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Archives of Insect Biochemistry and Physiology 54:3745 (2003)

Carminic Acid Dye From the Homopteran Dactylopius coccus Hemolymph Is Consumed During Treatment With Different Microbial Elicitors
Fidel de la Cruz Hernndez-Hernndez,1* Fernando Garca-Gil de Muoz,1,3 Alberto Rojas-Martnez,3 Salvador Hernndez-Martnez,2 and Humberto Lanz-Mendoza2
The activation of Dactylopius coccus (Costa) hemolymph with microbial polysaccharide molecules was studied. Hemolymph incubated in the presence of laminarin, zymosan, and N-acetyl glucosamine produced a dark fibrillar precipitated, and the red pigment (carminic acid) was consumed (measured spectrophotometrically at 495 nm). Lipopolysaccharide (LPS) did not induce any response. The reaction was inhibited with millimolar concentrations of serine and cysteine protease inhibitors, EGTA and phenyl thiourea. It was also diminished by prostaglandin synthesis inhibitors: dexamethasone, acetylsalicylic acid, and indomethacin. However, Mg2+ chelator EDTA did not inhibit hemolymph activation. Hemolymph proteins were depleted from soluble phase during treatment with laminarin, but a group of around 34 kDa remained unmodified. These results showed that D. coccus hemolymph is activated by microbial elicitors, its activation depends on eicosanoids, and suggest participation of a prophenoloxidase (PPO)-like activation system that could consume carminic acid. We are currently dissecting the molecular factors involved in D. coccus hemolymph activation to determine homologies and differences with other arthropods immune response pathways. Arch. Insect Biochem. Physiol. 54:3745, 2003. 2003 Wiley-Liss, Inc.
KEYWORDS: Dactylopius; hemolymph; carminic acid; prophenoloxidase; prostaglandins

INTRODUCTION
Dactylopius coccus (Costa) is a homopteran, parasite of prickly pears. It is native from Mexico and is the natural source of the cochineal dye (carminic acid). This pigment is present in most parts of the body, and high quantities are found in the hemolymph. The synthesis site is unknown, but it has been suggested that it is produced in T granulocytes present in hemolymph (Joshi and Lambdin, 1996). Other observations assigned its production to the fat body (Llanderal and Nieto, 1999). Car-

minic acid is an anthraquinone compound with phenolic groups similar to those present in dihydroxyphenylalanine (L-DOPA), tyrosine, and other intermediaries of prophenoloxidase (PPO) pathway. However, a PPO system or similar pathway has not been described in this homopteran specie. Arthropods have a wide spectrum of defense mechanisms (Strand and Peck, 1995). During the defense reaction, the attacking foreign object is often found encapsulated and darkened. The dark pigment is melanin. Therefore, melanin and the enzymes responsible of their synthesis, namely

1 2 3

Experimental Pathology Department, CINVESTAV-IPN, Mxico CISEI-INSP, Cuernavaca Morelos, Mxico School of Biology, Simn Bolvar University, Mxico

Contract grant sponsor: National Polytechnic Institute; Contract grant number: 990294; Contract grant sponsor: Bureau of Industry Services of Simon Bolivar University, Mexico. *Correspondence to: Fidel de la Cruz Hernndez-Hernndez, Departamento de Patologa Experimental, CINVESTAV-IPN, Av. IPN 2508, Sn Pedro Zacatenco. G.A. Madero, Mxico D.F. 07360. E-mail: cruzcruz@mail.cinvestav.mx Received 22 October 2002; Accepted 14 May 2003

