Minireview Adaptations against heme toxicity in blood-feeding arthropods

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InsectBiochemistry

andMolecularBiology

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doi:10.1016/j.ib

Abbreviations

tathione; GSSG

thioredoxin; SO

GR, glutathion

nius heme-bind�CorrespondE-mail addr

Insect Biochemistry and Molecular Biology 36 (2006) 322–335

www.elsevier.com/locate/ibmb

Minireview

Adaptations against heme toxicity in blood-feeding arthropods

Aurelio V. Grac-a-Souzaa, Clarissa Maya-Monteirob, Gabriela O. Paiva-Silvaa,Gloria R.C. Brazc, Marcia C. Paesd, Marcos H.F. Sorginea,

Marcus F. Oliveiraa, Pedro L. Oliveiraa,�

aInstituto de Bioquımica Medica, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude, Av. Brigadeiro Trompowsky, s/n, Cidade

Universitaria, Ilha do Fundao, 21941-690 Rio de Janeiro, RJ, BrazilbDepartamento de Fisiologia e Farmacodinamica, Instituto Oswaldo Cruz, Fundac- ao Oswaldo Cruz, Manguinhos, 21045-900 Rio de Janeiro, RJ, Brazil

cDepartamento de Bioquımica, Instituto de Quımica, Universidade Federal do Rio de Janeiro, 21945-570 Rio de Janeiro, RJ, BrazildInstituto de Biologia, IBRAG, Universidade Estadual do Rio de Janeiro, 210551-030 Rio de Janeiro, RJ, Brazil

Abstract

A blood-sucking habit appeared independently several times in the course of arthropod evolution. However, from more than a million

species of insects and arachnids presently living on earth, only about 14,000 species developed the capacity to feed on vertebrate blood.

This figure suggests the existence of severe physiological constraints for the evolution of hematophagy, implying the selective advantage

of special adaptations related to the use of blood as a food source. Digestion of vertebrate hemoglobin in the midgut of blood-feeding

arthropods results in the production of large amounts of heme, a potentially cytotoxic molecule. Here we will review mechanisms by

which heme can exert biological damage, together with a wide spectrum of adaptations developed by blood-feeding insects and ticks to

counteract its deleterious effects. In spite of the existence of a great molecular diversity of protective mechanisms, different

hematophagous organisms developed convergent solutions that may be physiologically equivalent.

r 2006 Elsevier Ltd. All rights reserved.

Keywords: Hematophagy; Oxidative stress; Heme; Antioxidant

1. Introduction

Arthropods are the most successful group of metazoa.Insects alone account for more than one million species.From this enormous pool of genetic diversity, about 14,000species, scattered among 400 different genera, havedeveloped the capacity to feed on vertebrate blood. It isalso clear from available evidence that hematophagy hasarisen independently several times during the course of theevolution of arthropods (Ribeiro, 1995). Although theprecise time when hematophagous arthropods first ap-peared is still uncertain, there is a tendency to set the origin

e front matter r 2006 Elsevier Ltd. All rights reserved.

mb.2006.01.009

: Hb, hemoglobin; Hz, hemozoin; GSH, reduced glu-

, oxided glutathione; Trx, thioredoxin; Trx(h2), oxided

D, superoxide dismutase; GPx, glutathione peroxidase;

e reductase; TrxR, thioredoxin reductase; RHBP, Rhod-

ing protein; HO, heme oxygenase

ing author. Fax: +55 2125626755.

ess: [email protected] (P.L. Oliveira).

of most groups in a cretaceous scenario (Lukashevich andMostovski, 2003; Mans and Neitz, 2004). This possibilityhas the attractive feature of suggesting that the firstappearance of hematophagy occurred in parallel to theearly split of mammals and birds, followed by diversifica-tion and spreading of these soft-skin terrestrial animals,which took the place of dinosaurs as dominant vertebrateson earth (65 MYA). Whatever be the exact date, severalfeatures of ancestors of modern bloodsuckers havefrequently been pointed as pre-conditions to hematophagy.Some of these pre-adaptations were of ecological natureand are related to living in close proximity to vertebrates,including feeding on skin remains, dung or even on fluidsof dead animals. More obvious was the need of morpho-logical adaptations such as mouthparts capable of piercingand cutting (Lehane, 1991). Although not usually recog-nized as pre-existing adaptations, several other features ofthe physiology of blood-sucking arthropods have beenindicated as playing an important role in conferring fitness

ARTICLE IN PRESSA.V. Grac-a-Souza et al. / Insect Biochemistry and Molecular Biology 36 (2006) 322–335 323

to hematophagy. By far, the most elegant and comprehen-sive body of work in entomological literature on thissubject is the description of the vast array of biologicalactive molecules designed to circumvent vertebrate hostresponse against arthropod biting, constituting an arsenalof molecules that enables blood-sucking creatures tointerfere on hemostasis, inflammation and immunity(Ribeiro and Francischetti, 2003).

Most blood-sucking animals ingest huge amounts ofblood in a single meal. Mosquitoes and triatomine bugstake in between three and ten times their body weights ineach meal (Friend et al., 1965). In the case of hard ticks, thevolume of blood ingested by a female can be as much asone hundred times the initial weight of the ectoparasite(Romoser, 1996). Hemoglobin (Hb), the most abundantprotein in mammalian blood, reaches concentrations ashigh as 150mg/mL and accounts alone for about 60% ofblood protein content. For that reason Hb degradation inthe digestive system of these animals will result in therelease of very high concentrations of heme, the prostheticgroup of Hb. In this review we are especially concernedabout the impact of the heme from the blood meal on thephysiology of blood-sucking arthropods.

