and Bt in vegetative form was found in the hemolymph. After a diet containing Bt, the number of total hemocytes, mainly plasmatocytes, in the RPW larva hemolymph declined sharply (12%) and then remained at a low level, while the number of other circulating cells was almost unchanged. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae), known as the Red Palm Weevil (RPW), causes significant damage to a wide genera of palms, and this makes it advisable to control it as a quarantine pest (EPPO, 2008). Nevertheless, the introduction of RPW has been reported from different European countries including Italy. In Europe, where infestation is mainly in urban areas, there is a strong emphasis on the development of IPM based on pheromone traps and biological control (El-Sufty et al., 2007; EPPO, 2008; Longo and Colazza, 2009). Indeed, there have been several attempts to isolate self-pathogens of RPW, with the goal of employing them in biological control: bacterial pathogens such as Pseudomonas aeruginosa and different species of Bacillus (Bunerjee and Dangar, 1995; Salama et al., 2004) and yeast (Dangar, 1997). Nevertheless, only Alfazariy (2004) reported a good pathogenicity level of naturally occurring Bacillus thuringiensis (Bt). A commercial product based on Bt, and registered against other coleopteran species, has been tested against R. ferrugineus and was found to show pathogenicity, but the concentration ⇑ Corresponding author. Address: Dipartimento di Biologia Animale ‘‘G. Reverberi’’, Università di Palermo, via Archirafi, 18, 90123 Palermo, Italy. Fax: +39 091 23891847. E-mail address: [email protected] (B. Manachini). 0022-2011/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2010.12.006
needed was quite high (Manachini et al., 2009a,c). The reasons for the difficulty in finding a good pathogen against RPW could be many. One important aspect to be examined is the insect’s ability to recover from infection (Parmakelis et al., 2008). Insects contain hemocytes in their hemolymph, which is mainly responsible for cell and humoral immune responses including phagocytosis and encapsulation, as well as inducible antimicrobial peptides, cell adhesion molecules, lysozyme, lectins, and the pro-phenoloxidase system (Hoffmann, 2003; Kanost et al., 2004). Cellular response in the insect immune system has been shown to be an important barrier to the infection process (Hoffmann, 1995, 2003). Several classes of hemocytes have been morphologically and functionally characterized, mainly in Diptera and Lepidoptera, and little is known about the coleopteran species (Lavine and Strand, 2002; Giulianini et al., 2003; Costa et al., 2005; Giglio et al., 2008). No data are available concerning hemocytes and the prompt innate hemocyte response of R. ferrugineus to foreign materials. Thus, it is of interest to study the cellular innate defence of RPW larvae against B. thuringiensis and yeast. We chose Bt because its mechanism of action in pests, mainly in Lepidoptera, has been extensively described (Gill et al., 1992; de Maagd et al., 2001; Crickmore, 2005), whereas there have been few studies on target insect defence responses, including experimental bacteria inoculation into the larval haemocoel (Mostafa et al., 2005). Studies on the immune
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response of insects infected with bacteria through the gut are rare (Mostafa et al., 2005; Dubovskiy et al., 2008), and the Coleopteran immune system has not yet been fully described. For yeast some data are available on potential pathogenicity, thus yeast could also be used as non-self organisms to study cellular immune reactions. The present paper contributes to today’s knowledge of Curculionidae immunity, and discusses larva hemocyte response to infection. To achieve this aim, RPW hemolymph larvae were treated in vitro with the yeast Saccharomyces cerevisiae Meyen and RPW larvae were fed with the entomopathogenic bacterium B. thuringiensis. 2. Materials and methods 2.1. Insects During the spring and summer 2008 and 2009, R. ferrugineus larvae were collected from infested Phoenix canariensis Chabaud in Palermo (Italy). The palms were cut down following phytosanitary measures for the control and eradication of R. ferrugineus (Regional Decree 6 March 2007), and RPW specimens were collected in collaboration with the Regional Department of Phytosanitary Structural Intervention for Regional Service Unit No. 53, Sicily Region and Sicilian Regional Agency Forestry. In the laboratory, the larvae were reared in a climatic chamber at 27 ± 0.9 °C, 75% relative humidity, and a 24 h night photoperiod. The development of the different larval instars was observed by detecting the molted head capsules. Last instar larvae, characterized by a high feeding rate, gained weight and high hemolymph content (Manachini et al., 2009a,b), were used in the experiments. 2.2. Bacteria and bioassay A commercial product containing 26.000 UI L.d. of mg B. thuringiensis var. kurstaki pathotype H-3A, 3b registered against Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) was used. According to the technical schedule provided by the producer (Intrachem), this strain (EG 2424) is a transconjugant strain with the genes of two different Bt strains, expressing extremely active crystal proteins against coleopteran pests. The food formulation consisted of apple (1 g) enriched with 1.27 mg/ml Bt product. This dose was chosen in accordance with previous results (Manachini et al., 2009b,c). One hundred larvae were selected for the experiment: 50 larvae were fed with untreated diet (control), and 50 with a diet containing Bt (treated). Each larva was put in a petri dish (7 cm height 2.8 cm diameter) with a live, spore-forming Bt-treated diet. After the experimental feeding, at 6, 12, 19, 24 and 48 h, six living larvae were randomly selected each time and their hemolymph was examined. 2.3. Bleeding procedure and hemocyte counting Hemolymph was collected from each RPW larva and kept separately to avoid allorecognition reactions. The larval surface was sterilized with 70% ethanol for a few seconds and rinsed with sterile water and then dried. The specimens were anaesthetized by placing them at 20 °C for 7 min, and hemolymph was withdrawn from the dorsal blood vessel with a sterile glass Pasteur pipet. About 1 ml hemolymph from each one larva was collected in a sterile Eppendorf tube in the presence (v/v) of anticoagulant solution (98 mM NaOH, 186 mM NaCl, 17 mM Na2 EDTA, 41 mM Citric acid and 10 mM Phenylthiourea pH 4.5). To carry out the in vitro tests and hemocyte slide preparation, the hemolymph was centrifuged at 1000g for 10 min. at 4 °C, and the pellet suspended to 1 106 cells ml 1 phosphate saline buffer (PBS). To count the hemocytes, pure hemolymph was put, immediately after bleeding, in a 0.0025 mm2 Neubauer improved hemocytometer
