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On the physico-chemical and physiological requirements of hemozoin formationpromoted by perimicrovillar membranes in Rhodnius prolixus midgut

Renata Stiebler a,b, Bruno L. Timma, Pedro L. Oliveira c,f, Giovanni R. Hearne d,Timothy J. Egan e, Marcus F. Oliveira a,b,*

a Laboratório de Bioquímica Redox, Programa de Biologia Molecular e Biotecnologia, Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazilb Laboratório de Inflamação e Metabolismo, Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem (INBEB),Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazilc Laboratório de Bioquímica de Artrópodes Hematófagos, Programa de Biologia Molecular e Biotecnologia, Instituto de Bioquímica Médica,Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, BrazildDepartment of Physics, University of Johannesburg, Johannesburg, South AfricaeDepartment of Chemistry, University of Cape Town, Private Bag, Rondebosch, South Africaf Instituto Nacional de Ciência e Tecnologia em Entomologia Molecular, Brazil

a r t i c l e i n f o

Article history:Received 29 September 2009Received in revised form21 December 2009Accepted 22 December 2009

Keywords:PlasmodiumSchistosomaTrypanosomaBiocrystallization

a b s t r a c t

Triatomine insects are obligatory blood-feeders that detoxify most of the hemoglobin-derived hemethrough its crystallization into hemozoin (Hz). Previous evidence demonstrates the key role of midgutperimicrovillar membranes (PMVM) on heme crystallization in triatomines. Here, we investigated someof the physico-chemical and physiological aspects of heme crystallization induced by Rhodnius prolixusPMVM. Hz formation in vitro proceeded optimally at pH 4.8 and 28 �C, apparently involving threekinetically distinct mechanisms along this process. Furthermore, the insect feeding status and ageaffected PMVM-induced heme crystallization whereas pharmacological blockage of PMVM formation byazadirachtin, reduced hemoglobin digestion and Hz formation in vivo. Mössbauer spectrometry analysesof R. prolixus midgut showed that Hz represents the only measurable iron species found four days aftera blood meal. Autocatalytic heme crystallization to Hz is revealed to be an inefficient process and thisconversion is further reduced as the Hz concentration increases. Also, PMVM-derived lipids were able toinduce rapid Hz formation, regardless of the diet composition. These results indicate that PMVM-drivenHz formation in R. prolixus midgut occurs at physiologically relevant physico-chemical conditions andthat lipids derived from this structure play an important role in heme crystallization.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

The midgut cells of insects belonging to the Orders Thysa-noptera and Hemiptera lacks a peritrophic matrix, a chitin-basedextracellular structure that separate the midgut cells from the foodbolus in most insect Orders (Lane and Harrison, 1979; Terra, 1988;Terra et al., 1996). Instead, the midgut epithelia are coveredby a double-layered phospholipid membrane known as peri-microvillar membranes (PMVM) (Terra et al., 1996), which have fewintegral proteins and extends toward the midgut lumen (Lane andHarrison, 1979; Gutiérrez and Burgos, 1986; Terra et al., 1996).

Much of the knowledge about PMVM came from studies of thehemipteran hemimetabolous kissing bug Rhodnius prolixus, one ofthe vectors of Chagas' disease and which has been used as a modelfor biochemical and physiological studies since the seminal work ofWigglesworth (Wigglesworth, 1934; Atella et al., 2005). In thisspecies, production and secretion of PMVM by midgut cells occursin response to the blood meal (Lane and Harrison, 1979; Billingsleyand Downe, 1983; Albuquerque-Cunha et al., 2004). Also, the fulldevelopment of microvilli and PMVM in the epithelial cellsdepends on the abdominal distension, ingestion of hemoglobin,and the release of ecdysone (Gonzales et al., 1998; Albuquerque-Cunha et al., 2004).

Many distinct roles have been proposed for PMVM, includingthe compartimentalization of the digestive process (Houseman andDowne, 1983; Billingsley and Downe, 1983, 1985, 1988; Ferreiraet al., 1988; Terra, 1990; Terra and Ferreira, 1994; Terra et al., 1996,2006), immobilization of digestive enzymes to avoid excretion

* Corresponding author at: Laboratório de Bioquímica Redox, Programa deBiologia Molecular e Biotecnologia, Instituto de Bioquímica Médica, UniversidadeFederal do Rio de Janeiro, Cidade Universitária, Rio de Janeiro, RJ 21941-590, Brazil.Tel.: þ55 21 25626755; fax: þ55 21 22708647.

E-mail address: maroli@bioqmed.ufrj.br (M.F. Oliveira).