2. Hb digestion and heme toxicity

Heme is a toxic molecule due to its ability to generatereactive oxygen species (Gutteridge and Smith, 1988).Furthermore, being a lipophilic anion, it has the capacityto alter membrane permeability and selectivity (Schmittet al., 1993). References to heme cytotoxicity can be foundfar back in the literature. Heme lysis of the bloodstreamforms of Trypanosoma brucei was described in 1977(Meshnick et al., 1977). Studies of the malaria parasite haveshown that these cells were promptly lysed by addition of

Heme -Fe+2 + ROOH Heme -Fe+3

Heme -Fe+3 + ROOH Heme -Fe+2 +

Fe+2 + H2O2 Fe+3 +

FENTON REACTION

LIPID PEROXIDATION

Heme -Fe+2 + ROOH Heme -Fe+3

Heme -Fe+3 + ROOH Heme -Fe+2 +

Fe+2 + H O2 Fe+3 +

+

RH + OH H2O + R

R + O2 ROO

ROO + RH ROOH +

Fig. 1. Heme and iron promote lipid peroxidation by different mechanisms.

reaction which generates hydroxyl radicals (OHd) that can initiate lipid pero

unsaturated fatty acid (RH), generating an alkyl radical (Rd ). In contrast, h

reactive organic hydroperoxides (ROOH) into highly reactive alkoxyl (ROd) a

10mM heme to the culture medium (Orjih et al., 1981). Therelevance of heme potential deleterious effects for Plasmo-

dium is reinforced by the inhibition of parasite growth bydiseases that are associated to intracellular Hb denaturationor increased oxidative stress, such as sickle cell anemia(Friedman et al., 1979) or glucose-6-phosphate dehydrogen-ase deficiency (Roth et al., 1983). Chloroquine, the most usedand studied antimalarial drug, acts on Plasmodium cellsthrough binding to heme in its food vacuoles, preventingheme aggregation into hemozoin (Hz) (Goldberg et al.,1990). In mammals, heme has been implicated in oxidativedamage to LDL and hence in the onset of pathologicalconditions such as hemolysis and atherosclerosis (Hebbeland Eaton, 1989; Balla et al., 1991).The molecular basis of heme toxicity, however, has been

frequently overlooked in the literature. Heme has beenshown to act as a promoter of the formation of free radicals,leading to the oxidation of lipids (Tappel, 1955; Gutteridgeand Smith, 1988), proteins (Aft and Mueller, 1984) andDNA (Aft and Mueller, 1983). Although hydroxyl radicalproduction in a Fenton-type reaction has been reported forheme free in solution or bound to hemeproteins such as Hb(Sadrzadeh et al., 1984), there is consistent evidenceindicating that heme-induced lipid peroxidation is exertedmainly by decomposition of organic hydroperoxides—instead of H2O2—into alkoxyl and peroxyl radicals (Kalya-naraman et al., 1983; Van der Zee et al., 1996) (Fig. 1). Onthe other hand, lipid hydroperoxides are formed during lipidperoxidation chain initiated by another source of reactivespecies such as superoxide formed by NADPH oxidase oreven by mitochondrial respiration, therefore suggesting thatheme pro-oxidant action might be better described asamplifying the formation of reactive species, depending onthe occurrence (and magnitude) of those de novo sources offree radicals to fully exert its toxicity.

H+ + ROO

OH + OH

RO

S T R E S S

H+ + ROO

OH + OH

OXIDATIVES T R E S S

OH +

R

Iron-induced oxidative stress is thought to be mediated by the Fenton

xidation chains by abstracting electrons from other molecules such as an

eme-induced formation of radical species relies on the conversion of low-

nd peroxyl (ROOd) radicals.

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Besides the radical-mediated toxicity described above,heme is an amphiphilic compound and is capable ofjumping into phospholipid membranes, leading to exten-sive leakage due to physical disturbance of the bilayerstructure (Schmitt et al., 1993). Heme-induced oxidativedamage to phospholipid membranes is already maximal atconcentrations in the range 50–100 mM and this (non-radicalar) effect on membrane permeability occurs above100 mM (Schmitt et al., 1993). Blood heme (bound to Hb) isaround 10mM, implying that midgut cells might beexposed to both components of heme toxicity in vivoduring digestion of a blood meal.

In contrast with these two general mechanisms thatpotentially can affect any type of cell, lysis of red bloodcells by heme has been attributed to the inhibition ofNa+K+ ATPase of the plasma membrane (Chou andFitch, 1981), raising the possibility that some effects ofheme may be restricted to a particular cell type. Alltogether, the data above support the proposition thatmassive release of heme in the digestive apparatus ofblood-feeding arthropods represents a selective pressurethat should have been counteracted—during the course ofthe evolution of hematophagy—by the generation of anarray of protective adaptations.

3. Heme aggregation

While bound to the globin polypeptide, heme is in asoluble form. However, the destabilization of Hb tertiarystructure during proteolysis eventually results in the releaseof the heme, in a process that has been well characterized inthe digestive vacuole of Plasmodium (Slater et al., 1991;Francis et al., 1997). Since heme solubility is low inaqueous solutions, it may have two distinct fates afterdissociation from Hb: insertion into hydrophobic sites suchas phospholipid bilayers and hydrophobic pockets ofproteins (if they are exposed) or, alternatively, hememolecules can get out of the solution by the formation ofheme aggregates (which demands recruitment of severalmolecules to generate an initial nucleation site) (Slater etal., 1991; Oliveira et al., 1999; Lara et al., 2003).Partitioning into membranes would lead to the deleteriouseffects of heme described before. Aggregation, on the otherhand, results in reduction of free radicals formation as aconsequence of simple sterical hindrance of heme mole-cules, once organic hydroperoxides have access only toheme at the surface of large aggregates (Oliveira et al.,2002). Therefore, increasing the rate of formation of hemeaggregates should be, in principle, beneficial to Hb-eatingorganisms. Old literature has neglected this point and hemeaggregates found in hematophagous animals were alwaysdescribed under the generic name of hematin, inaccuratelysuggesting that they are non-specific deposits of heme(Wigglesworth, 1943). However, for Plasmodium there is acomplete set of data characterizing a particular kind ofheme aggregate which is called ‘malaria pigment’ or Hz(Pagola et al., 2000). The seminal work of Slater and

colleagues determined the nature of the interaction betweenthe heme molecules in Hz, associating with each other bymeans of iron-carboxylate bonds (Slater et al., 1991).Finally, the complex structure of Hz was solved bysynchrotron radiation X-ray diffraction and consisted insingle heme molecules linked by reciprocal iron carboxylatebonds, producing dimers of heme which interact with otherdimers through hydrogen bonds between the propionateside chains of the porphyrin ring (Pagola et al., 2000).For a long time, Hz was considered to be unique to

malaria parasites, but its existence has been also demon-strated in other blood-feeding organisms such as thetriatomine insect Rhodnius prolixus (Oliveira et al., 1999),in the helminth Schistosoma mansoni, responsible forhuman schistosomiasis (Oliveira et al., 2000b) and in theparasitic protozoan Haemoproteus columbae (Chen et al.,2001). Thus, aggregation of heme into Hz turned out to berecurrent in nature. Several hematophagous organismsmake use of this strategy as an efficient way to disposelarge amounts of heme and protect themselves againstheme toxicity.Formation of Hz is favored by acid pH values near the