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(Assistant, Germany) under a light microscope DLMB Leica, (Wetzlar, D). Total hemocyte numbers (THC), expressed as 106/ml, and the hemocyte types from untreated and Bt treated larvae, were recorded at 6, 12, 19, 24 and 48 h. 2.4. Preparation and identification of hemocytes A drop of hemolymph was placed on a coverslip and allowed to dry at room temperature for 20–30 min. After hemocyte adhesion to the coverslip, the debris-containing hemolymph was discarded and the cell monolayer was repeatedly washed by dipping the coverslip in 100 ml PBS. Hemocytes were observed by a Nomarski interferential contrast microscope (Diaplan, Leika, Wetzlar, D). To examine the hemocytes, cell monolayers were fixed with 5% methanol in anticoagulant solution for 5 min, and treated with a 10% Giemsa (Fluka) aqueous solution for 10 min, then washed in distilled water and mounted in Acquovitrex (Carlo Erba, Milan, Italy) on glass slides. To reveal mucous-substances and glucosaminoglycans, fixed hemocytes were incubated with a filtered aqueous solution of 0.5% toluidine blue (Fluka) and 0.5% sodium tetraborate for 10 s. This stain shows pink–violet metachromasia, and was also suitable to stain Bt. For vital staining, after hemocyte adhesion to the coverslips, the anticoagulant solution was substituted with 60 ll of neutral red (Merck, Darmstadt, Germany) solution (8 mg/ml) in anticoagulant solution, and observed under a DLMB Leica (Wetzlar, D) microscope. This dye specifically stains the acidic compartments (e.g., lysosomes or acid vacuolar contents) of living cells (Bancroft and Gamble, 2002). Insect hemocyte classification is currently a matter of debate. However, according to recent literature (Lavine and Strand, 2002; Price and Ratcliffe, 2004; Costa et al., 2005; Ribeiro and Brehelin, 2006; Giglio et al., 2008; Strand, 2008) the following hemocytetypes have been identified: (1) plasmatocytes, (2) granular hemocytes or granulocytes, (3) oenocytes, (4) prohemocytes, (5) spherule cells or spherulocytes. In addition, observations have been made of not well identified circulating cells. 2.5. Scanning electron microscopy (SEM) The hemolymph, in the presence of anticoagulant, was dropped directly on a coverslip pretreated with 0.1% poly-l-lysine. After adhesion the monolayer was fixed in cacodylate buffer (0.1 M, pH 7.3) containing 2.5% glutaraldehyde, post-fixed in osmium tetroxide 1%, dehydrated in graded alcohol and dried at the critical point. The preparations were mounted on stubs, gold coated in a sputter coater and analyzed by SEM (LEO 420). 2.6. Yeast preparation and challenge with hemocytes A yeast Saccharomyces cerevisiae (baker’s yeast, type II) suspension was prepared in distilled water at 0.25% w/v (approx. 1 108 yeasts/ml), autoclaved for 15 min, and washed twice by centrifuging at 2000g (5 min, 4 °C). After the repeated washing, the yeasts were suspended at 0.125% final concentration in sterile PBS and immediately used. For the phagocytosis assay, 200 ll hemocyte suspension (1 10 6/ml) were mixed with 100 ll yeast preparation (10:1 yeast/hemocyte ratio), and incubated in 1 ml test plastic tubes with gentle stirring for 90 min at 20 °C. A drop of this suspension was smeared onto a slide and examined at 400 magnification under a Nomarski interferential contrast microscope (Diaplan, Leika, Wetzlar, D). 2.7. Statistical analysis Values of hemocyte sizes were expressed as the mean of 10 cells, randomly taken, of each type ± standard deviation (SD). The
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differences in the hemocyte counts between the treated and larvae (control) were compared for significance by one-way analysis of the variance test and post hoc Dunnett’s t-test for multiple comparisons. All statistical analyses were conducted using the Statistical Package for Social Science v. 8.0 software (SPSS Inc., Chicago, IL). 3. Results 3.1. Identification of hemocytes In untreated larvae hemolymph monolayers, all the known hemocyte-types were identified for their size, morphology and dye-staining properties (Fig. 1A and B). The most abundant hemocyte-types were plasmatocytes (50.79 ± 2.23% of THC corresponding to 2.27 106/ml) and granulocytes (34.61 ± 2.02% of THC). Plasmatocytes were large/medium sized hemocytes (in average long 38.30 ± 8.54 lm and large 16.32 ± 5.22 lm with a ratio 2.38 ± 0.28). Just after bleeding, plasmatocytes were seen as oval (elliptic) cells with a typical spindle shape. In the monolayers, after a few minutes, the plasmatocytes spread rapidly over the coverslip and became large and thin cells, developing pseudopodia and long and wide lamellipodia. A large round nucleus lay almost in the middle of the cell (Fig. 1). The granulocytes were medium/large (in average 22.15 ± 4.23 lm diameter) sized hemocytes with a rather rounded shape and many small granules, and were often congested together. Spherulocytes (3.34 ± 0.23% of THC) were round -medium sized hemocytes (13.12 ± 1.41 lm diameter) containing inclusions (spherules). Oenocytes (3.32 ± 0.99% of THC) were non adhesive hemocytes, which, after fixing, appeared regular in shape (37.96 ± 12.08 lm diameter) and containing an eccentric nucleus. Prohemocytes (7.94 ± 1.31% of THC) were round and small hemocytes (5.55 ± 2.32 lm diameter) with a hgh nucleus/cytoplasm ratio. The hemocytes were examined under scanning electron microscopy (Fig. 1C–G). Round prohemocytes could be identified by their small size (Fig. 1E). Two sizes of round cell groups, smaller cell (Fig. 1F) and larger cell (Fig. 1G), showed a rough surface with short philopodia; the surface of all these cells was found to be covered with small projections and very small membranous blobs. Plasmatocytes, with a typical spindle-shape feature and long spike-like philopodia extending from the end zones, were characterized by a smooth surface (Fig. 1C and D). 3.2. Hemocytes involved in immune responses To check for hemocyte immune activity, an in vitro assay was performed by mixing hemocytes and S. cerevisiae. Observations under a Nomarski interferential contrast microscope showed that, in the presence of hemocytes, the yeast became agglutinated, and some clusters can be seen (Fig. 2A and B).