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(Cristofolletti et al., 2003) and amino acid absorption (Terra, 1988).PMVM are also implicated in Trypanosoma cruzi epimastigotemultiplication and development in triatomines (Garcia et al., 1989;Gonzalez et al., 1989; Gonzalez and Garcia, 1992) and it also acts asa physical and chemical protection against toxic products of blooddigestion (Oliveira et al., 1999). In this regard, in R. prolixus theingested blood is stored in the anterior midgut, where red cells arelysed and water is absorbed. The blood content is then directed tothe posterior midgut, where carbohydrates, lipids and proteins areeventually digested (Terra et al., 1996). Especially, hemoglobindigestion occurs through the action of cysteine and asparticproteases (Terra, 1988), releasing peptides, amino acids and theprosthetic group heme. Heme in aqueous solutions is able topromote oxygen-derived free radicals formation (Davies, 1988; Vander Zee et al., 1996), lipid peroxidation (Tappel, 1955; Gutteridgeand Smith, 1988), as well as protein (Aft and Mueller, 1984) andDNA (Aft and Mueller, 1983) oxidation. Blood-feeding organismscounteract heme toxicity using an array of adaptations to amelio-rate or prevent heme-induced damage (Graça-Souza et al., 2006)and one of these mechanisms consists on the crystallization ofheme into a dark brown pigment named hemozoin (Hz), which wasfirstly identified in the digestive vacuole of the malaria parasites(Slater et al., 1991; Pagola et al., 2000). Besides malaria parasites,heme crystallization was also reported in other organisms such asin the helminth Schistosoma mansoni as well as in triatomineinsects, representing one of the major heme detoxification path-ways in these organisms (Oliveira et al., 1999, 2000a,b, 2002;Corrêa Soares et al., 2007, 2009).

There is a general consensus in the literature that amphipathicstructures, such as lipid droplets (Oliveira et al., 2005; CorrêaSoares et al., 2007; Pisciotta et al., 2007) and phospholipidmembranes (Oliveira et al., 1999, 2000a, 2005; Silva et al., 2007)play a key role in heme crystallization. In this regard, heme crys-tallization in the helminth S. mansoni occurs on the surface ofextracellular lipid droplets present in the gut lumen (Oliveira et al.,2000b; Corrêa Soares et al., 2007). Our group demonstrated theintimate association between Hz crystals and PMVM in fourtriatomine species (Oliveira et al., 1999, 2007). Strikingly, we alsodemonstrated that PMVM are able to catalyze heme crystallizationin vitro in temperature and chloroquine sensitive reactions (Oliveiraet al., 2000a). Recently, it was demonstrated that artificiallygenerated hydrophilicehydrophobic interfaces promote very rapidand efficient heme crystallization (Egan et al., 2006). These data leddistinct research groups to converge on the role of lipids in Hzformation. In fact, lipid-driven heme crystallization to Hz has beenreported since 1995, when Bendrat et al. (1995) showed that anacetonitrile extract from Plasmodium food vacuoles were able toproduce this heme crystal. Investigations over themechanism of Hzformation by PMVM were further carried out by Silva et al. (2007),which demonstrated that both a lipid extract and a protein fractionfrom PMVM were able to promote heme crystallization in vitro.However, Hz formation driven by PMVM-derived lipids in thatwork was significantly lower compared to the activity supported bynative PMVM. Also, there is recent compelling evidence that anenzymatic marker of PMVM, the a-glucosidase activity, is involvedin Hz formation by this structure possibly at the nucleation step(Mury et al., 2009).

Here, we investigated how physico-chemical and physiologicalconditions affect heme crystallization promoted by R. prolixusPMVM. The data presented here indicate that Hz formation inducedby PMVM occurs more favorably under physico-chemical condi-tions very similar to those found in the insect midgut, and thatlipids of PMVM are essential to increase the rate of heme crystal-lization. We also show that Hz formation in R. prolixus midgut isa very efficient process, since at least 97% of all iron-containing

species are present as Hz in this compartment four days aftera blood meal.

2. Materials and methods

2.1. Chemicals and reagents

Hemin chloride was purchased from Sigma Chemical Co.(St. Louis, MO, USA). Pyridine, sodium acetate, sodium bicarbonate,sodium carbonate, sodium hydroxide, glacial acetic acid, SDS,sodium citrate and others reagents were obtained from Merck(Darmstadt, Germany). All other reagents were of analytical grade.All water used in the study was of ultrapure grade.

2.2. Animals

For most of the experiments conducted here, adult R. prolixusfemales were reared at 28 �C and 80% relative humidity and fed onrabbit blood or plasma using artificial feeders (Garcia et al., 1975)andmaintained in our colony at Federal University of Rio de Janeiro.In this colony, all instars are fed every 30 days, while adults arereared every 21 days. To investigate the role of aging on Hzformation, seven groups of insects of different ages were separatedthree weeks after the last blood-feeding. Then, all groups were fedwith rabbit plasma in order to eliminate most of the Hz content inthe midgut (Oliveira et al., 2000a) which would potentially inter-fere with our measurements. Four days later, all insects weredissected to obtain the midgut content.