pKa of propionic side chains of the porphyrin ring(4.8–5.0), a condition close to the pH found in the digestivevacuole of Plasmodium and in the midgut of Rhodnius andSchistosoma (Terra et al., 1988; Bogitsh and Davenport,1991; Slater and Cerami, 1992). Beyond this point ofconsensus, the precise mechanism by which heme isaggregated into Hz has not yet been completely understoodand has been a point of much controversy in the literature(Ridley, 1996). Inside the food vacuole of Plasmodium itwas identified an activity capable of promoting hemeaggregation that was sensitive to antimalarial quinolinedrugs, which led Slater and Cerami (1992) to postulate aprotein with an ‘heme polymerase’ activity. This hypothesisreceived additional support with the demonstration thatthe histidine-rich proteins (HRPs) of Plasmodium were ableto induce Hz synthesis in vitro (Sullivan et al., 1996). Incontrast, it was shown that the activity present in the foodvacuoles of P. falciparum was resistant to heat and toproteinase treatments and would aggregate heme even inthe absence of any protein (Dorn et al., 1995, 1998), bymeans of an autocatalytic process, promoted by the pre-formed Hz itself and not by an enzyme. Bendrat et al.(1995), on the other hand, demonstrated that lipid fractionspurified from the food vacuole content, also could promoteheme aggregation into Hz, a conclusion reinforced by theobservation of Hempelmann et al. (2003) that Hz wasformed in close association with the food vacuolemembrane.

R. prolixus midgut is a privileged system to study thepossible correlation between membranes and heme poly-merization. Triatomine insects have perimicrovillar intest-inal membranes (PMV), which are extracellularphospholipid membranes that separate the midgut epithe-lial cells from the luminal content (Lane and Harrison,1979). Data from our laboratory demonstrated the

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involvement of the PMV on heme aggregation in R.

prolixus (Oliveira et al., 2000a). Therefore, a commonpattern for both Rhodnius and the Plasmodium is that hemeaggregation in vivo takes place in or near to hydrophobicenvironments, an observation that can also be extended toSchistosoma (Oliveira, M.F., unpublished results). On theother hand, the heating of the PMV membrane fractionprevents Hz formation, suggesting that a protein would beinvolved in this reaction (Oliveira et al., 2000a).

Possibly, all those three different catalytic mechanismsthat have been proposed may act together in vivopromoting Hz formation. Once a ‘primer’ of Hz crystal isformed upon adsorption of a protein bearing multipleheme-binding sites (such as the HRP of Plasmodium), thisinitial aggregate would be elongated by means of anautocatalytic reaction, and the rate of this second reactionmight be increased by hydrophobic environments (such asphospholipid membranes) (Dorn et al., 1995; Bendratet al., 1995; Hempelmann et al., 2003).

Regardless of the nature of the catalyst, heme aggrega-tion seems to be an extremely efficient process. In R.

prolixus, Hz may account for approximately 70% of thetotal heme content in the midgut lumen (Dansa-Petretski,personal communication), whereas in S. mansoni about50% of the heme released from Hb is converted into Hz(Oliveira et al., 2000b). As observed in Plasmodium, Hzformation in Rhodnius and Schistosoma was inhibited bychloroquine (an antimalarial quinoline drug), both in vitroand in vivo (Oliveira et al., 2000a, 2004). Quinoline drugsbind heme and this association diverts heme moleculesfrom the aggregation pathway, resulting in increased hemelevels, which leads to severe cell damage either through freeradical generation or by the interaction of heme withbiological membranes. These drugs lead to the death of thePlasmodium and to a drastic reduction of viability ofSchistosoma (Orjih et al., 1981; Sugioka and Suzuki, 1991;Oliveira et al., 2004). In R. prolixus a different profile wasobserved. These insects do not die upon feeding with bloodenriched with chloroquine, showing only a relatively mildpathologic condition, characterized by the presence in thehemolymph of increased heme titers together with elevatedlipid peroxidation (Oliveira et al., 2000a). Tolerance to theinhibition of Hz formation in Rhodnius is probably theconsequence of the concerted action of other protectivemechanisms that cooperate with heme aggregation in thegut lumen. Hence, treatment with chloroquine seems toinduce the upregulation of other defenses, such as hemedetoxification by a heme oxygenase (HO)-like activity(Paiva-Silva, unpublished results).

As mentioned before, all the organisms described to datethat are able to form Hz, digest Hb in acidic conditions,around pH 5, which is close to the pKa of heme carboxylategroups. However, vesicles with acidic pH and proteolyticenzymes do not seem to be a condition that leads to Hzformation by itself, because in the cattle tick Boophilus

microplus heme from Hb digestion is accumulated as a non-crystalline aggregate that clearly is not Hz (Lara et al.,

2003). Distinctly from other arthropods, where digestiontakes place in an extracellular environment (the lumen ofmidgut), Hb digestion is intracellular in ticks, occurring bymeans of proteolytic enzymes with an optimum acidic pH(Mendiola et al., 1996), inside a digestive vesicle of a specialcell lineage, the so-called digest cell (Tarnowski and Coons,1989; Gough and Kemp, 1995). In ticks, Hb seems to betaken up by receptor-mediated endocytosis and directed toa specific population of acidic vesicles (Lara et al., 2003,2005). A unique feature of the tick is that after Hbdigestion the heme moiety is transferred from the digestivevacuole to the cytosol and then accumulated into anothermembrane-delimited organelle, the hemosome (Lara et al.,2003, 2005). Inside the hemosome, in spite of not being acrystal structure as Hz, heme is packed as an organizedaggregate, within a structure made from the assembly ofseveral 40 nm-particles. For this reason, also in this case,the non-specific denomination of hematin, frequently usedin the past, is not an appropriate description for the tickheme aggregate. This point is reinforced by the fact thatthis material has a characteristic FTIR spectrum, distinctfrom that of hematin, which suggests the occurrence ofspecific molecular interactions in the structure of those 40-nm heme particles (Lara et al., 2003).Blood degradation in mosquitoes presents a completely

different picture. A first distinction is that heme solubility isincreased under neutral to slightly alkaline pH, whichcharacterizes the digestive tract of these insects. Moreover,from the arguments outlined above, this pH in principleshould preclude Hz formation. In Aedes aegypti, heme isalso insolubilized as a non-crystalline heme aggregate, butassociated to the peritrophic matrix (Pascoa et al., 2002),an extracellular layer with a complex composition ofproteins and polysaccharides (Shao et al., 2001). Theseheme aggregates can be isolated and also show spectro-scopic properties that are distinct from simple hematin,suggesting close association of heme moiety with othermolecules in the midgut of mosquitoes (Oliveira, P.L.,unpublished results).The formation of insoluble aggregates of heme inside the

digestive system of hematophagous animals seems there-fore to constitute a first line of defense against hemetoxicity. Although the formation of heme aggregates is acommon feature to all blood-feeding organisms discussedin this review, the diversity of aggregate structures andtheir mechanisms of formation put in evidence a highdegree of biological specificity. Hence, heme detoxificationthrough its aggregation may represent an ‘‘Achilles heel’’ ofblood-feeders and an important target for development ofdrugs that would selectively inhibit this process leading tooxidative stress conditions (as already found for both thePlasmodium and Schistosoma).