An encapsulation process due to the granulocytes and plasmatocytes was observed. At the early phase, granulocytes came into contact with yeast, and small and large nodules were formed (Fig. 2A), these then degranulated and free granules were seen in the medium (Fig. 2B). Fig. 2B shows a late (60 min) large nodule in which granulocytes and some plasmatocytes surrounded yeast clusters. In the meantime, single yeasts were phagocytised by plasmatocytes (Fig. 2C and D) and granulocytes (Fig. 2E). Other circulating hemocyte-types were not found to be involved in this response. 3.3. Effect of Bt ingestion on hemocytes To check the effect of Bt ingestion by RPW larvae, the changes in the circulating hemocytes from larvae fed with a diet containing Bt (treated), the total hemocyte number per ml of hemolymph was evaluated. In the treated larvae, 19 h from the feeding bioassay, the mean value of total circulating hemocytes (THC) was significantly lower than that found in hemolymph collected from the control larvae (Fig. 3). In these last, THC value ranged from 3.85 106 to 5.2 106. No statistical differences were recorded in the THC control larvae over time (Fig. 3). In the treated larvae there was a little decrement of THC already at 6 h but this was not statistically significant, while at 19 h it was statistically lower than the THC recorded for the control larvae. In addition, a statistical significant decrement in the number of plasmatocytes was recorded 19 h after treatment. The values decreased from 2.27 106/ml ± 0.08 of the controls to 0.35 106/ ml ± 0.08 of the treated larvae, reaching up to 12.56 ± 0.66% of total hemocytes (50% in the controls) in the hemolymph (Fig. 4). Also the number of prohemocytes was found to be lower in the treated larvae (Fig. 4). 3.4. Hemocytes–Bt interactions Fig. 5 shows the hemolymph from the control (A) and the larvae fed with Bt (B and C). In the untreated larvae, all types of hemocytes were recorded. It was revealed in the treated larvae that, after ingestion of the diet treated with a commercial Bt product (containing spores and Cry toxins), the Bt was able to invade and to replicate in the RPW hemolymph, where it was found in its vegetative form (Btv). In fact after 19 h of experimental feeding, the hemolymph contained a great amount of Btv (Fig. 5B), and after 24 h the hemocyte populations were dramatically changed probably due to the presence of Btv (Fig. 5C). Destruction of cells was also evident and a lot of cellular debris was found (Fig. 5B and C1). Fig. 5B shows how the bacteria are in contact with several hemocytes, however they mainly adhere to the plasmatocyte surface (Fig. 5B arrow).
Fig. 1. Hemocytes from hemolymph of Rhynchophorous ferrugineus larva. (A) Neutral red stain; (B) Giemsa stain. Pl, plasmatocyte; G, granular hemocyte with refractive granules; Oe, oenocytoid; S, spherulocytes; Pr, prohemocytes; n, nucleus. Bar = 5 lm. (C–G): Scanning electron microscopy: (C and D) plasmatocytes, bar: 5 lm; (E) prohemocyte; (F–G) round cells of varying size; (E and G) bar: 2 lm.
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Fig. 2. Yeast (S. cerevisiae) encapsulation and phagocytosis by hemocytes from Rhynchophorous ferrugineus larvae as shown by an in vitro assay and phase contrast microscopy observations. (A) Early phase, granulocytes bound to target particles after 20 min incubation (bar = 30 lm). (B) Capsule formed after 60 min incubation (bar = 10 lm). (C–E) Phagocytosis of yeasts by hemocytes. Bar = 10 lm. Pl: plasmatocyte; G, granulocytes; Oe: oenocytoid; Ph: early phagocytosis; gg: free granules; n: small and large yeast nodules; F: control yeast in PBS observed under a light microscope (bar = 30 lm).
4. Discussion
Fig. 3. Total hemocyte count (THC) in the circulating hemolymph from RPW larvae collecting 6, 12, 19, 24 and 48 h after feeding untreated diet (control) and diet containing Bt (treated). Results are given as mean (n = 6), and standard deviation. Bars with different letter are statistically significant different at post hoc Dunnett’s t-test for multiple comparisons significant (P < 0.01).
Fig. 4. Hemocyte differential counts recorded in the hemolymph from RPW larvae fed with untreated diet (control) and with diet containing Bt (treated) after 19 h. Results are given as mean (n = 6), and standard deviation. (P < 0.05), (P < 0.01).
This paper provides a first description of hemocytes from hemolymph collected from late instar larvae of R. ferrugineus (RPW). Based on recent insect hemocyte literature (Lavine and Strand, 2002; Price and Ratcliffe, 2004; Ribeiro and Brehelin, 2006; Giglio et al., 2008; Strand, 2008), we identified in RPW larvae five major hemolymph cell types: plasmatocytes, granular hemocytes or granulocytes, oenocytes, prohemocytes and spherulocytes. SEM observations helped reveal the cell surface features of prohemocytes, small and large hemocytes and plasmatocytes. Although morpho-functional data are not available, it is of interest that the prohemocytes, round small and round large cells, presented a rough surface, whereas the plasmatocytes (of varying size) were characterized by a smooth surface and long philopodia. Due to taxonomic differences, insect hemocyte classification, and the terminology used for their identification, can be controversial. Furthermore, total hemocyte count (THC) and hemocyte population composition can differ according to insect taxa and developmental stages (Ribeiro and Brehelin, 2006; Giglio et al., 2008). In RPW larva, the most abundant hemocytes were plasmatocytes (50%) followed by granulocytes (30%). A similar proportionality was found in Carabus lefebvrei larvae (Coleoptera Carabidae) (Giglio et al., 2008), and in Melipona scutellaris (Hymenoptera, Apidae) (Amaral et al., 2010), whereas, in Lepidoptera larvae, granulocytes have been reported as the most abundant hemocytes (Strand, 2008). To understand the role of hemocytes and their involvement in the defence reactions of RPW larvae, a non pathogenic yeast and a pathogenic agent were used. Hemocytes incubated in vitro with yeast form nodules and capsules. Granular hemocytes bind promptly to target cells and degranulate; plasmatocytes then cooperate to form a multilayer sheath, suggesting an in vitro encapsulation process. Granulocyte degranulation could be responsible for several inflammatory factors that can be involved in agglutinating yeast and
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Fig. 5. Hemocytes from RPW larvae stained with toluidine blue. (A) Control hemolymph; A1 control Bacillus thuringiensis vegetative form (Btv) in PBS; larvae feed with Bt after 12 h (B) and after 19 h (C). C1 hemocyte debris after bacterial interaction. Pl: plasmatocyte; G: granular haemocyte; S: spherulocytes; Pr: prohemocyte; Db: cellular debris. Bar = 10 lm.