2.3. Heme crystallization assay

R. prolixus adult femalesmidguts were incubated in plastic tubescontaining 4 mL of phosphate buffered saline and protease cocktailinhibitors (Sigma, MO, USA) at 4 �C and gently shaken for 5 min.Then, the tubes were left undisturbed for 5 min to sediment thetissue debris and a supernatant aliquot was centrifuged 20,000 � gfor 10 min at 4 �C. The amount of protein in the pellet fraction wasmeasured following the method of Lowry (Lowry et al., 1951). Thisfractionwas used tomeasure heme crystallization activity (Sullivanet al., 1996). The reactions were carried out in polypropylene tubesin the presence of 0.5 M sodium acetate buffer, pH 4.8, 100 mMhemin, previously prepared in 0.1 M NaOH as 10 mM stock solu-tions, with a final volume of 1.0 mL. A sample corresponding to20 mg of protein was incubated for different times at 28 �C. Afterincubation, the reaction mixture was centrifuged at 17,500 � g for10 min at room temperature. The pellet was washed three timeswith “extraction buffer” (0.1 M sodium bicarbonate and SDS 2.5%,pH 9.1), and twice with milliQ water. The final pellet was solubi-lized in 0.1 M NaOH and the amount of heme was determinedspectrophotometrically at 400 nm in a GBC-920 spectrophotometer(GBC, Australia).

2.4. Lipids

Total lipids were extracted from fresh intestinal contents fromplasma or blood-fed insects using a chloroformemethanoleaqu-eous solution (2:1:0.8) mixture (Bligh and Dyer, 1959).

2.5. FTIR spectrometry

Fourier-Transformed Infrared (FTIR) spectroscopy was used toconfirm the identity of Hz made in the presence of PMVM-derivedlipids. For the experiments presented in Fig. 5, dried samples werehomogenized in Nujol mulls and spectra were recorded between2000 cm�1 and 1000 cm�1 in a PerkineElmer Paragon 1000 FT

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Infrared Spectrophotometer. FTIR analysis was also carried out ina Nicolet, Magna 550 in KBr pellets as previously described (Oli-veira et al., 2000a).

2.6. Preparation of b-hematin in lipid/water systems

Heme was first dissolved in 0.1 M NaOH and then mixed withacetone:metanol (1:9) to form a 2 mg/mL solution. Lipids weredissolved in acetone:methanol (1:9) in a final concentration0.5 mg/mL. Typically, we used 0.05 M citrate buffer pH 4.8 as anaqueous solution (50 mL) in a cylindrical vessel with an internaldiameter of 6.5 cm. A sample corresponding to 500 mL of hemesolution was mixed with 1.0 mL of lipid solution in acetone:methanol (1:9) and as then applied onto the top of 50 mM citratebuffer, pH 4.8. This mixture was incubated at 37 �C for 1 h. Hemecrystals were isolated by centrifugation of the reaction mixturefollowed by shaking the pellet in 2 M Hepes pH 7.5 bufferedaqueous solution containing 50% acetone and 5% pyridine.The final products were then characterized by FTIR spectroscopy(Egan et al., 2006).

2.7. Mössbauer spectroscopy analysis

To evaluate the iron-containing species found in R. prolixusmidgut, we dissected about 200 blood-engorged females four daysafter feeding and the midguts collected in a 1.2 mL cold poly-propylene tube and SpeedVac dried (Savant, model SC110). Becauseof the low iron content, a sample of as much as w880 mg of driedmidgut tissue was clamped in copper holders for low temperaturemeasurements in a Mössbauer spectrometer. A 25 mCi 57Co(Rh)source was used in conjunction with a KreCO2 proportionalcounter detector to record Mössbauer spectra in the normaltransmission geometry using a WISSEL constant acceleration drive.For measurements at w4 K, both sample and 57Co(Rh) Mössbauersource were top-loaded into a customized liquid-helium bathcryostat, where they were immersed in liquid helium. The sourcewas in close proximity to the sample and therefore at nearly thesame temperature. Measurements at 90 K were performed ina cold-finger cryostat with the source kept at room temperature.Typical count rate was w20,000 c-p-sec and data acquisition timewas for 18e24 h to obtain w106 counts per channel. The spec-trometer was calibrated with a natural iron-metal absorber(sample) held at room temperature, with the source kept at roomtemperature or at the same low temperature conditions as thesample. Line-widths of 0.24 mm/s were obtained for the innermost

lines of a 25-mm Fe-foil calibration spectrum. Isomer shifts arequoted relative to a-Fe at room temperature. Each spectrum, and itsmirror image, was acquired in 1024 channels, and then folded toremove geometrical base-line curvature. Adjacent channels wereadded to reduce statistical scatter before theoretical fitting waseffected on the final processed spectrum in 256 channels using theMössbauer fitting program NORMOS (WISSEL-GmbH). The spectrahave been fitted with components having Lorentzian profiles fromwhich the hyperfine interaction parameters (isomer shift IS/Fe,quadrupole splitting QS, and profile line-width LW)were derived. Itis mainly these parameter values (indicative of the Fe local envi-ronment) that serve to identify the iron-based species involved.