4. Antioxidant enzymes

Antioxidant enzymes play a major role in the protectionof cells against free radical damage. Traditionally, most of

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the studies involving antioxidant enzymes were concen-trated on: (i) Cu, Zn and Mn superoxide dismutases(SOD), enzymes responsible for the conversion of thesuperoxide anion into hydrogen peroxide in the cytoplasmand in the mitochondrial matrix, respectively (McCord andFridovich, 1969; Fridovich, 1995); (ii) catalase, thatcatalyzes the dismutation of hydrogen peroxide to oxygenand water (Chance et al., 1979); and (iii) glutathioneperoxidase (GPx), a peroxidase responsible for the reduc-tion of hydrogen peroxide and organic hydroperoxideswhich uses reduced glutathione (GSH) as a hydrogendonor (Ursini et al., 1995). Oxidized glutathione formed byGPx can be reduced by the action of glutathione reductase(GR), a disulfide-reducing enzyme that requires NADPHas a donor of reductive potential to maintain steady-statelevels of GSH.

Although GR is widespread in nature and is highlyconserved from yeast to mammals, Kanzok et al. (2001)demonstrated its absence in Drosophila melanogaster.

Instead, thioredoxin reductase (TrxR)—which catalysesthe reduction of disulfide thioredoxin (TrxS2) to dithiol[Trx(SH)2]—can functionally substitute GR. Thioredoxins(Trxs) are small, ubiquitous monomeric proteins with amolecular mass of 12 kDa and a redox active cysteine pairthat cycles between TrxS2 to [Trx(SH)2]. Therefore, thepair Trx/TrxR functions as a major intracellular disulfide-reducing system that could work together or replacefunctions that are assigned to the GR/GSH system inmammalian cells (Sztajer et al., 2001). The same authorsalso presented evidence that thioredoxins can regenerateGSH from GSSG non-enzymatically, an essential assump-tion of their model (Kanzok et al., 2000, 2001). TheDrosophila genome contains two different TrxR genes:TrxR-1 and TrxR-2, but only TrxR-1 mutation had aneffect on the viability of the organism. This single TrxR-1gene codes for two forms of the enzyme, a cytoplasmic andmitochondrial one, and both isoforms are essential forDrosophila viability. Genetic evidence from mutationalanalysis has shown that the thioredoxin system isfunctionally complemented by SOD and catalase in thedetoxification of free radicals (Missirlis et al., 2001, 2002).

In mosquitoes, the TrxR/Trx system also appears to behighly relevant in the antioxidant machinery. During theAnopheles gambiae genome project, a comparison of cDNAlibraries constructed from sugar- and blood-fed females ledto identification of several genes whose expression wasaltered 24 h after the blood meal, some of them potentiallyassociated to oxidative stress and heme metabolism (Holtet al., 2002; Ribeiro and Francischetti, 2003). At the sametime, Sanders et al. (2003) generated a gene chip with 1778clones obtained from one adult and one larval A. aegypti

midgut cDNA library. This chip was further used inmicroarray experiments to investigate blood meal-inducedmetabolic changes in the mosquito, focusing on the midgutand providing a more detailed profile of the time course ofgene expression (non-fed, 0, 3, 24 and 72 h ABM). Bothsets of data indicated increased expression of stress

proteins, in particular proteins related to the TrxR/Trxsystem, giving additional support to the proposition thatthese proteins would have a central role in oxygen radicaldetoxification (Kanzok et al., 2001). Several other stress-related proteins also came out from the microarray studieswith A. aegypti, delineating a general stress responsepattern after blood meal ingestion. Proteins such asGRP94 and Heat shock 70 family proteins were induced.Reduced expression of importins and transportins (nucleartransport) and different translation initiation factors suchas eIF4a, 5C and 3 were observed. The synthesis ofhelicases and ribosomal genes was also reduced and genesrelated to detoxification enzymes such as cytochrome p450sand ABC transporters were also modulated (Sanders et al.,2003).Regarding intermediate metabolism, one important

finding that may have relevance in the oxidative stresspicture was the observation of a pronounced decrease ofseveral glycolytic enzymes and an increase in mRNAs forcomponents of the gluconeogenesis pathway following ablood meal in Aedes. This fact was attributed to the changeof the main energetic source from sugar (prior to the bloodmeal) to proteins and lipids during blood digestion.Interestingly, in the report of Sanders et al. (2003) therewas an increase in hexokinase, but not in any otherglycolytic enzyme following the blood meal. It has recentlybeen shown (da-Silva et al., 2004) that in rat brain,hexokinase can associate to mitochondria, performing akey role as a preventive antioxidant against oxidativestress. This enzyme has been reported to reduce mitochon-drial generation of reactive oxygen species through anADP-recycling mechanism, avoiding a dangerous increasein the accumulation of reduced intermediates in themitochondria electron transport chain, which is one ofthe most important sources of oxygen radicals in mostaerobic cells. In this way, the maintenance of the midguthexokinase levels PBM could be another mechanism toreduce oxidative stress in this tissue which is at thismoment handling with the massive amounts of releasedheme.In R. prolixus, among all tissues, midgut ranked on top

for specific activities of SOD and catalase (Paes et al.,2001). SOD detoxifies superoxide but produces H2O2,scavenged by catalase, and therefore, both enzymestogether may be regarded as directed to face a ‘‘Fenton’’problem: hydroxyl radicals being produced from H2O2 andFe2+ (Fig. 1), and Fe2+ being regenerated from Fe3+ bysuperoxide (the so-called Haber–Weiss reaction). As hemeitself does not produce hydroxyl radical (Fig. 1) and doesnot seem to be a Fenton reagent (Ryter and Tyrrell, 2000),this finding suggested the production of a significantamount of iron. This was consistent with the presence offerritin in the midgut cells (Nichols et al., 2002) togetherwith the intense accumulation of a biliverdin-derivedpigment from a HO-like enzyme, which must generateiron as a by-product (Paiva-Silva, unpublished results).Evidence for intense production of H2O2 after a blood