2003 Wiley-Liss, Inc. DOI: 10.1002/arch.10099 Published online in Wiley InterScience (www.interscience.wiley.com)

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phenoloxidases, are considered essential components of the arthropods defense mechanisms (Chase et al., 2000). Phenoloxidase (PO) usually occurs as an inactive proenzyme in the hemolymph and it is activated during the immune response by a serine protease. Microbial products and phospholipids can induce the activation (Stanley, 1998). Activated phenoloxidases generate primarily quinones by acting on diphenolic substrates found in the hemolymph. Quinones can be toxic to foreign organisms (Vilar-Rojas et al., 1996) or undergo polymerization during encapsulation (Sderhall and Cerenius, 1998). On the other hand, in several studies it has been proposed and demonstrated that prostaglandins can modulate activities of insects immune system (Stanley, 1998); of particular interest for this work is the previous study on the cicadas Magicicada septendecim and M. cassini , which share with D. coccus an unusual life history (heterometabolous development) (Tunaz et al., 1999). In preliminary studies, we have observed that carminic acid present in D. coccus hemolymph was sequestered from the soluble phase during the incubation with zymosan (Gonzlez et al., 2002). In this report, we extended our investigation with the hypothesis that carminic acid is consumed during defense reactions to immune challenge, and that the consumption is mediated by prostaglandins. We developed a spectrophotometric protocol to record the depletion of carminic acid from insect hemolymph and used this protocol to test our hypothesis. Carminic acid depletion was recorded following incubations with laminarin, zymosan, and N-acetyl galactosamine (GalNac), but not in the presence of lipopolysaccharide (LPS). Carminic acid depletion was inhibited in reactions conducted in the presence of serine and cysteine protease inhibitors.

Insects
D. coccus female insects were reared on cut cladodia of Opuntia ficus-indica and maintained in a greenhouse at 2426C. Under these conditions, the life cycle lasted approximately 3 months and the wingless adult females, which are attached to the cactus by their proboscis, were up to 6.0 mm long.

Hemolymph Collection
Adult female insects were carefully scraped from the prick pear pads and perfused through with 50 ml of saline solution (NaCl 0.9%). Hemolymph was transferred to a polypropylene tube and centrifuged at low speed (600g) to remove debris. Supernatant was recovered and kept on ice until use. Hemocytes were recovered from the supernatants and their integrity was analyzed under phase contrast optics (Nikon E-600 microscope).

Hemolymph Activation Assays


Zymosan stock solution (100 mg glucose equivalents/ml) was prepared as described by LanzMendoza et al. (1993). Laminarin, GalNac, and LPS were prepared in saline solution (0.9% NaCl) in a concentration between 0.1 to 100 mg/ml. Twenty-five microliters of hemolymph prepared as described above (3.8 mg/ml of protein) were mixed with different carbohydrates concentrations and volume was completed to 50 ml with saline solution. Samples were incubated at 37C during different times according to the experiment. At the end of the incubations, reactions were stopped with 10 ml of anticoagulant solution (0.61 M NaOH, 1.56 M NaCl, 0.16 M EDTA, 0.10 M citric acid, pH. 4.5; Graham et al., 1986) and samples were centrifuged at 2,000g during 2 min at 4C to eliminate the fibrillar precipitate. Supernatants were transferred to new tubes for spectrophotometric or electrophoretic analysis. Carminic acid consumption was measured in a spectrophotometer (Beckman DU 640) at 495 nm. The results represent the average of three different experiments done by duplicate.
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MATERIALS AND METHODS Reagents


Reagents of the highest purity grade available were obtained from Sigma Chemical Company (St. Louis MO).

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Statistical Analysis
The mean and standard deviations of different treatments were compared between experimental against control groups using one-way ANOVA, while Fc values were compared using Tukey test (P < 0.05) with one degree of freedom (Fry, 1996).