2.8. Data analysis

Kinetics of b-hematin reactions were analyzed by using linearleast-squares fitting methods with the program GraphPad� Prism5.0. Comparisons between groups were done by the non-pairedStudent's t-test or one-way ANOVA analysis of variance anda posteriori Tukey's test for pair-wise comparisons. The results wereexpressed as mean � standard error and considered significantlydifferent at p < 0.05 as indicated in figure legends. Student's t-test,ANOVA, Tukey's test and correlation analysis were performed byGraphPad Prism 5.0 software.

3. Results

3.1. Heme crystallization induced by PMVM occursat strict physico-chemical conditions

It is well known that heme forms highly insoluble aggregates atbiologically relevant conditions, such as those found inside Plas-modium parasites food vacuoles (Falk, 1964; Klonis et al., 2007).Among the physico-chemical requirements for heme crystalliza-tion, a pH close to the heme propionate pKa, measured between 4.8and 5.0, is of foremost importance (Slater and Cerami, 1992; Eganet al., 2001). As shown in Fig. 1A, the optimum pH for heme crys-tallization induced by PMVM in vitro was near 4.8, which is inagreement with results obtained with Hz formation in both Plas-modium and in artificial systems (Slater and Cerami, 1992; Eganet al., 2001) and is close to the estimations of R. prolixus midgut pH(about 5.5, according to Terra, 1988). Fig. 1B shows that PMVM-induced Hz formation is temperature-dependent, being maximalbetween 28 and 37 �C. It is important to notice that both pH andtemperature conditions were expressed as relative to control values

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Fig. 1. Physico-chemical characterisation of heme crystallization induced by R. prolixus PMVM. Adult females of R. prolixus were fed with plasma and four days later the midgutcontent was collected and centrifuged 20,000 � g for 10 min to obtain PMVM. Heme crystallization was assayed using 12 mg (A,B) or 20 mg (C) of proteins from PMVM with 100 mMof heme. (A) Heme crystallization was performed at different pHs at 28 �C for 24 h. The buffers utilized were 0.5 M sodium acetate (for pHs 3.5e6.0) and 0.5 M sodium phosphate(for pHs above 6.0). (B) Heme crystallization was performed at different temperatures at pH 4.8, with 0.5 M sodium acetate buffer, during 24 h. (C) Kinetic assessment of hemecrystallization induced by PMVM during 72 h using 0.5 M sodium acetate buffer, pH 4.8 at 28 �C. Data are expressed as mean � SEM, of two different experiments with replicates(n ¼ 3) in A, (n ¼ 4) in B, and (n ¼ 12) in C.

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since at pH values below 4 and temperatures over 37 �C, sponta-neous Hz crystallization occurs without PMVM (data not shown).Therefore, we chose a pH ¼ 4.8 and a temperature of 28 �C forall subsequent experiments. A kinetic analysis of Hz formationinduced by PMVM (Fig. 1C) show that three different stages can beidentified during Hz formation, the first one being nucleation thattakes place at early periods (up to 6 h). The second componentoccurs between 6 h and 24 h and is the period that we can observemost of the heme crystallization activity. Finally, an intermediate-efficient mechanism is present as the third component occurringfrom 24 h to 72 h.

3.2. Physiological aspects of heme crystallizationinduced by R. prolixus PMVM

To gain insight into the physiological requirements of Hzformation in R. prolixus, we observed the effect of diet, age, sex andthe pharmacological interference of PMVM production in thisinsect. Thus, in Fig. 2A, we evaluated the effect of feeding status onthe ability of midgut luminal content to promote heme crystalli-zation. We observed that midgut content from starved insectswere much less efficient at inducing heme crystallization whencompared with blood-fed insects (4 days after meal). We alsoinvestigated the effect of insect's aging on the capacity of PMVM toinduce Hz formation. Fig. 2B shows that PMVM isolated from

insects with different ages were capable to form Hz. Interestingly,the young adult insects about 150 days old were the most efficientat promoting heme crystallization compared to any other period ofthe insect's life (Fig. 2B). Also, a Fourier-Transformed InfraredSpectrometry analysis of pigments isolated from the midgut ofblood-fed males of R. prolixus indicates the presence of true Hz andsuggested that heme crystallization occurs in R. prolixus regardlessof the sex (Fig. 2C). Based on previous data showing that Hzformation is stimulated in the presence of isolated PMVM obtainedfrom R. prolixus midgut, we next evaluated whether pharmaco-logical interference of PMVM formation, by supplementing theinsect's blood meal with azadirachtin, would affect Hz formation invivo. Azadirachtin is known to be a potent natural insecticide(Morgan, 2009) and interferes with PMVM formation in tri-atomines, leading to alterations in the midgut ultrastructuralorganization, clustering of microvilli and disorganization of thePMVM (Nogueira et al., 1997). Thus, we testedwhether azadirachtinwould interfere with heme crystallization induced by differentcatalysts. In fact, azadirachtin did not interferewith Hz formation invitro induced by both b-hematin or PMVM (data not shown). Also,spectrophotometric analyses revealed that there was no changes inthe light absorption spectra of heme in the presence of azadir-achtin, suggesting that these compounds do not interact (data notshown). We observed in Fig. 2D, that supplementation of insect'sblood meal with azadirachtin caused a significant reduction not