allowing plasmatocyte recruitment. These findings confirm what has been reported by other authors (Costa et al., 2005; Ribeiro and Brehelin, 2006; Strand, 2008; Amaral et al., 2010). Moreover both cellular types appear to have a role in the phagocytosis of yeast. The feeding experiments showed that B. thuringiensis ingested as spores by RPW larvae invades the hemolymph, and the total circulating hemocytes decreased, mainly the plasmatocytes, after 19 h. Moreover, for the first time, many Bt vegetative forms were recorded in the hemolymph of RPW after Bt commercial product ingestion. Bt is a gram-positive spore-forming bacterium with entomopathogenic properties. In Lepidoptera, toxicity occurs after Bt is ingested, solubilised in the alkaline midgut, and then proteolytically cleaved to release the active endotoxin/s (Cry) that can bind their receptors (De Maagd et al., 2001; Soberón, 2005). The mode of action of Cry toxins has been characterized mainly in lepidopteran insects, and it is widely accepted that the primary action of Cry toxins is to lyse insect midgut epithelial cells (Bravo et al., 2007), the cell content and other components then promoting spore germination. This process leads to severe septicemia and insect death. It has been reported that the vegetative form is involved in insect septicemia processes, but the killing mechanism has not yet been described. During the vegetative growth stage, Bt is known to release a new family of insecticidal proteins (Salamitou et al., 2000; De Maagd et al., 2001; Soberón, 2005; Bravo et al., 2007). The finding that bacteria, as a vegetative form, are in RPW hemolymph suggests that Bt is able to bypass the various above described steps to reach the hemolymph and affect the defense system. THC were dramatically reduced, especially the plasmatocytes. Many parasites must avoid hemocyte-mediated immune responses to growth in host larvae, and many species achieve this by suppressing one or more components of the host immune defense system, e.g. alteration of THC, inhibition of hemocyte spreading, apoptosis in circulating hemocytes (Adamo, 2005; Eleftherianos et al., 2008; Ericsson et al., 2009). The interaction between entomopathogen bacteria and hemocytes is little studied in insects, and the available literature is mainly on Lepidoptera. However similar results to our findings have been found in insect response to other entomopathogenic bacteria, for example Btk in Trichoplusia ni (Ericsson et al., 2009) and Photorhabdus in fifth-stage larvae of Manduca sexta (Eleftherianos et al., 2008). Differences in antibacterial response have been attributed to bacterial species and virulence levels (Dettloff et al., 2001; Giannoulis et al., 2007), however some quite important gaps in understanding the general mode of action of Bt still exist (Then, 2009). Indeed, there are several contradictions among the different models (Then, 2009). Thus, the question of how bacteria act in RPW larvae is still open. In RPW, several cells interact with Bt, but we found that it was the circulating plasmatocytes that were mainly affected by Bt, as shown by their decreased proportion. In any case, the role of
plasmatocytes remains controversial. They have been reported as either phagocytes (Tojo et al., 2000; Ling and Yu, 2006) or not (Giulianini et al., 2003). In the scarabeid, Cetonischema aeruginosa, Giulianini et al. (2003) also showed the phagocytic activity of oenocytes. We found that both plasmatocytes and granulocytes can exert phagocytosis. Our observations also show that Bt interacts with the plasmatocytes, probably causing damage to them and a decrease in the number of circulating cells. A similar effect was exerted by Xenorhabdus nematophila against the hemocytes of the lepidopteran species Malacosoma disstria (Giannoulis et al., 2007). A greater knowledge of RPW biology and, in particular, of the interaction between potential pathogens and immunocytes would be useful to improve RPW-IPM programs, which should focus on the identification of more virulent natural pathogen strains and on improving the virulence capacity of Bt. Acknowledgments This study was partially supported by a grant from the Italian Ministero dell’Istruzione, dell’Università e della Ricerca (Research Program PRIN 2008 ‘‘Discovery and evaluation of new microbial and vegetable bio-pesticides for natural insect pest control’’. Coordinator: Prof. Ignazio Floris, University of Sassari – Italy). The authors wish to thank the Regional Department of Phytosanitary Structural Intervention for Regional Service Unit No. 53 Region of Sicily and the Sicilian Regional Forestry Agency, particularly Giovanni Lo Sasso, for helping with the sampling and collecting of the RPW larvae. A special thank you to Dr. S. Franceschini and E. Ladurner for their critical comments and supplying the Bt strain. Thanks also go to Cost 862 ‘‘Bacterial Toxins for Insect Control’’. References Adamo, A.S., 2005. Parasitic suppression of feeding in the tobacco hornworm, Manduca sexta: parallels with feeding depression after an immune challenge. Arch. Insect Biochem. Physiol. 60, 185–197. Alfazariy, A.A., 2004. Notes on the survival capacity of two naturally occurring entomopathogens on the red palm weevil Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae). Egypt. J. Biol. Pest Control 14, 423. 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. Bancroft, J.D., Gamble, M., 2002. Theory and Practice of Histological Techniques. Churchill Livingstone, London. Bravo, A., Gill, S.S., Soberón, M., 2007. Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon 49 (4), 423–435. Bunerjee, A., Dangar, T.K., 1995. Pseudomonas aeruginosa, a facultative pathogen of red palm weevil, Rhynchophorus ferrugineus. World J. Microbiol. Biotechnol. 11, 618–620. Costa, S.C., Ribeiro, C., Girard, P.A., Zumbihl, R., Brehelin, M., 2005. Modes of phagocytosis of gram-positive and gram-negative bacteria by Spodoptera littoralis granular hemocytes. J. Insect Physiol. 51, 39–46.