Effect of Prostaglandin, Proteases, and Phenoloxidase System Inhibitors on D. coccus Hemolymph Activation
Fifty microliters of hemolymph, alone or activated with 25 mg/ml of laminarin, were incubated in the presence of prostaglandin production inhibitors, protease inhibitors, and phenoloxidase competitive inhibitor, Phenylthiourea (PTU), during 3 min. Absorbance at 495 nm was recorded and the values normalized to 100% with respect to control without activator. Prostaglandin inhibitors were prepared in NaCl 0.95%: water-soluble dexamethasone (250 mg/ml), indomethacin (50 mg/ ml), and acetylsalicylic acid (40 mg/ml) (REASOL Analytic reagents, Mexico). Protease inhibitors stocks were prepared in ethanol and were added to hemolymph samples to reach the following final concentrations: Tosyl lysyl chloromethyl ketone (TLCK) 10 mM; tosyl phenylalanyl chloromethyl ketone (TPCK) 10 mM; ethylene glycol-bis (baminoethyl-ether) N,N,N,N-tetraacetic acid (EGTA) 50 mM; ethylenediaminetetraacetic acid (EDTA) 50 mM; o-phenanthroline 50 mM (Benyon and Salvesen, 1989; Lee and Anstee, 1995; Novillo et al., 1999). Phenoloxidase competitive inhibitor, phenylthiourea (PTU), was prepared in ethanol and added to sample reactions to obtain 2 mM final concentration. The results represent the average of three different experiments done by duplicate.

ham et al ., 1986). Samples were centrifuged (2,000g during 2 min at 4C) to remove the fibrillar precipitate. Thirty-five micrograms of protein of samples were mixed with 25 ml electrophoresis sample buffer (2), boiled 10 min, and loaded on individual wells of a 10% polyacrylamide gel for SDS-PAGE electrophoresis. Gels were resolved in standard reducing conditions and stained with Coomassie brilliant blue (Smith, 1989).

RESULTS Red Pigment of D. coccus Hemolymph Is Consumed During Activation With Different Microbial Compounds
Hemolymph preparations from D. coccus females contained hemocytes and carminic acid after low-speed centrifugation. Hemocytes were able to attach to glass (data not shown). Carminic acid was detected by microscopic observation and spectrophotometric measures (maximal absorbance peak at 495 nm) (Fig. 1). Zymosan (50 mg/ml), laminarin (25 mg/ml), and galNac (50 mg/ml) produced a diminution of absorbance at 495 nm, indicating that carminic acid was consumed during reaction (Fig. 1), and a black fibrillar aggregate was produced. LPS neither induce dye consumption nor black fibrillar aggregate. Direct analysis of the proteins forming the precipitate was not possible, because fibrillar aggregates were insoluble in saline buffers, anticoagulant solution, SDS electrophoresis buffers with b mercaptoethanol, boiling, or urea. Since the carminic acid absorbance consistently decreased during hemolymph activation, in the following experiments absorbance at 495 nm was used to evaluate it. The maximal reaction occurred after 60 sec (Fig. 2). The temperature increase from 4 to 22C accelerates slight but significantly the dye consumption, but other step to 37C did not affected the reaction (Fig. 2). To determine the concentration effect of the microbial immune response elicitors on hemolymph activation, samples were incubated with different concentrations of zymosan, GalNac, laminarin, and LPS. As seen in Figure 3, the system is very sensitive

Protein Analysis by Polyacrylamide-SDS Gels


Protein concentration in hemolymph samples was determined by the Bradford method, using bovine serum albumin as standard (Smith, 1989). Anticoagulant solution was added to stop the reaction of hemolymph samples alone or treated with laminarin (25 mg/ml) at different times (GraSeptember 2003

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Fig. 1. Red pigment of D. coccus hemolymph is consumed during clotting reaction. Hemolymph of D. coccus was incubated alone or in the presence of clotting activators and absorption spectra, in the range 400 to 700 nm, and was measured in soluble phase (plasma) after 3-min incubation time. Solid squares, Plasma alone; Open squares, GalNac 50 mg/ ml; X, Laminarin 25 mg/ml; Triangles, LPS 1 mg/ml; Open circles, Zymosan 50 mg/ml. Mean S.E. n = 4.

to zymosan; concentrations as low as 0.1 mg/ml were able to reach the maximum hemolymph activation. Laminarin and GalNac were unable to activate the hemolymph in concentrations <1 mg/ml. In contrast, no reaction was observed with LPS in concentrations ranging from 0.1 to 5 mg/ml (Fig. 3) and not even at 100 mg/ml (data not shown).