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Fig. 2. Physiological aspects of heme crystallization induced by PMVM. (A) Adult females of R. prolixus that were kept for at least 20 days of starvation or four days after plasmafeeding were dissected. The midgut content was collected and centrifuged 20,000 � g for 10 min to obtain PMVM. Heme crystallization was assayed using 0.5 M sodium acetatebuffer, pH 4.8, 100 mM of heme, 12 mg of proteins from the pellet fractions of both insect groups (starved or plasma-fed) and incubated at 28 �C for 24 h (*p < 0.05). (B) Hemecrystallization activity of PMVM obtained from insects at different ages (ap < 0.001, 161 days old vs. all groups; bp < 0.01, 186 days old vs. 64, 124 and 236 days old; cp < 0.001, 211days old vs. 64, 94, 161, 186 and 236 days old). (C) Adult R. prolixus males were fed with blood and dissected four days later to obtain the midgut content. This sample was thensubjected to Hz isolation procedure, as described in methods, and the final material analyzed by FTIR to confirm the identity of Hz, exhibiting the characteristic transmittance peaksat 1666 cm�1 and 1215 cm�1. (D) Adult females of R. prolixus were fed with blood supplemented with 1 mg/mL of azadirachtin and samples of midgut content were obtained fourdays later to quantify both Hz (**p < 0.001) and total heme content (**p < 0.01) in the midgut. Data are expressed as mean � SEM, of at least two experiments (n ¼ 6 in A, n ¼ 5 in B,n ¼ 16 in D).

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only on the Hz content but also on the total heme present in theinsect midgut. This effect of azadirachtinwas not caused by reducedblood intake since theweight of insects right after a bloodmeal wasnot distinct among the groups (data not shown).

3.3. Hz represents the dominant iron-containingcompound in R. prolixus midgut

In order to identity the iron-containing species present inR. prolixus midgut, we performed Mössbauer spectroscopymeasurements of posterior midguts from insects dissected fourdays after blood-feeding. Using this technique, which is a probe ofthe local environment around the iron atom, it is possible to identifythe exact iron-containing species even in a complex biologicalmixture. This is usually accomplished by comparisonwith referenceMössbauer spectra of known iron species for which the hyperfineinteraction parameters are known or have been reported previ-ously. The spectrum of dried samples from the midgut measured at4 K was analyzed and show unambiguously the presence of a singleiron-containing species (Fig. 3A). This dominant iron-containingspecies has the characteristic fingerprints of Hz since the hyperfineinteraction parameters derived from the analysis of Mössbauer

spectra of both midgut samples and b-hematin (Fig. 3A and B) areessentially identical. This is also corroborated by the spectrameasured at 90 K (Combrinck et al., 2002). The Mössbauer analysisprovides compelling confirmation that at least 97% of all iron-containing species present in R. prolixus midgut four days after theblood meal is Hz. Other iron-containing species such as deoxy-hemoglobin, oxyhemoglobin, methemoglobin, heme monomers,heme m-oxo dimers and free iron are absent or below the detectionlimit of a few percent (Fig. 3A).

3.4. Lipids derived from R. prolixus midgut are efficientcatalysts of heme crystallization

A number of catalysts of heme crystallization have beenproposed and one of them is the autocatalytic crystal extensionmediated by Hz itself (Dorn et al., 1995). In order to investigate theautocatalytic activity, we incubated different concentrations of Hzisolated from R. prolixus with heme and then observed its effect onthe crystal's extension in vitro. Fig. 4A shows that R. prolixuspurified Hz was capable of promoting crystal growth after incu-bation with heme for 24 h. However, quantification of the amountof crystals formed through autocatalysis relative to the amount of

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Fig. 3. Hz represents the dominant iron-containing compound in R. prolixus midgut. Adult females of R. prolixus were fed with blood and four days later the midgut was collectedand dried under vacuum. 57Fe Mössbauer spectra of dried midgut sample (A) and b-hematin (B) reference measured at w4 K and 90 K. The solid line through the data points (opencircles) is the overall theoretical fit. Spectra at 4 K have been fitted with a single Lorentzian doublet component which in both cases yields hyperfine interaction parameters ofIS/Fe ¼ 0.42 mm/s, QS ¼ 0.58 mm/s and a line-width of 0.30 mm/s. Spectra at 90 K are each constituted of a doublet in which both relative intensities and relative widths of thedoublet have been allowed to vary and involve IS/Fe ¼ 0.40 mm/s, QS ¼ 0.58 mm/s.

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Fig. 4. Low efficiency of autocatalytic heme crystallization triggered by R. prolixus Hz. Different quantities of Hz (1, 2, 5, 10 or 50 nmol) were incubated at pH 4.8 with 0.5 M sodiumacetate buffer and 100 mM heme during 24 h at 28 �C. (A) Hz crystals produced by different quantities of pre-formed Hz. (B) Specific autocatalytic activity of R. prolixus Hz relative tothe amount of pre-formed Hz crystals. Data are expressed as mean � SEM of two experiments (n ¼ 4). *p < 0.05, statistically different from 1 nmol Hz.