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Hemocytes of Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae) and their response to Saccharomyces cerevisiae and Bacillus thuringiensis Barbara Manachini ⇑, Vincenzo Arizza, Daniela Parrinello, Nicolò Parrinello Dipartimento di Biologia Animale ‘‘G. Reverberi’’, Università di Palermo, Palermo, Italy
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Article history: Received 15 September 2010 Accepted 4 December 2010 Available online 13 December 2010 Keywords: Red Palm Weevil Infection Insect immunity Hemolymph Biological control
a b s t r a c t Originally from tropical Asia, the Red Palm Weevil (RPW Rhynchophorus ferrugineus (Olivier)) is the most dangerous and deadly pest of many palm trees, and there have been reports of its recent detection in France, Greece and Italy. At present, emphasis is on the development of integrated pest management based on biological control rather than on chemical insecticides, however the success of both systems is often insufficient. In this regard, RPW appears to be one pest that is very difficult to control. Thus investigations into the natural defences of this curculionid are advisable. RPW hemocytes, the main immunocompetent cells in the insect, are described for the first time. We identified five hemocyte cell types from the hemolymph of R. ferrugineus: plasmatocytes (50%), granulocytes (35%), prohemocytes (8%), oenocytes (4%) and spherulocytes (3%). SEM observations were also carried out. Some aspects of RPW interaction with non-self organisms, such as Saccharomyces cerevisiae and the entomopathogen bacterium, Bacillus thuringiensis (Bt), are discussed. Plasmatocytes and granulocytes were involved in nodules and capsule formation as well as in the phagocytosis of yeast. The hemocyte response of RPW larvae to sub-lethal doses of commercial products containing Bt was examined. In vivo assays were carried out and Bt in vegetative form was found in the hemolymph. After a diet containing Bt, the number of total hemocytes, mainly plasmatocytes, in the RPW larva hemolymph declined sharply (12%) and then remained at a low level, while the number of other circulating cells was almost unchanged. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae), known as the Red Palm Weevil (RPW), causes significant damage to a wide genera of palms, and this makes it advisable to control it as a quarantine pest (EPPO, 2008). Nevertheless, the introduction of RPW has been reported from different European countries including Italy. In Europe, where infestation is mainly in urban areas, there is a strong emphasis on the development of IPM based on pheromone traps and biological control (El-Sufty et al., 2007; EPPO, 2008; Longo and Colazza, 2009). Indeed, there have been several attempts to isolate self-pathogens of RPW, with the goal of employing them in biological control: bacterial pathogens such as Pseudomonas aeruginosa and different species of Bacillus (Bunerjee and Dangar, 1995; Salama et al., 2004) and yeast (Dangar, 1997). Nevertheless, only Alfazariy (2004) reported a good pathogenicity level of naturally occurring Bacillus thuringiensis (Bt). A commercial product based on Bt, and registered against other coleopteran species, has been tested against R. ferrugineus and was found to show pathogenicity, but the concentration ⇑ Corresponding author. Address: Dipartimento di Biologia Animale ‘‘G. Reverberi’’, Università di Palermo, via Archirafi, 18, 90123 Palermo, Italy. Fax: +39 091 23891847. E-mail address: [email protected] (B. Manachini). 0022-2011/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2010.12.006
needed was quite high (Manachini et al., 2009a,c). The reasons for the difficulty in finding a good pathogen against RPW could be many. One important aspect to be examined is the insect’s ability to recover from infection (Parmakelis et al., 2008). Insects contain hemocytes in their hemolymph, which is mainly responsible for cell and humoral immune responses including phagocytosis and encapsulation, as well as inducible antimicrobial peptides, cell adhesion molecules, lysozyme, lectins, and the pro-phenoloxidase system (Hoffmann, 2003; Kanost et al., 2004). Cellular response in the insect immune system has been shown to be an important barrier to the infection process (Hoffmann, 1995, 2003). Several classes of hemocytes have been morphologically and functionally characterized, mainly in Diptera and Lepidoptera, and little is known about the coleopteran species (Lavine and Strand, 2002; Giulianini et al., 2003; Costa et al., 2005; Giglio et al., 2008). No data are available concerning hemocytes and the prompt innate hemocyte response of R. ferrugineus to foreign materials. Thus, it is of interest to study the cellular innate defence of RPW larvae against B. thuringiensis and yeast. We chose Bt because its mechanism of action in pests, mainly in Lepidoptera, has been extensively described (Gill et al., 1992; de Maagd et al., 2001; Crickmore, 2005), whereas there have been few studies on target insect defence responses, including experimental bacteria inoculation into the larval haemocoel (Mostafa et al., 2005). Studies on the immune
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response of insects infected with bacteria through the gut are rare (Mostafa et al., 2005; Dubovskiy et al., 2008), and the Coleopteran immune system has not yet been fully described. For yeast some data are available on potential pathogenicity, thus yeast could also be used as non-self organisms to study cellular immune reactions. The present paper contributes to today’s knowledge of Curculionidae immunity, and discusses larva hemocyte response to infection. To achieve this aim, RPW hemolymph larvae were treated in vitro with the yeast Saccharomyces cerevisiae Meyen and RPW larvae were fed with the entomopathogenic bacterium B. thuringiensis. 2. Materials and methods 2.1. Insects During the spring and summer 2008 and 2009, R. ferrugineus larvae were collected from infested Phoenix canariensis Chabaud in Palermo (Italy). The palms were cut down following phytosanitary measures for the control and eradication of R. ferrugineus (Regional Decree 6 March 2007), and RPW specimens were collected in collaboration with the Regional Department of Phytosanitary Structural Intervention for Regional Service Unit No. 53, Sicily Region and Sicilian Regional Agency Forestry. In the laboratory, the larvae were reared in a climatic chamber at 27 ± 0.9 °C, 75% relative humidity, and a 24 h night photoperiod. The development of the different larval instars was observed by detecting the molted head capsules. Last instar larvae, characterized by a high feeding rate, gained weight and high hemolymph content (Manachini et al., 2009a,b), were used in the experiments. 2.2. Bacteria and bioassay A commercial product containing 26.000 UI L.d. of mg B. thuringiensis var. kurstaki pathotype H-3A, 3b registered against Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) was used. According to the technical schedule provided by the producer (Intrachem), this strain (EG 2424) is a transconjugant strain with the genes of two different Bt strains, expressing extremely active crystal proteins against coleopteran pests. The food formulation consisted of apple (1 g) enriched with 1.27 mg/ml Bt product. This dose was chosen in accordance with previous results (Manachini et al., 2009b,c). One hundred larvae were selected for the experiment: 50 larvae were fed with untreated diet (control), and 50 with a diet containing Bt (treated). Each larva was put in a petri dish (7 cm height 2.8 cm diameter) with a live, spore-forming Bt-treated diet. After the experimental feeding, at 6, 12, 19, 24 and 48 h, six living larvae were randomly selected each time and their hemolymph was examined. 