Effect of Inhibitors of PPO and Prostaglandin Synthesis on D. coccus Plasma Clotting


Serine-cysteine protease inhibitors TLCK and TPCK stopped hemolymph reaction at millimolar concentrations (20 mM) (Fig. 4). The metalloprotease inhibitors o-phenantroline (a Zn2+ and

Fig. 2. Temperature effect on D. coccus clotting kinetics. Samples of D. coccus hemolymph were activated with laminarin (25 mg/ml) and incubated at different temperatures. The reactions were stopped at indicated times with anticoagulant solution as described in Materials and Methods and absorbance was determined at 495 nm. Squares, plasma alone; circles, plasma prepared with anticoagulant; X, laminarin-activated hemolymph at 4 C; open diamonds, 22 C; solid diamonds, 37C. Mean S.E. n = 3.

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Fig. 3. Effect of concentration of laminarin, zymosan, and LPS concentrations on clotting activity. Hemolymph of D. coccus was incubated during 3 min in the presence of GalNac (squares), laminarin (X), LPS (triangles), and zymosan (circles), all in the range from 0.15 mg/ml, and the absorbance at 495 nm was measured in plasma. Mean S.E. n = 3.

Ca2+ chelator) and EDTA (a Mg2+ chelator) significantly diminished the dye consumption (Fig. 4). PTU, which interferes with the PPO activity, was also tested and total inhibition was reached. In contrast, the calcium chelator EGTA did not show

any effect (Fig. 4). Prostaglandin production inhibitors also diminished activation. Dexamethasone (20 mg/ml) and Indomethacin (50 mg/ml) inhibited it significantly (50%), and acetylsalicylic acid (40 mg/ml) abolished it (Fig. 5).

Fig. 4. Effect of pPO route inhibitors on clotting of D. coccus. D. coccus hemolymph alone (HL), activated for clotting with 25 mg/ml of laminarin (HL + Lam) and activated in the presence of pPO route inhibitors, were incubated during 3 min and absorbance at 495 nm measured in plasma and absorbance values were expressed as percentage with respect to noninduced control. Inhibitors added were: TLCK (10 mM), TPCK (10 mM), EGTA (50 mM), EDTA (50 mM), O-phenantrolin (Phenan) (50 mM), and phenylthiourea (PTU) (2 mM). F = 1.72, P < 0.05, n = 7).

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Fig. 5. Effect of prostaglandin production inhibitors on clotting of D. coccus. D. coccus hemolymph (HL) was activated with 25 mg/ml of laminarin alone or in the presence of prostaglandin production inhibitors: dexamethasone (Dexa) (20 mg/ml), indomethacin (Indo) (50 mg/ ml), and acetylsalycilic acid (ASA) (40 mg/ml). Absorbance at 495 nm was measured in plasma and absorbance values were expressed as percentage with respect to noninduced control. F = 32.91, P < 0.05, n = 4.

Most of the Plasma Proteins Are Sequestered During Hemolymph Activation


The proteins pattern of non-activated hemolymph was complex, with polypeptides of molecular sizes ranging from up to 200 to less than 17 kDa (Fig. 6). After activation with laminarin, most proteins diminished significantly from soluble phase in less than 20 sec, but a group of proteins of around 34 kDa remained. This group persisted at least until 2 h after the hemolymph activation.

Fig. 6. Most of the plasma proteins are sequestered from the plasma during the clotting process. D. coccus hemolymph was incubated with laminarin (25 mg/ml) at 37C during different times. At each time, reaction was stopped with anticoagulant (Materials and Methods), centrifuged, and 35 mg of the protein of the resultant plasma was loaded on wells of a 10% polyacrylamide gel for PAGESDS analysis. Lane 1: Plasma proteins obtained with anticoagulant. Lanes 27: Plasma proteins obtained after hemolymph incubation with laminarin during: 10, 20, 30, 45, 60, and 90 sec. Molecular size marker migration is indicated at left.