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pre-formed Hz shows that the reaction's rate was significantlyreduced at higher Hz concentrations (Fig. 4B). Previous observa-tions show that in R. prolixus Hz crystals are found in close asso-ciation with PMVM (Oliveira et al., 2005, 2007; Silva et al., 2007)in such a way that its formation depends on the presence of thisstructure (Oliveira et al., 2000a). Fig. 5A shows that total lipidsisolated from midgut content of blood-fed insects (40 mg) stronglystimulated heme crystallization. Interestingly, stimulation of hemecrystallization was also observed in an artificial hydro-philicehydrophobic interface system, provided by lipids isolatedfrom either blood-fed (Fig. 5B) or plasma-fed (Fig. 5C) insectsapplied over the top of an acidic citrate buffer solution. Theidentity of Hz crystals produced through this method wasconfirmed by FTIR spectroscopy, showing the presence ofdistinctive absorption at 1210 cm�1 and 1663 cm�1 (Oliveira et al.,1999). It is important to notice that measurable amounts of Hzproduced by the interface method occur as early as 1 h after thereaction is initiated. Also, quantification of some components of R.prolixus midgut in adult females four days after the blood meal(Table 1) shows that the lipids content (about 239 mg/midgut) iscompatible with the conditions tested in Fig. 5 which may allowthese lipids to support Hz formation in vivo (Fig. 5).

4. Discussion

Hematophagous organisms developed very specialized adapta-tions to use vertebrate blood as a food source and also to avoidheme and iron toxicity (Graça-Souza et al., 2006; Oliveira and Oli-veira, 2002). In this regard, Hz formation represents a key hemedetoxification mechanism in several blood-feeding organisms(Egan, 2008). In the triatomine insect R. prolixus Hz formation ispromoted by midgut PMVM (Oliveira et al., 1999, 2000a, 2005,2007; Silva et al., 2007). Very recently, Hz formation was shown tobe associated with an a-glucosidase activity in such a way that itmay participate in the nucleation process of nascent Hz crystals(Mury et al., 2009). In the present work, we describe some of the

physico-chemical and physiological parameters that regulatePMVM-induced heme crystallization in R. prolixus. The resultspresented here indicate that PMVM-driven Hz formation occurs atphysiologically relevant physico-chemical conditions and thatlipids derived from this structure are important players of thisprocess, which are able to nucleate Hz formation. We alsodemonstrate that Hz represents the unique iron species detectablein R. prolixus midgut, which strongly indicates that heme crystal-lization in vivo is a very efficient process.

Previous evidence has shown that pH values near the hemepropionate pKa (about 4.8), plays a fundamental role to allow bothbiological (Slater and Cerami, 1992) and spontaneous (Egan et al.,2001) heme crystallization. Interestingly, pH values near 5 werefound in the digestive environments of hematophagous organismsthat produce Hz as a main heme detoxification route such as thePlasmodium parasites (Goldberg et al., 1990; Slater and Cerami,1992), S. mansoni flukes (Bogitsh and Davenport, 1991) and intriatomine insect Chagas' disease vectors (Terra, 1988). However,acidic hemoglobin digestion is not the unique requirement to allowheme crystallization since other blood-feeding organisms, such asthe cattle tick Boophilus microplus and the parasitic protozoaBabesia are unable to produce Hz (Lara et al., 2003; Stahl et al.,1978). The pH dependence of heme crystallization near the hemepropionate pKa may be explained by the structural requirement ofHz in which only one of the carboxylate groups must be de-protonated (Pagola et al., 2000), a condition that is only achieved atthe heme propionate pKa. Thus, other physiological factors arerequired during acidic blood digestion to allow biological hemecrystallization. A physiological interpretation of the strict depen-dence on temperature of PMVM-induced heme crystallization maybe related to the conditions where insects are kept in our colony(at 28 �C), which may determine the optimum composition of keyPMVM components (lipids and a-glucosidase) that support Hzformation. Furthermore, data from our group have previouslydemonstrated that pre-treatment of PMVM at higher temperatures(over 70 �C) caused a drastic loss of Hz formation activity (Oliveiraet al., 2000a). The data presented in Fig. 1C suggest that threedistinct mechanisms are involved in crystal growth, in such a waythat in each phase the rates of crystal growth are distinct.Conceivably, in early periods (0e6 h), nucleation of the first hemecrystals could be supported by the recently described a-glucosidaseactivity (Mury et al., 2009), which may be consistent with spots ofheme-peroxidase stainingwithin the PMVM as shown by Silva et al.(2007). Then, a second highly efficient process, acts between 6 hand 24 h. Possibly, during this period, extension of small Hz crystalscould be promoted by the hydrophilicehydrophobic interfacesprovided by lipids from PMVM. Finally, the later mechanism