2.3. Bleeding procedure and hemocyte counting Hemolymph was collected from each RPW larva and kept separately to avoid allorecognition reactions. The larval surface was sterilized with 70% ethanol for a few seconds and rinsed with sterile water and then dried. The specimens were anaesthetized by placing them at 20 °C for 7 min, and hemolymph was withdrawn from the dorsal blood vessel with a sterile glass Pasteur pipet. About 1 ml hemolymph from each one larva was collected in a sterile Eppendorf tube in the presence (v/v) of anticoagulant solution (98 mM NaOH, 186 mM NaCl, 17 mM Na2 EDTA, 41 mM Citric acid and 10 mM Phenylthiourea pH 4.5). To carry out the in vitro tests and hemocyte slide preparation, the hemolymph was centrifuged at 1000g for 10 min. at 4 °C, and the pellet suspended to 1 106 cells ml 1 phosphate saline buffer (PBS). To count the hemocytes, pure hemolymph was put, immediately after bleeding, in a 0.0025 mm2 Neubauer improved hemocytometer
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(Assistant, Germany) under a light microscope DLMB Leica, (Wetzlar, D). Total hemocyte numbers (THC), expressed as 106/ml, and the hemocyte types from untreated and Bt treated larvae, were recorded at 6, 12, 19, 24 and 48 h. 2.4. Preparation and identification of hemocytes A drop of hemolymph was placed on a coverslip and allowed to dry at room temperature for 20–30 min. After hemocyte adhesion to the coverslip, the debris-containing hemolymph was discarded and the cell monolayer was repeatedly washed by dipping the coverslip in 100 ml PBS. Hemocytes were observed by a Nomarski interferential contrast microscope (Diaplan, Leika, Wetzlar, D). To examine the hemocytes, cell monolayers were fixed with 5% methanol in anticoagulant solution for 5 min, and treated with a 10% Giemsa (Fluka) aqueous solution for 10 min, then washed in distilled water and mounted in Acquovitrex (Carlo Erba, Milan, Italy) on glass slides. To reveal mucous-substances and glucosaminoglycans, fixed hemocytes were incubated with a filtered aqueous solution of 0.5% toluidine blue (Fluka) and 0.5% sodium tetraborate for 10 s. This stain shows pink–violet metachromasia, and was also suitable to stain Bt. For vital staining, after hemocyte adhesion to the coverslips, the anticoagulant solution was substituted with 60 ll of neutral red (Merck, Darmstadt, Germany) solution (8 mg/ml) in anticoagulant solution, and observed under a DLMB Leica (Wetzlar, D) microscope. This dye specifically stains the acidic compartments (e.g., lysosomes or acid vacuolar contents) of living cells (Bancroft and Gamble, 2002). Insect hemocyte classification is currently a matter of debate. However, according to recent literature (Lavine and Strand, 2002; Price and Ratcliffe, 2004; Costa et al., 2005; Ribeiro and Brehelin, 2006; Giglio et al., 2008; Strand, 2008) the following hemocytetypes have been identified: (1) plasmatocytes, (2) granular hemocytes or granulocytes, (3) oenocytes, (4) prohemocytes, (5) spherule cells or spherulocytes. In addition, observations have been made of not well identified circulating cells. 2.5. Scanning electron microscopy (SEM) The hemolymph, in the presence of anticoagulant, was dropped directly on a coverslip pretreated with 0.1% poly-l-lysine. After adhesion the monolayer was fixed in cacodylate buffer (0.1 M, pH 7.3) containing 2.5% glutaraldehyde, post-fixed in osmium tetroxide 1%, dehydrated in graded alcohol and dried at the critical point. The preparations were mounted on stubs, gold coated in a sputter coater and analyzed by SEM (LEO 420). 2.6. Yeast preparation and challenge with hemocytes A yeast Saccharomyces cerevisiae (baker’s yeast, type II) suspension was prepared in distilled water at 0.25% w/v (approx. 1 108 yeasts/ml), autoclaved for 15 min, and washed twice by centrifuging at 2000g (5 min, 4 °C). After the repeated washing, the yeasts were suspended at 0.125% final concentration in sterile PBS and immediately used. For the phagocytosis assay, 200 ll hemocyte suspension (1 10 6/ml) were mixed with 100 ll yeast preparation (10:1 yeast/hemocyte ratio), and incubated in 1 ml test plastic tubes with gentle stirring for 90 min at 20 °C. A drop of this suspension was smeared onto a slide and examined at 400 magnification under a Nomarski interferential contrast microscope (Diaplan, Leika, Wetzlar, D). 2.7. Statistical analysis Values of hemocyte sizes were expressed as the mean of 10 cells, randomly taken, of each type ± standard deviation (SD). The
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differences in the hemocyte counts between the treated and larvae (control) were compared for significance by one-way analysis of the variance test and post hoc Dunnett’s t-test for multiple comparisons. All statistical analyses were conducted using the Statistical Package for Social Science v. 8.0 software (SPSS Inc., Chicago, IL). 3. Results 3.1. Identification of hemocytes In untreated larvae hemolymph monolayers, all the known hemocyte-types were identified for their size, morphology and dye-staining properties (Fig. 1A and B). The most abundant hemocyte-types were plasmatocytes (50.79 ± 2.23% of THC corresponding to 2.27 106/ml) and granulocytes (34.61 ± 2.02% of THC). Plasmatocytes were large/medium sized hemocytes (in average long 38.30 ± 8.54 lm and large 16.32 ± 5.22 lm with a ratio 2.38 ± 0.28). Just after bleeding, plasmatocytes were seen as oval (elliptic) cells with a typical spindle shape. In the monolayers, after a few minutes, the plasmatocytes spread rapidly over the coverslip and became large and thin cells, developing pseudopodia and long and wide lamellipodia. A large round nucleus lay almost in the middle of the cell (Fig. 1). The granulocytes were medium/large (in average 22.15 ± 4.23 lm diameter) sized hemocytes with a rather rounded shape and many small granules, and were often congested together. Spherulocytes (3.34 ± 0.23% of THC) were round -medium sized hemocytes (13.12 ± 1.41 lm diameter) containing inclusions (spherules). Oenocytes (3.32 ± 0.99% of THC) were non adhesive hemocytes, which, after fixing, appeared regular in shape (37.96 ± 12.08 lm diameter) and containing an eccentric nucleus. Prohemocytes (7.94 ± 1.31% of THC) were round and small hemocytes (5.55 ± 2.32 lm diameter) with a hgh nucleus/cytoplasm ratio. The hemocytes were examined under scanning electron microscopy (Fig. 1C–G). Round prohemocytes could be identified by their small size (Fig. 1E). Two sizes of round cell groups, smaller cell (Fig. 1F) and larger cell (Fig. 1G), showed a rough surface with short philopodia; the surface of all these cells was found to be covered with small projections and very small membranous blobs. Plasmatocytes, with a typical spindle-shape feature and long spike-like philopodia extending from the end zones, were characterized by a smooth surface (Fig. 1C and D). 3.2. Hemocytes involved in immune responses To check for hemocyte immune activity, an in vitro assay was performed by mixing hemocytes and S. cerevisiae. Observations under a Nomarski interferential contrast microscope showed that, in the presence of hemocytes, the yeast became agglutinated, and some clusters can be seen (Fig. 2A and B).