activation resemble the PPO pathway in several aspects: 1. D. coccus hemolymph is activated by microbial elicitors. The PPO system is triggered by different microbial compounds including laminarin, zymosan and peptidoglycans (Sderhall and Cerenius, 1998). Other microbial products such as LPS are also active as elicitors of the PPO (Charalambidis et al., 1996; Lanz-Mendoza et al., 1993). However, it has been reported that LPS does not seem to activate the PPO cascade in all arthropod tested. Ratcliffe et al. (1991) showed that LPS could trigger the PPO cascade in whole homogenate of locust hemolymph, but not in
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DISCUSSION
D. coccus hemolymph activation was studied with different microbial elicitors. The first evident reaction was that carminic acid dye present in the hemolymph disappeared from the soluble phase after treatment with laminarin, zymosan and GalNac. These results of D. coccus hemolymph

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the hemocyte lysate of this insect. In contrast, in the wax moth, Galleria mellonella (Pye, 1974), PPO activation was not detected after injection of Gram-negative bacteria into hemocoel. These data indicate that, between insect species, differences in LPS sensitivity could exist. The lack of response to LPS by D. coccus hemolymph may indicate that hemolymph activation is not the mechanism to eliminate Gram-negative bacteria. 2. D. coccus hemolymph activation is blocked by protease inhibitors. Activation of PPO involves a serine proteinase cascade that cleaves PPO at specific peptide bond near its amino terminus (Sderhall et al., 1994). D. coccus hemolymph activation was inhibited by TLCK and TPCK (serine protease inhibitors). Moreover, Locusta migratoria (Boigegrain et al., 1992) and Pacifastacus leniusculus (Sderhall et al., 1990) PPO was inhibited with these same inhibitors. Inhibition of D. coccus activity was reached with millimolar concentrations of inhibitors, as occurs in Drosophila (Chosa et al., 1997). On the other hand, the process is only partially inhibited at 4C, which could be related to the survival of these organisms in extreme environmental conditions. We have observed in the field that other species of the Dactylopius genus that cohabitate with D. coccus, namely D. confusus and D. confertus, are able to survive even at temperatures < 4C. 3. PTU, a direct competitive inhibitor of phenoloxidase (Barrett and Andersen, 1981), blocked the hemolymph activation, indicating a connection with the PPO pathway (Li et al., 2002). 4. D. coccus hemolymph activation is blocked by eicosanoids production inhibitors. It has been shown that phospholipids, especially lysolecithin via phosphoplipase A2 action, activate PPO at mg levels (Sugumaran and Nellaiappan, 1991; Mandato et al., 1997). In Periplaneta americana and Limulus polyphemus, several lipids could activate the phenoloxidase enzyme (Sugumaran and NellaiSeptember 2003

appan 1991, Nellaiappan and Sugumaran, 1990). On the other hand, evidence has been presented suggesting that eicosanoids are able to activate adenylyl cyclases, which, in turn, can up-regulate, via cAMP production, the serine proteases required for the activation of PPO (Stanley-Samuelson, 1994) and other immune responses (Tunaz et al., 1999). This hypothesis is consistent with our results, supporting a role for PPO during D. coccus activation and its inhibition by prostaglandin inhibitors. In agreement with this, D. coccus hemolymph activation was blocked in the presence of eicosanoids production inhibitors, indicating an eicosanoid-dependent route similar to those described for other insects (Mandato et al., 1997; Stanley-Samuelson et al., 1991; Stanley, 1998). There is a high diversity both, in number and in molecular size, in the proteins present in D. coccus plasma observed by PAGE-SDS. Hemolymph activation of D. coccus was quick, happening in less than 2 min. On the other hand, an unspecific and massive diminution of proteins from the soluble phase was observed except for the 34-kDa molecules. This result suggested that the molecules consumed from the soluble phase could be trapped in the fibrillar precipitate. Interesting new characteristics of the D. coccus response to immune elicitors are presented here. During D. coccus hemolymph activation, carminic acid was consumed from the soluble phase. It may be possible that this molecule works as a substrate during hemolymph activation, forming part of the insoluble fibrillar precipitate. No information regarding a possible direct role of anthraquinones as a substrate for melanizing enzymes has been reported. However, the formation of ortho-quinones during the activation in vitro of oxidative immune response with neutrophil extracts, which bind to proteins, and damage them, has been described previously (Dean et al., 1993; Vilar-Rojas et al., 1996). On the other hand, phenoloxidase uses as substrate tyrosine, L-Dopa, other catecholamines, hydroxylated compounds, and quinones