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Fig. 5. Total lipids from R. prolixus midgut content promote Hz formation in vitro. Blood or plasma-fed adult R. prolixus females were dissected four days after feeding to obtain themidgut and total lipids were extracted following the method described earlier. (A) Heme crystallization activity was performed by incubating 40 mg of total lipids extracted fromblood-fed insects in the presence of 0.5 M sodium acetate buffer pH 4.8 with 100 mM heme at 28 �C for 24 h. Data are expressed as mean � SEM, of four experiments (n ¼ 15)(p < 0.05). Assessment of heme crystallization induced by a hydrophilicehydrophobic interface produced by layering total lipids extracted from blood (B) or plasma (C) fed insectsonto the top of 50 mM citrate buffer, pH 4.8 and kept at 37 �C for 1 h. The final reaction products were then characterized by FTIR spectroscopy. The large Nujol peak transmittancein the region between 1320 cm�1 and 1550 cm�1 are depicted in light gray whereas the key Hz peaks are shown at 1663 cm�1 and 1210 cm�1.

Table 1Quantification of components in R. prolixus midgut content. The levels of Hz, totallipids and proteins were quantified in the midgut content of 2nd feeding adultfemales of R. prolixus, 4 days after a blood meal. Results are expressed as mean andstandard deviation (SD).

Mean SD n

Hemozoin (nmol/midgut) 255.5 122.4 6Total lipids (mg/midgut) 239.3 25.3 3Total proteins (mg/midgut) 1.45 0.16 3

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(24e72 h) showed a less efficient heme crystallization activity,which could be supported in vitro by the spontaneous extension ofpre-formed crystals by autocatalysis. Experiments designed toinvestigate the synergistic action of both a-glucosidase and lipidson heme crystallizationwould be quite interesting to determine thereal contribution of each component on Hz formation in this insect.

Considering that blood digestion in R. prolixus is a gradualprocess, lasting about 15 days, heme toxicity due to the ingestion oflarge quantities of blood in a single meal is overcome by severaladaptivemechanisms fromagradual release of heme to its rapid andefficient crystallization into Hz. This would explain the data pre-sented in Fig. 3 that Hzwas the only iron species found in R. prolixusmidgut, while no other heme or iron species was detectable in thiscompartment. Thus, whatever the exact mechanisms involved,heme crystallization in R. prolixusmidgut is a very efficient process.Interestingly, a similar kinetic pattern of Hz formationwas observedin vivo, which suggest three different mechanisms involved in thisprocess (Silva et al., 2007).

Silva et al., 2007 have shown that development of PMVMchanges during blood digestion, in which maximum PMVM levelsare detected seven days after a blood meal (Silva et al., 2007). Theyalso demonstrate that PMVM levels are correlated with a-glucosi-dase activity (Silva et al., 2007) and it seems that part of the hemecrystallization activity is due to the nucleation action of a-gluco-sidase on Hz formation (Mury et al., 2009). Regarding the role ofinsect aging, since R. prolixus is an obligatory hematophagousinsect, it is not surprising that all the insect's stages are capable ofproducing this heme crystal. Nevertheless, we cannot explain whyPMVM from first adult stages are more prompt in crystallizingheme than in any other period of insect life. Possibly, higher levelsof a-glucosidase activity and/or a higher lipideprotein ratio presentin the PMVM of first adult stages would explain their highercapacity to produce Hz.

Azadirachtin is a natural compound that has been used toinhibit the development of PMVM, affecting several aspects ofinsect's physiology such as the adhesion of T. cruzi epimastigoteforms in the midgut (Garcia et al., 1989, 1991; Nogueira et al., 1997;Gonzales et al., 1998). Based on the data shown in Fig. 2D, inter-ference with PMVM formation decreases total heme content withinthe midgut suggesting that PMVM are important for blood diges-tion (Ferreira et al., 1988). Since Hz formation depends on the hemecontent in the midgut, it is not surprising that heme crystallizationin the midgut of R. prolixus is affected when blood digestion isimpaired. However, whether reduction of Hz content was a directconsequence of reduced PMVM content or indirectly throughinterference of hemoglobin digestion, is currently unknown. In thisregard, the complete development of PMVM depends on severalfactors such as midgut stretching caused by blood engorgement,hemoglobin content and secretion of specific hormones. In thisregard, the insect secretes the steroid hormone ecdysone aftera blood intake, which promotesmidgut epithelium growth and alsoPMVM formation and secretion (Albuquerque-Cunha et al., 2004).Full development of bothmicrovilli and PMVM in R. prolixusmidgutdepends not only on the abdominal distension but also to ingestionof hemoglobin, and subsequent release of ecdysone (Albuquerque-Cunha et al., 2004). Identification of glycosaminoglycans inR. prolixusmidgut pointed out the presence of measurable amountsof both heparan sulfate and chondroitin sulfate on PMVM (Costa-Filho et al., 2004). Preliminary data obtained by our group suggestthat chondroitin sulfate depletion of PMVM by chondroitinasetreatment caused no effect on their ability to promote hemecrystallization in vitro (data not shown). This indicates that Hzformation in R. prolixus midgut does not occur by any PMVMcomponent and strengths the concept that specific molecules/structures are directly involved in this process.