An encapsulation process due to the granulocytes and plasmatocytes was observed. At the early phase, granulocytes came into contact with yeast, and small and large nodules were formed (Fig. 2A), these then degranulated and free granules were seen in the medium (Fig. 2B). Fig. 2B shows a late (60 min) large nodule in which granulocytes and some plasmatocytes surrounded yeast clusters. In the meantime, single yeasts were phagocytised by plasmatocytes (Fig. 2C and D) and granulocytes (Fig. 2E). Other circulating hemocyte-types were not found to be involved in this response. 3.3. Effect of Bt ingestion on hemocytes To check the effect of Bt ingestion by RPW larvae, the changes in the circulating hemocytes from larvae fed with a diet containing Bt (treated), the total hemocyte number per ml of hemolymph was evaluated. In the treated larvae, 19 h from the feeding bioassay, the mean value of total circulating hemocytes (THC) was significantly lower than that found in hemolymph collected from the control larvae (Fig. 3). In these last, THC value ranged from 3.85 106 to 5.2 106. No statistical differences were recorded in the THC control larvae over time (Fig. 3). In the treated larvae there was a little decrement of THC already at 6 h but this was not statistically significant, while at 19 h it was statistically lower than the THC recorded for the control larvae. In addition, a statistical significant decrement in the number of plasmatocytes was recorded 19 h after treatment. The values decreased from 2.27 106/ml ± 0.08 of the controls to 0.35 106/ ml ± 0.08 of the treated larvae, reaching up to 12.56 ± 0.66% of total hemocytes (50% in the controls) in the hemolymph (Fig. 4). Also the number of prohemocytes was found to be lower in the treated larvae (Fig. 4). 3.4. Hemocytes–Bt interactions Fig. 5 shows the hemolymph from the control (A) and the larvae fed with Bt (B and C). In the untreated larvae, all types of hemocytes were recorded. It was revealed in the treated larvae that, after ingestion of the diet treated with a commercial Bt product (containing spores and Cry toxins), the Bt was able to invade and to replicate in the RPW hemolymph, where it was found in its vegetative form (Btv). In fact after 19 h of experimental feeding, the hemolymph contained a great amount of Btv (Fig. 5B), and after 24 h the hemocyte populations were dramatically changed probably due to the presence of Btv (Fig. 5C). Destruction of cells was also evident and a lot of cellular debris was found (Fig. 5B and C1). Fig. 5B shows how the bacteria are in contact with several hemocytes, however they mainly adhere to the plasmatocyte surface (Fig. 5B arrow).
Fig. 1. Hemocytes from hemolymph of Rhynchophorous ferrugineus larva. (A) Neutral red stain; (B) Giemsa stain. Pl, plasmatocyte; G, granular hemocyte with refractive granules; Oe, oenocytoid; S, spherulocytes; Pr, prohemocytes; n, nucleus. Bar = 5 lm. (C–G): Scanning electron microscopy: (C and D) plasmatocytes, bar: 5 lm; (E) prohemocyte; (F–G) round cells of varying size; (E and G) bar: 2 lm.
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Fig. 2. Yeast (S. cerevisiae) encapsulation and phagocytosis by hemocytes from Rhynchophorous ferrugineus larvae as shown by an in vitro assay and phase contrast microscopy observations. (A) Early phase, granulocytes bound to target particles after 20 min incubation (bar = 30 lm). (B) Capsule formed after 60 min incubation (bar = 10 lm). (C–E) Phagocytosis of yeasts by hemocytes. Bar = 10 lm. Pl: plasmatocyte; G, granulocytes; Oe: oenocytoid; Ph: early phagocytosis; gg: free granules; n: small and large yeast nodules; F: control yeast in PBS observed under a light microscope (bar = 30 lm).
4. Discussion
Fig. 3. Total hemocyte count (THC) in the circulating hemolymph from RPW larvae collecting 6, 12, 19, 24 and 48 h after feeding untreated diet (control) and diet containing Bt (treated). Results are given as mean (n = 6), and standard deviation. Bars with different letter are statistically significant different at post hoc Dunnett’s t-test for multiple comparisons significant (P < 0.01).
Fig. 4. Hemocyte differential counts recorded in the hemolymph from RPW larvae fed with untreated diet (control) and with diet containing Bt (treated) after 19 h. Results are given as mean (n = 6), and standard deviation. (P < 0.05), (P < 0.01).
This paper provides a first description of hemocytes from hemolymph collected from late instar larvae of R. ferrugineus (RPW). Based on recent insect hemocyte literature (Lavine and Strand, 2002; Price and Ratcliffe, 2004; Ribeiro and Brehelin, 2006; Giglio et al., 2008; Strand, 2008), we identified in RPW larvae five major hemolymph cell types: plasmatocytes, granular hemocytes or granulocytes, oenocytes, prohemocytes and spherulocytes. SEM observations helped reveal the cell surface features of prohemocytes, small and large hemocytes and plasmatocytes. Although morpho-functional data are not available, it is of interest that the prohemocytes, round small and round large cells, presented a rough surface, whereas the plasmatocytes (of varying size) were characterized by a smooth surface and long philopodia. Due to taxonomic differences, insect hemocyte classification, and the terminology used for their identification, can be controversial. Furthermore, total hemocyte count (THC) and hemocyte population composition can differ according to insect taxa and developmental stages (Ribeiro and Brehelin, 2006; Giglio et al., 2008). In RPW larva, the most abundant hemocytes were plasmatocytes (50%) followed by granulocytes (30%). A similar proportionality was found in Carabus lefebvrei larvae (Coleoptera Carabidae) (Giglio et al., 2008), and in Melipona scutellaris (Hymenoptera, Apidae) (Amaral et al., 2010), whereas, in Lepidoptera larvae, granulocytes have been reported as the most abundant hemocytes (Strand, 2008). To understand the role of hemocytes and their involvement in the defence reactions of RPW larvae, a non pathogenic yeast and a pathogenic agent were used. Hemocytes incubated in vitro with yeast form nodules and capsules. Granular hemocytes bind promptly to target cells and degranulate; plasmatocytes then cooperate to form a multilayer sheath, suggesting an in vitro encapsulation process. Granulocyte degranulation could be responsible for several inflammatory factors that can be involved in agglutinating yeast and
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Fig. 5. Hemocytes from RPW larvae stained with toluidine blue. (A) Control hemolymph; A1 control Bacillus thuringiensis vegetative form (Btv) in PBS; larvae feed with Bt after 12 h (B) and after 19 h (C). C1 hemocyte debris after bacterial interaction. Pl: plasmatocyte; G: granular haemocyte; S: spherulocytes; Pr: prohemocyte; Db: cellular debris. Bar = 10 lm.