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meal has been provided both for Rhodnius (Paes et al.,2001) and Anopheles (Kumar et al., 2003). As expected,control of redox balance was achieved by the concertedaction of SOD and catalase, which displays increasedexpression after a blood meal in the Anopheles midgut(Kumar et al., 2003). In the case of Rhodnius, glutathione-dependent mechanisms have been shown to operatetogether with catalase as inhibition of both glutathionesynthesis and catalase activity synergically increased H2O2

production in the midgut (Paes et al., 2001). As importantas the antioxidant enzymes, the preventive antioxidantaction of ferritin, a powerful iron-chelator, should not beunderestimated. In most organisms, ferritin is the majorintracellular iron-binding protein. Ferritin is widely dis-tributed in insect tissues being also secreted to hemolymph(reviewed in Nichols et al., 2002). Not by chance, midgut isan important site of ferritin synthesis in insects (Georgievaet al., 2002; Dunkov et al., 2002). In addition, bloodfeeding increases ferritin message and subunits abundancein A. aegypti midgut (Dunkov et al., 2002; Sanders et al.,2003). We will not discuss these data in further detail asother reviews in this same volume will discuss this subject(Dunkov and Georgieva, 2006).

Not less important than knowing which antioxidantdefenses are operating in the insect midgut, is to identifythe sources of oxygen radicals that, as mentioned before,act synergically with heme from the diet, generating aradical amplification chain. Natural candidates to beincriminated as oxygen radical producers are the alreadymentioned mitochondrial respiration and an NADPH-oxidase and the xanthine oxidase (XO). This last enzymecan exist both as an NADP-dependent xanthine dehydro-genase (XD)—in which circumstance it is not a cause ofoxidative stress, but only a source of uric acid, a powerfulantioxidant (Ames et al., 1981)—or alternatively theenzyme can be converted to the oxidase form (XO), whichuses O2 instead of NADP (Kuwabara et al., 2003), and,besides uric acid, results in superoxide radical production.Midgut cells play an essential role in the innate immuneresponse of insects, as they constitute their most exposedinterface to candidate pathogens. Oxidative burst resultingin the production of free radicals by the action of NADPH-oxidase has been implicated as one major microbe-killingdefense mechanism, but in arthropods until now, onlyhemocytes have been described to contain such an enzymeactivity (Pereira et al., 2001). However, there is noinformation on these essential questions and this shouldbe regarded as an important direction for future research.

5. Heme-binding proteins

In spite of heme detoxification by aggregation and bydegradation by HO in the midgut (discussed below), asignificant amount of heme from the blood meal stillreaches the hemocoel of blood-feeding arthropods (Dansa-Petretski et al., 1995; Lara et al., 2003). Coherently, both inRhodnius and Boophilus heme-binding proteins have been

found in the hemolymph (Oliveira et al., 1995; Maya-Monteiro et al., 2000). The adaptative value of heme-binding proteins can be understood as a way to control thepotentially dangerous reactivity of free heme and attenuateits toxicity. This assumption proved to be true both forthe Rhodnius heme-binding protein (RHBP) and for theHemelipoprotein (HeLp) of ticks (Dansa-Petretski et al.,1995; Maya-Monteiro et al., 2004), where a preventiveantioxidant role has been demonstrated. In both cases,association of heme to these proteins resulted in a markedreduction of its capacity to promote lipid peroxidation. Ananalogous picture is found in vertebrate plasma, where freeheme is immediately scavenged by hemopexin, a proteinthat binds heme with high affinity and inhibits heme-mediated peroxidative reactions (Seery and Muller-Eber-hard, 1973; Smith and Morgan, 1984; Gutteridge andSmith, 1988; Ponka, 1999). However, binding to a proteinby itself is not enough to restrict heme reactivity towardorganic peroxides, as some other proteins that have thecapacity to bind heme do not have this antioxidantproperty. As a matter of fact, some of them even stimulateheme-induced lipid peroxidation, as L-FABP, bovinealbumin, myoglobin and Hb (Vincent et al., 1989). In fact,what allows heme to act as a prosthetic group of a vastspectrum of enzymes that catalyzes very different reactionsis precisely the modulation of its reactivity by the chemicalenvironment of the protein binding site, rather than asimple silencing of chemical activity of the iron porphyrin.Therefore, an important question to which there is nosimple answer is how the structure and composition ofheme-binding sites of HeLp and RHBP can prevent freeradical formation by heme. Both proteins are heme-bindingproteins with antioxidant properties that also act as hemetransporters. In this way, RHBP and HeLp represent aremarkable example of functional convergence of comple-tely different molecular solutions. RHBP is a 12 kDamonomeric protein, capable of binding a single hememolecule, having no other prosthetic group and with aheme pocket resembling that of mammalian hemopexin,probably with two histidine residues as axial ligands(Oliveira et al., 1995). HeLp, in contrast, is a complexhigh molecular mass lipoprotein, with 354 kDa, twoapoproteins of 103 and 92 kDa, 33% of lipids and a smallamount of carbohydrate (Maya-Monteiro et al., 2000). Inthe tick hemolymph, HeLp has two heme moleculesalready bound to the protein and is capable of bindingsix additional heme molecules. Its light absorptionspectrum suggests a hydrophobic heme-binding site withno axial ligand, similar to human albumin (Beaven et al.,1974).It is tempting to raise the hypothesis that these two

heme-binding proteins arose from existing proteins, pre-viously assigned to other functions. From RHBP proteinsequence (Paiva-Silva et al., 2002), although rather spec-ulative because overall similarity is not high (20–35% ofidentical residues), we can hypothesize that they wereoriginated from an odorant-binding protein ancestor. This

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suggestion is supported by the similarity in size(12–15 kDa), position of conserved cysteines, presence ofa hydrophobic ligand binding site and also by the fact thatboth are secreted proteins (Xu et al., 2003). Moreover, thefact that odorant-binding proteins comprise a polygenicfamily would allow changes of function of one of itsmembers without a large loss of fitness.

HeLp homologs have been found in other species ofticks, Dermacentor variabilis and Ornithodoros parkeri

(Guderra et al., 2001) and partial sequence from Rhipice-

phalus appendiculatus has been obtained from an ESTproject (Nene et al., 2004). Its sequence indicates thatHeLp belongs to the very high-density lipoprotein family,which also includes vitellogenins (although HeLp itself wasnot detected in the eggs of Boophilus). This homologyimplies the presence in the ancestor protein of binding sitescapable of recognizing hydrophobic ligands and fits wellwith physical properties and chemical composition ofHeLp (Maya-Monteiro et al., 2000).