The autocatalytic activity of Hz has been known since the firststudies of Dorn et al. (1995), which demonstrated that bothsynthetic b-hematin and Plasmodium-purified Hz were able toextend crystal growth in vitro in heat-resistant reactions. The datashown in Fig. 4 clearly demonstrates that R. prolixus Hz is also ableto extend crystal growth, but this reaction is drastically affected athigher Hz concentrations (Fig. 4B). It is important to notice that,considering the limited volume of the midgut lumen, estimated inabout 8 mL (data not shown), and the amount of Hz present in thiscompartment (w270 nmol of Hz, Fig. 2D and Silva et al., 2007), theestimated Hz “concentration” (calculated as if it were in solution) inthe midgut lumen four days after a blood-feeding is about 30 mM(data not shown). Thus, it is unlikely that autocatalysis in R. prolixusplays an important role in Hz formation in vivo. A similar trend wasobserved S. mansoni lipid droplets (LD), extracellular structures thatmediate Hz formation within the parasite gut (Corrêa Soares et al.,2007). In S. mansoni, the higher the Hz content in LDs the lower isthe capacity to promote further Hz formation. Nevertheless,although the autocatalytic reaction is a low efficient process, Hzcrystals are promptly formed upon blood digestion (Fig. 3),implying the involvement of other catalysts, such as lipids in bio-logical heme crystallization.

Several lines of evidence have converged on the idea that lipidsplay amajor catalytic role in biological heme crystallization in acidicenvironments (Bendrat et al., 1995; Dorn et al., 1998; Fitch et al.,1999; Egan et al., 2001, 2006; Pisciotta et al., 2007; Jackson et al.,2004; Oliveira et al., 2004, 2005, 2007; Corrêa Soares et al., 2007). Inline with these findings, lipid bodies associated with Plasmodiumfood vacuoles are efficient catalysts of Hz formation in vitro (Jacksonet al., 2004). Also, rapid and efficient heme crystallization can beachieved in vitro in an artificial hydrophilicehydrophobic interfaceindicating that Hz formation could be performed through self-assembly of monomeric heme into nascent crystals (Egan et al.,2006). Supporting these observations, neutral lipids surroundingHz crystals in Plasmodiumwere able to promoteHz crystallization invitro (Pisciotta et al., 2007) and is closely linked to the lipids presentin the digestive vacuole of the parasite (Coppens and Vielemeyer,2005). Finally, in S. mansoni, heme crystallization occurs on thesurface of LDs in the intestinal lumen of the parasite (Corrêa Soareset al., 2007). The data shown in Fig. 5 are in agreementwith all thesefindings indicating that lipids are not only efficient catalysts of Hzformation but are also able to nucleate this process. However, theexact contribution of the a-glucosidase or lipid-driven Hz media-tion is unknown. Although Hz formation driven by PMVM-derivedlipids shown in Silva et al., 2007 exhibits negligible activitycompared to both PMVM-supported (Figs. 2 and 3 of Silva et al.,2007) and PMVM-derived lipids heme crystallization in ourexperiments (Fig. 5A), this apparent discrepancy can be explainedsince the amounts of lipids tested in our work were considerablyhigher compared to the amount tested in Silva's work (40 mg vs.15 mg). Thus, despite compelling evidence in favor of a-glucosidaseactivity as a promoter of Hz formation (Mury et al., 2009), PMVM-derived lipid are capable of supporting substantial heme crystalli-zation in vitro (Fig. 5). Interestingly, eventual changes in lipidscomposition of PMVM caused by different diets (Albuquerque-Cunha et al., 2004) did not affect heme crystallization activitysupported by lipids isolated from PMVM obtained from both bloodand plasma-fed insects (Fig. 5B and C).

In conclusion, the data presented here indicate that Hz forma-tion induced by R. prolixus PMVM occurs more favorably understrict physiologically relevant physico-chemical conditions, whichmay be efficiently promoted by lipids derived from this structure.We also provide strong evidence that Hz formation in this insect isa very efficient process, suggesting that, whatever the mechanism,heme crystallization occurs favorably within R. prolixus midgut.

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These findings provide important information concerning biolog-ical heme crystallization and open new perspectives for a betterunderstanding of this process in this Chagas disease vector.

Acknowledgements

We are grateful to Prof. Gloria R. Braz for the kind support duringmanuscript preparation and to Mr. João V. de Oliveira Neto, José deS. L. Junior, and Mrs. Litiane M. Rodrigues, for excellent technicalassistance. This investigation was supported by grants from Con-selho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq)(through Institutos Nacionais de Ciência e Tecnologia 2008),Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado doRio de Janeiro (FAPERJ) (MFO through Jovens Cientistas do NossoEstado 2007), International Centre for Genetic Engineering andBiotechnology (ICGEB), and Howard Hughes Medical Institute(HHMI). PLO and MFO are research scholars from CNPq.

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