allowing plasmatocyte recruitment. These findings confirm what has been reported by other authors (Costa et al., 2005; Ribeiro and Brehelin, 2006; Strand, 2008; Amaral et al., 2010). Moreover both cellular types appear to have a role in the phagocytosis of yeast. The feeding experiments showed that B. thuringiensis ingested as spores by RPW larvae invades the hemolymph, and the total circulating hemocytes decreased, mainly the plasmatocytes, after 19 h. Moreover, for the first time, many Bt vegetative forms were recorded in the hemolymph of RPW after Bt commercial product ingestion. Bt is a gram-positive spore-forming bacterium with entomopathogenic properties. In Lepidoptera, toxicity occurs after Bt is ingested, solubilised in the alkaline midgut, and then proteolytically cleaved to release the active endotoxin/s (Cry) that can bind their receptors (De Maagd et al., 2001; Soberón, 2005). The mode of action of Cry toxins has been characterized mainly in lepidopteran insects, and it is widely accepted that the primary action of Cry toxins is to lyse insect midgut epithelial cells (Bravo et al., 2007), the cell content and other components then promoting spore germination. This process leads to severe septicemia and insect death. It has been reported that the vegetative form is involved in insect septicemia processes, but the killing mechanism has not yet been described. During the vegetative growth stage, Bt is known to release a new family of insecticidal proteins (Salamitou et al., 2000; De Maagd et al., 2001; Soberón, 2005; Bravo et al., 2007). The finding that bacteria, as a vegetative form, are in RPW hemolymph suggests that Bt is able to bypass the various above described steps to reach the hemolymph and affect the defense system. THC were dramatically reduced, especially the plasmatocytes. Many parasites must avoid hemocyte-mediated immune responses to growth in host larvae, and many species achieve this by suppressing one or more components of the host immune defense system, e.g. alteration of THC, inhibition of hemocyte spreading, apoptosis in circulating hemocytes (Adamo, 2005; Eleftherianos et al., 2008; Ericsson et al., 2009). The interaction between entomopathogen bacteria and hemocytes is little studied in insects, and the available literature is mainly on Lepidoptera. However similar results to our findings have been found in insect response to other entomopathogenic bacteria, for example Btk in Trichoplusia ni (Ericsson et al., 2009) and Photorhabdus in fifth-stage larvae of Manduca sexta (Eleftherianos et al., 2008). Differences in antibacterial response have been attributed to bacterial species and virulence levels (Dettloff et al., 2001; Giannoulis et al., 2007), however some quite important gaps in understanding the general mode of action of Bt still exist (Then, 2009). Indeed, there are several contradictions among the different models (Then, 2009). Thus, the question of how bacteria act in RPW larvae is still open. In RPW, several cells interact with Bt, but we found that it was the circulating plasmatocytes that were mainly affected by Bt, as shown by their decreased proportion. In any case, the role of
plasmatocytes remains controversial. They have been reported as either phagocytes (Tojo et al., 2000; Ling and Yu, 2006) or not (Giulianini et al., 2003). In the scarabeid, Cetonischema aeruginosa, Giulianini et al. (2003) also showed the phagocytic activity of oenocytes. We found that both plasmatocytes and granulocytes can exert phagocytosis. Our observations also show that Bt interacts with the plasmatocytes, probably causing damage to them and a decrease in the number of circulating cells. A similar effect was exerted by Xenorhabdus nematophila against the hemocytes of the lepidopteran species Malacosoma disstria (Giannoulis et al., 2007). A greater knowledge of RPW biology and, in particular, of the interaction between potential pathogens and immunocytes would be useful to improve RPW-IPM programs, which should focus on the identification of more virulent natural pathogen strains and on improving the virulence capacity of Bt. Acknowledgments This study was partially supported by a grant from the Italian Ministero dell’Istruzione, dell’Università e della Ricerca (Research Program PRIN 2008 ‘‘Discovery and evaluation of new microbial and vegetable bio-pesticides for natural insect pest control’’. Coordinator: Prof. Ignazio Floris, University of Sassari – Italy). The authors wish to thank the Regional Department of Phytosanitary Structural Intervention for Regional Service Unit No. 53 Region of Sicily and the Sicilian Regional Forestry Agency, particularly Giovanni Lo Sasso, for helping with the sampling and collecting of the RPW larvae. A special thank you to Dr. S. Franceschini and E. Ladurner for their critical comments and supplying the Bt strain. Thanks also go to Cost 862 ‘‘Bacterial Toxins for Insect Control’’. References Adamo, A.S., 2005. Parasitic suppression of feeding in the tobacco hornworm, Manduca sexta: parallels with feeding depression after an immune challenge. Arch. Insect Biochem. Physiol. 60, 185–197. Alfazariy, A.A., 2004. Notes on the survival capacity of two naturally occurring entomopathogens on the red palm weevil Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae). Egypt. J. Biol. Pest Control 14, 423. 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. Bancroft, J.D., Gamble, M., 2002. Theory and Practice of Histological Techniques. Churchill Livingstone, London. Bravo, A., Gill, S.S., Soberón, M., 2007. Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon 49 (4), 423–435. Bunerjee, A., Dangar, T.K., 1995. Pseudomonas aeruginosa, a facultative pathogen of red palm weevil, Rhynchophorus ferrugineus. World J. Microbiol. Biotechnol. 11, 618–620. Costa, S.C., Ribeiro, C., Girard, P.A., Zumbihl, R., Brehelin, M., 2005. Modes of phagocytosis of gram-positive and gram-negative bacteria by Spodoptera littoralis granular hemocytes. J. Insect Physiol. 51, 39–46.
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