In the case of B. microplus, blood is the sole food sourcefor all larval stages and adult females (Sonenshine, 1993). Itis usually believed that all eukaryotic cells synthesize theirown heme (Ponka, 1997). Boophilus is an exception to thisgeneral rule, as this tick does not have a functional hemesynthesis pathway, and thus must rely exclusively on theheme obtained from digestion of the vertebrate blood inorder to make its own heme-proteins (Braz et al., 1999).The heme biosynthetic pathway is also defective in somepathogenic bacteria that depend on host blood as aheme source and, in these bacteria, proteins involved inheme transport were shown to have an essential role inheme reutilization (Lee, 1995; Wandersman and Stojiljko-vic, 2000). Following the same rationale, the dependence ofthe tick on heme from its diet must require specificmechanisms for heme absorption, transport and recycling,which have not been described for any other multi-cellularorganisms. HeLp has been shown to act as an inter-organheme transporter in Boophilus (Maya-Monteiro et al.,2000), being an essential adaptation to allow the tick todeal with this loss of the heme synthesis pathway.However, to efficiently reutilize heme an intracellularmachinery of heme-binding proteins must complement thisdescribed hemolymphatic heme transport system. At thispoint, one must stress that there is a critical lack ofknowledge on intracellular traffic of heme in all types ofeukaryotic cells (Smith, 1990). Heme is synthesized in themitochondria and several hemeproteins are assembled inthe cytosol (Ponka et al., 1973). HO is a microsomalenzyme but heme from lysis of red blood cells reachesmacrophages from the outside, both facts implying thatintracellular traffic of heme is widespread. In spite of ageneral recognition of the potential harmful effects of freeheme and the general assumption that inside the cells it isgenerally bound to proteins (Ponka, 1999), until now onlya few proteins have been related to intracellular hemetransport (Goldman et al., 1998; Immenschuh et al., 2003).Although speculative, it is plausible that these putative

pathways for heme cell traffic should be of remarkableimportance for blood-feeding arthropods. We have re-cently used a porphyrin substituted with palladium as afluorescent heme analog to follow the fate of this moleculein the digest cells of Boophilus (Lara et al., 2005). Aninteresting outcome of these studies was that the fluores-cence spectrum of this analog changed from a greenemission in water to a red emission upon binding toproteins, which allowed demonstration that heme in thecytosol of these cells was, in fact, associated to carrierproteins on its way from the digestive vesicle to thehemosome. Precise identification of the proteins involvedin heme transport inside these cells will be essential tounderstand how these animals are able to recycle hemefrom their diet.

6. Low molecular mass antioxidants

Low molecular weight antioxidants have a paramountimportance in protection against oxidative damage. Thesemolecules act not only as co-substrates in detoxifyingreactions, as glutathione and ascorbate do (Dickinson andForman, 2002), but also as radicals scavengers that convertreactive species into less harmful compounds. The generalconception is that these molecules are obtained from thediet and therefore antioxidant-rich or supplemented nour-ishment is desirable. Among the arthropods, it has beendocumented that plant-feeders can easily acquire potentantioxidant such as tocopherol, carotene and flavonoidsfrom their diet (Carlson et al., 1967; Rothschild et al.,1975). Accumulation of those molecules seems to be crucialin the protection against the oxidative insult that isgenerated by different natural-occurring molecules suchas benzofuranes, furanocumarines and quinines, whichultimately represent the plant defense against herbivores(Aucoin et al., 1995). Surprisingly, levels of these anti-oxidants were found to be extremely low in both Rhodnius

and tick hemolymph (unpublished).In insects, urate is known to be produced mainly in fat

body cells. After being released in the hemolymph, urate issequestered by the Malpighian tubules where it willconstitute the major component of the urine (Barrett andFriend, 1970). Urate is believed to be one of the mostimportant antioxidants in human plasma, acting both byforming redox inactive complexes with transition metalsand by intercepting hydroxyl radicals, singlet oxygen, lipidhydroperoxides and hypochlorous acid (Maples andMason, 1988). The demonstration that mutants of Droso-

phila that were not able to synthesize urate were moresusceptible to radiation-induced oxidative stress suggestedthat urate is also an important antioxidant in insects(Hilliker et al., 1992).We have found the presence of massive amounts of urate

in the hemolymph of Rhodnius, representing the mostpowerful low-molecular mass antioxidant in this fluid(Souza et al., 1997). Urate levels in the hemolymph ofRhodnius were approximately ten times higher than in

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human plasma, and blockage of urate synthesis in Rhodnius

increased both endogenous and heme-induced accumula-tion of lipid peroxidation products (Souza et al., 1997).This finding suggested a prominent role for this molecule inpreventing heme-mediated oxidative damage. Its increasedproduction in response to heme injection in vivo clearlyopposed the conception that low molecular weightantioxidants levels are controlled only at the diet level. Inline with these findings, Sanders et al. (2003) observed animportant increase in the levels of XD mRNA in themidgut of Aedes in response to the ingestion of blood.

7. Heme degradation

One important intracellular mechanism to control hemehomeostasis is its enzymatic degradation by the micro-somal HO. HO catalyzes the oxygen-dependent degrada-tion of heme to biliverdin, carbon monoxide (CO) and iron(Fe2+) (Tenhunen et al., 1969). In mammals, biliverdin isimmediately converted to bilirubin by a soluble enzymenamed biliverdin reductase (Kutty and Maines, 1981).Given that no convincing description of bilirubin-likepigments in insects has been done, it is accepted thatbiliverdin is the end product of heme catabolism in theseanimals.

HO has been extensively studied in the last few years indifferent models, not only by its obvious role as a heme-detoxifying enzyme, but also due to the biological activityof its products: biliverdin, carbon monoxide (CO) and iron.In mammals, CO presents anti-inflammatory and anti-apoptotic activities (Otterbein et al., 2000; Brouard et al.,2002). On the other hand, biliverdin and its reducedproduct bilirubin may work as important antioxidants(Stocker et al., 1987; Dore et al., 1999). Only recently thefirst insect HO homologue cDNA was cloned from the fruitfly Drosophila melanogaster (Zhang et al., 2004). Inopposition to mammalian HOs, the putative DrosophilaHO is not alpha-specific, producing also the isomers betaIX and delta IX of biliverdin from heme cleavage.