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Charalambidis N, Foukas L, Zervas C, Marmaras V. 1996. Hemocyte surface phenoloxidase (PO) and immune response to lipopolysaccharide (LPS) in Ceratitis capitata. Insect Biochem Mol Biol 26:867874. Chase R, Raina K, Bruno J, Sugumaran M. 2000. Purification, characterization and molecular cloning of prophenoloxidases from Sarcophaga bullata. Insect Biochem Mol Biol 30:953967. Chosa N, Fukumitsu T, Fujimoto K, Ohnishi E. 1997. Activation of prophenoloxidase A1 by an activating enzyme in Drosophila melanogaster. Insect Biochem Mol Biol 27:6168. Dean R, Gieseg S, Davies M. 1993. Reactive species and their accumulation on radical-damaged proteins. TIBS 18:437 441. Fry, J. 1996. One way analysis of variance. In: Fry J, editor. Biological data analysis. A practical approach series. Oxford: Oxford University Press. p 111. Gonzlez L, Alba M, Garca-Gil, F, Lanz H, Rojas A, del Rio I, Hernandez-Hernandez F. 2002. Evaluation of the clotting response of the hemolymph of cochineal (Dactylopius sp.) and its predator (Laetilia coccidivora). Society for Invertebrate Pathology. XXXIII Annual Meeting. Guanajuato, Mxico. Abstracts p 47. Graham P, Ratcliffe N, Renwrantz L. 1986. The separation of insect haemocyte types on percoll gradients: methodology and problems. J Insect Physiol 32:167177. Jiangyong L, Zhao X, Christensen B. 1994. Dopachrome conversion activity in Aedes aegypti: significance during melanotic encapsulation of parasites and cuticular tanning. Insect Bioch Mol Biol 24:10431049. Joshi P, Lambdin P. 1996. The ultraestructure of hemocytes in Dactylopius confusus (Cockerell), and the role of granulocytes in the synthesis of cochineal dye. Protoplasma 192:199216. Lanz-Mendoza H, Hernndez S, Garrido-Guerrero E, Tsutsumi V, Archiga H. 1993. Prophenoloxydase system activation in the Crayfish: Procambarus clarki. Dev Comp Immunol 17:399406. Lee M, Anstee J. 1995. Endoprotease from the midgut of larval Spodoptera littoralis include a chymotrypsin-like enzyme with an extended binding site. Insect Biochem Mol Biol 25:4961.

during the Raper-Mason pathway and it may be suggested that the o-quinone groups of carminic acid could be used in a similar way. The o-phenolic quinones, once formed, react with nucleophillic groups on protein molecules to produce protein-phenolic compounds that form part of the melanotic capsules (Jiangyong et al., 1994). In support of this scheme, our preliminary results using PCR with specific PO primers suggest the presence of PO genes in D. coccus (data not shown). All these data taken together lead us to propose the hypothesis that carminic acid, an anthraquinone that has o-quinone groups similar to those raised during PO cascade, could be consumed by PO during hemolymph activation. At present, we are dissecting the molecular factors involved in D. coccus hemolymph activation to determine homologies and differences with other arthropod PO pathways, i.e., its regulation by eicosanoids, as suggested by the experiments presented here.

ACKNOWLEDGMENTS
We thank Febe Cazares Raga, PhD, Ivonne Marquez Garca, PhD, and Celina Llanderal, PhD, for their advice on the manuscript, and Ing. Ignacio del Rio (Tlapanocheztli Foundation) for providing biological material and critical and encouraging comments. We wish to express special thanks to Lorena Gonzlez and Mnica Alba for their technical assistance.

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