Although bilepigments are widespread among differentinsect orders, heme degradation in these animals hasreceived little attention. Rare examples are some studiesshowing that radiolabeled heme biosynthesis precursorscould be converted in vivo into biliverdin IX andbiliverdin-derived pigments by different phytophagousinsects (Rudiger, 1970; Choussy et al., 1975; Kayseret al., 1982; Kayser, 1985). Bilepigments were found inepidermal cells, interlamellar space of wings, hemolymphand eggs of different species (reviewed by Kayser, 1985),mostly associated to insecticyanin (Goodman et al., 1985;Saito, 1998) or to different chromoproteins, typically high-density lipoproteins (Haunerland and Bowers, 1986; Joneset al., 1988). It is the presence of biliverdin, for example, incombination with carotenoids, that confers a typical greencolor to some caterpillar teguments (Kawooya et al., 1985).

The physiologic importance of heme degradation ininsects is not completely clear. Incontestably, biliverdin has

a role in insect pigmentation. It provides an effect that maybe crucial in nature, especially for plant-feeding insects, asit can give a protective camouflage. It has also beenproposed that bilepigments may protect insects againstlight-induced oxidative damage (McDonagh, 2001).Bringing these data to the question of oxidative stress in

blood-sucking insects, it is reasonable to speculate thatheme degradation may have an antioxidant role, not onlyby the removal of the pro-oxidant free heme but also due tobiliverdin antioxidant properties (McDonagh, 1972; Stock-er et al., 1987). Nonetheless, heme degradation is supposedto be protective only when coupled to efficient ironremoval by ferritin, in order to avoid reactive oxygenspecies production by Fenton reaction (Ryter and Tyrrell,2000). In the blood-sucking bug R. prolixus, deposits ofiron were described in pericardial cells and midgutepithelium, where intense heme degradation occurs (Wig-glesworth, 1943). Remarkably, these are major sites ofexpression of ferritin genes in this insect (Paiva-Silva,unpublished data).In a pioneer study regarding heme metabolism in

hematophagous arthropods, Wigglesworth described thepresence of green pigments in the gut epithelium and inheart pericardial cells of the blood-sucking bug R. prolixus,attributing the presence of these biliverdin-like pigments tothe breakdown of host heme-Hb (Wigglesworth, 1943). Infact, midgut epithelium cells seem to be responsible fordegradation of heme molecules that arise from host Hbdigestion in the insect gut lumen (Paiva-Silva, unpublisheddata). On the other hand, R. prolixus pericardial cells areable to degrade heme transported from insect hemolymphto the heart by heme-RHBP (Paiva-Silva, unpublisheddata). This condition closely resembles what is found invertebrates, where heme bound to the plasma heme-binding protein hemopexin is delivered to cells, beingreadily degraded by HO, thus preventing accumulation offree heme (Ponka, 1999).In the kissing bug R. prolixus and in the mosquito A.

aegypti, both blood-sucking insects, biliverdins are notassociated to proteins. In these insects, the end products ofheme degradation are biliverdins conjugated to specificamino acids, cysteine and glutamine, respectively (Paiva-Silva, unpublished data). Amino acids attachment tobiliverdin structure turns this molecule into a more solublecompound, which is therefore easier to be excreted. Thesefindings suggest that heme degradation in blood-feedinginsects may have unique characteristics related to therequirement of an efficient disposal of massive amounts ofbiliverdin, produced after blood digestion. In fact, mam-mals perform bilirubin excretion by its conjugation toglucuronic acid residues. Failure in this process causesnewborn jaundice and neurological disorders in Crigle-r–Najar Syndrome (McDonagh, 2001). Accordingly, thesebilepigment modifications could represent a convergentevolutive case in a common effort to avoid membranedisturbance and cytotoxicity by these hydrophobic hemedegradation products.

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8. Regulatory effects of heme

The classical view that reactive oxygen species are onlyinvolved in the damage of biomolecules has changed withthe demonstration that these species can regulate impor-tant physiological processes such as cellular proliferation,differentiation, apoptosis and immune response (Poli et al.,2004).

Most of the work done on the role of oxygen radicals ininsect physiology is related to insect immunity where theyplay an important role. In Drosophila, the destruction ofthe eggs of the parasitoid Leptopilina boulardi seems toinvolve superoxide, hydrogen peroxide and nitric oxideproduction by plasmatocytes, probably interacting in away that generates the highly reactive species peroxynitriteand hydroxyl radicals (Nappi et al., 1995). The role of NOas a signaling molecule was recently addressed in Droso-

phila where this gas seems to play a pivotal role in theinnate immune response against Gram-negative bacteriathrough modulation of the Imd pathway (Foley and O’Farrell, 2003; Ratcliffe and Witthen, 2004).

We showed that induction of urate synthesis in R.

prolixus is triggered by the presence of heme, indicating theinvolvement of this blood component in the synthesis ofthis protective antioxidant. We found later that this processrelied on the activation of a protein kinase C (PKC) (Grac-a-Souza et al., 1999), suggesting that the synthesis of a lowmolecular weight antioxidant can be regulated by theavailability of a pro-oxidant molecule.

The demonstration that heme could modulate PKCactivity, raised the hypothesis that this molecule could beinvolved in other functions not only related to its pro-oxidant activity. In fact, we showed that heme is able toactivate human neutrophils, short-term living cells thathave central importance for the innate immunity inmammals (Grac-a-Souza et al., 2002). Different functionsin these cells were activated by heme, such as migration,oxidative burst and cytoskeleton reorganization. Anotherimportant aspect of this pro-inflammatory responsetriggered by heme is that this molecule is able to modulateneutrophils apoptosis, an important finding that suggeststhat heme might play a vital role in the development ofchronic inflammation by interfering with the longevity ofneutrophils, our first cellular defense line against invadingmicroorganism (Scapini et al., 2000; Arruda et al., 2004).Matzinger (2002) has proposed a unifying model forimmunity that applies equally for vertebrates and inverte-brates with the basic assumption that the immune responseis essentially concerned with cell and tissue damage, insteadof the old paradigm of self�non-self. Taking into accountthe potential toxic effects and signaling capacity of heme,we hypothesize that this molecule is an ancient ‘‘dangersignal’’, shared by different organisms as a signalingmolecule ultimately related to cellular injury, such ashemolysis or massive muscular damage. Blood-feedinginsects might experiment a unique situation as theaugmented levels of extracellular heme do not signal for

tissue damage, but are instead regular physiological events.As a consequence—although speculative—one may expectto find a heme-induced immunomodulatory response at themidgut-blood meal interface, capable of preventing ex-acerbation of innate immunity by heme. It is worthwhile tomention here that at least part of this regulatory loop couldbe fulfilled by the products of HO, biliverdin and CO,which were shown to have potent anti-inflammatoryactivities (Otterbein et al., 2003).

9. Parasites transmitted by insect vectors and heme toxicity

Pathogens transmitted by disease vectors go throughcritical steps of their life cycles inside the midgut ofhematophagous arthropods. This implies that an impor-tant part of their adaptation to a parasitic condition mayconsist in adapting to deal with the high heme concentra-tions and oxidative stress found in the midgut of theirinvertebrate host. In line with this concept is the fact thatmost studies related to flavivirus vector competence showthat infecting of the midgut cells is the most importantbarrier to successful infection of the insect vector (Black IVet al., 2002). Strong evidence supporting this hypothesiscame also from the work of Kumar et al. (2003) whichshowed that a strain of A. gambiae refractory toPlasmodium experiments a chronic state of oxidative stress,characterized by elevated levels of H2O2 and superoxide inthe hemolymph. Survival of the malaria parasite in the gutwas increased upon ingestion of antioxidants—ascorbateand uric acid—in the refractory strain, and these com-pounds have been shown to block the melanotic encapsu-lation of ookynetes. The same group also presentedevidence that an ookinete invasion of midgut cells inducedexpression of both NO synthase and a peroxidase that usednitrite and hydrogen peroxide to promote protein tyrosinenitration, eventually leading to apoptosis of the epithelialcell (Kumar et al., 2004; Kumar and Barillas-Mury, 2005).On the other hand, Thomas et al. (2002) showed thathemin alone is a powerful promoter of protein nitrationfrom just NO2 and H2O2, suggesting a non-enzymaticmechanism that would work in the same direction of theperoxidase-dependent pathway mentioned before. Theheme from blood meal may have a key role both as adirect participant on the generation of reactive species andas a regulator of redox equilibrium and immune response.The existence of a complex metabolism of oxygen and

nitrogen radical species points to the paramount impor-tance of an adequate balance between balance radicalproduction and protective mechanisms, a point clearlyobserved in a recent and elegant work (Ha et al., 2005) thatdescribed an extracellular catalase in Drosophila whoseknockout led to the death of the fly upon ingestion ofbacteria as a consequence of uncontrolled oxidative stress.Interestingly, even heat-killed bacteria induced seriousoxidative damage, providing evidence of a radical-mediated inflammatory-like immune response operatingin insect midgut. Therefore, mechanisms related to

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maintenance of redox balance of midgut cells and hemedetoxification mechanisms should play a main role in thedynamics of parasite–invertebrate host interaction andshould be situated in the center of development of vectorialcompetence.

10. Conclusion and perspectives

Taken together, the evidences discussed in this reviewsupport the concept that counteracting the deleteriouseffects of heme is an important driving force in theevolution of blood-feeding arthropods. Major pathwaysleading to protection against high nutritional intake ofheme in blood-feeding insects and ticks are summarized inFig. 2. Although part of these adaptations against hemetoxicity is constituted by defense mechanisms found also innon-hematophagous animals, the polyphyletic origin ofblood-feeding habit in the different groups of arthropods

Heme

HemeHeme bind

proteins

ROS

Peritrophic Matrix

(mosquitoes)

Hemosome(B.microplus)

Hemozoin(R. prolixus)

Biliverdinderivatives

+ CO + Fe+2

Fenton Reacti

2O +

H

HemeHb

Aggregation

Degradation(R.prol ixusA. aegypt i)

Degradation(R.prol ixusA. aegypt i)

LOWOXIDATIVE

STRESSS

Fig. 2. Major defenses of blood-feeding animals against heme toxicity are conc

by a combination of the formation of heme aggregates, heme degradation,

hemolymph with low molecular antioxidants and heme-binding proteins tha

resulting in a low level of oxidative stress in the other tissues. Hb—hemoglobin

peroxide; SOD—superoxide dismutase; TrxR—thioredoxin reductase; TrxPx—

thioredoxin; GSH—reduced glutathione; GSSG—oxidized glutathione; LMW

has generated a large amount of genetic diversity concern-ing the molecular nature of the protective mechanisms(Fig. 2). On the other hand, as the agent of naturalselection—heme toxicity—is the same for all groups, it isnot surprising to find several convergent solutions commonto species with large phylogenetic distances, regardless thediverse ways of reaching the same solutions. The generalpresence of mechanisms to promote aggregation of heme asa primary defense, the presence of heme-binding proteinswith antioxidant properties in the hemolymph of Rhodnius

and Boophilus and biliverdin pigments coupled to aminoacids to facilitate excretion in Rhodnius and Aedes, areexamples of convergence. Further research in the genomicsof these animals will reveal how much of evolutive noveltyhas been created during this process and probably severalnew adaptations to protect against heme deleterious effectswill be revealed. The recent recognition of the capacity ofheme—and its degradation products, CO, iron and

Biliverdinderivatives

+ CO + Fe+2 Ferritin

Antioxidantenzymes

ing

Ferritin

on

LMWantioxidants

(Urate, GSH)

HeLp(B. microplus)

RHBP(R. prolixus)

LMWantioxidants

(Urate)

Midgut

Hemolymph

Peripheral tissues

2H+

O + H O

H O + ½ O

GSSG

GSH

NADP+

ROOH ROH

H O H O + ½ O

Trx ox

NADPH

Trx red

HIGHOXIDATIVE

STRESS

O

Catalase

Antioxidantenzymes

SOD

TrxR

TrxPx

entrated in the midgut. Control of heme toxicity in the gut is accomplished

antioxidant enzymes and low molecular weight radical scavengers. The

t prevent generation of free radicals constitute a second line of defense,

; ROS—reactive oxygen species; O2d—superoxide anion; H2O2—hydrogen

thioredoxin peroxidase; Trx red—reduced thioredoxin; Trx ox—oxidized

—low molecular weight.

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biliverdin—to modulate protein function and gene expres-sion may be a major trend for future research. These maybe especially relevant to the study of hematophagousdisease vectors, due to the marked effects of heme onsignaling pathways related to innate immunity that mightconstitute an important new player on the game ofvector–parasite interaction.

Acknowledgements

We wish to express our gratitude to Ana Paula AbreuFialho Campos da Paz for helping with the figures. Thiswork was supported by the Third World Academy ofSciences (TWAS), CNPq, FAPERJ, FINEP, PRONEX,PADCT, FUJB and Howard Hughes Medical Institute.

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