Biochemical properties of the major proteins from Rhodnius prolixus eggshell

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Biochemical properties of the major proteins fromRhodnius prolixus eggshell

Denise M.D. Boutsa,1, Ana Claudia do Amaral Melob,c,�,1,Adriana Lyn Hunter Andradea,1, Mario A.C. Silva-Netoa, Gabriela de Oliveira Paiva-Silvaa,

Marcos Henrique Ferreira Sorginea, Lılian Soares da Cunha Gomesa,Heloısa S. Coelhoa, Adriano Penha Furtadoc, Eduardo C.M. Aguiara,Luciano Neves de Medeirosa, Eleonora Kurtenbacha, Sonia Rozentald,

Narcisa Leal Cunha-E-Silvad, Wanderley de Souzad, Hatisaburo Masudaa

aInstituto de Bioquımica Medica, Programa de Biologia Molecular e Biotecnologia, Universidade Federal do Rio de Janeiro,

21941-902 Rio de Janeiro/RJ, BrazilbInstituto de Quımica, Departamento de Bioquımica, Universidade Federal do Rio de Janeiro, 21941-909 Rio de Janeiro/RJ, Brazil

cCentro de Ciencias Biologicas, Departamento de Patologia, Universidade Federal do Para, 66075-110 Belem/PA, BrazildInstituto de Biofısica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, 21941-902 Rio de Janeiro/RJ, Brazil

Received 8 June 2007; accepted 17 July 2007

Abstract

Two proteins from the eggshell of Rhodnius prolixus were isolated, characterized and named Rp30 and Rp45 according to their

molecular masses. Purified proteins were used to obtain specific antiserum which was later used for immunolocalization. The antiserum

against Rp30 and Rp45 detected their presence inside the follicle cells, their secretion and their association with oocyte microvilli. Both

proteins are expressed during the final stage of vitellogenesis, preserved during embryogenesis and discarded together with the eggshell.

The amino terminals were sequenced and both proteins were further cloned using degenerated primers. The amino acid sequences appear

to have a tripartite arrangement with a highly conserved central domain which presents a repetitive motif of valine–proline–valine (VPV)

at intervals of 15 amino acid residues. Their amino acid sequence showed no similarity to any known eggshell protein. The expression of

these proteins was also investigated; the results demonstrated that this occurred strictly in choriogenic follicles. Antifungal activity

against Aspergillus niger was found to be associated with Rp45 but not with Rp30. A. niger exposed to Rp45 protein induced growth

inhibition and several morphological changes such as large vacuoles, swollen mitochondria, multi-lamellar structures and a disorganized

cell wall as demonstrated by electron microscopy analysis.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: Follicle cells; Eggshell proteins; Chorion formation; Antifungal activity; Rhodnius prolixus; Aspergillus niger

1. Introduction

The blood-sucking bug Rhodnius prolixus is an impor-tant vector of Chagas Disease in Central and South

e front matter r 2007 Elsevier Ltd. All rights reserved.

mb.2007.07.010

ing author. Instituto de Quımica, Departamento de

iversidade Federal do Rio de Janeiro, 21941-909 Rio de

zil. Tel.: +5521 2556 6867; fax: +5521 2562 7266.

ess: [email protected] (A.C.A. Melo).

rs contributed equally to this work.

s article as: Bouts, D.M.D., et al., Biochemical properties of th

oi:10.1016/j.ibmb.2007.07.010

America. The number of people infected with Trypanosoma

cruzi, the etiological agent of Chagas Disease, wasestimated at between 16 and 18 million, with a further100 million considered at risk (TDR report, 2002).Consequently all research concerning R. prolixus isconsidered an opportunity in the direction of findingsolutions for disease control. One very important aspect ofthe life cycle of this insect involves a period of embryodevelopment in the eggs that are deposited in theenvironment. At oviposition, the eggs contain all the

e major proteins from Rhodnius prolixus eggshell. Insect Biochem. Mol.

ARTICLE IN PRESSD.M.D. Bouts et al. / Insect Biochemistry and Molecular Biology ] (]]]]) ]]]–]]]2

nutrients and energy necessary for embryonic growth andhave to be able to protect themselves of all natural dangers.The evolutionary success of this group undoubtedlyinvolves the acquisition of this ability. R. prolixus eggsare formed in a telotrophic meroistic ovary that consists oftwo semi-ovaries connected by a common oviduct. Eachhemi-ovary contains seven ovarioles and each ovariole iscomposed of the vitellarium and of the terminal filament, alanceolate structure (the trophary), which contains thegermarium, the oocytes and pre-follicular tissues. Thevitellarium is composed of oocytes in different stages ofdevelopment that are surrounded by follicle cells (Vander-berg, 1963; Huebner and Anderson, 1972a–c; Lutz andHuebner, 1980, Atella et al., 2005) and each oocyte isconnected to nurse cells by trophic cords, until stage 8(1000–1500 mm in length) when the trophic cord closes(Pratt and Davey, 1972; Bjornsson and Huebner, 2004).Oogenesis can be divided in three phases: (1) pre-vitellogenesis, (2) vitellogenesis and (3) choriogenesis.Pre-vitellogenesis corresponds to a period of slow growthrate when the oocytes receive nutrients primarily from thenurse cells. Following pre-vitellogenesis the oocytes initiatea rapid growing phase (vitellogenesis) by the uptake ofproteins synthesized by fat body and ovary to form theyolk granules (Pan et al., 1969; Engelmann, 1979;Hagedorn and Kunkel, 1979; Postlethwait et al., 1980;Bownes, 1982; Brennan et al., 1982; Fourney et al., 1982;Harnish et al., 1982; Zhai et al., 1984; Bianchi et al., 1985;Peferoen and De Loof, 1986; Zongza and Dimitriadis,1988; Raikhel et al., 1990; Melo et al., 2000; Tufail et al.,2004). Choriogenesis corresponds to the period of synthesisof the protective eggshell (Beament, 1946b; King andAggarwal, 1965; Telfer and Anderson, 1968; Mazur et al.,1982; Berg, 2005). Some evidences have showed that thetransition between the vitellogenesis to choriogenesisdepends on the involvement of cyclic nucleotides (Wangand Telfer, 1996; Medeiros et al., 2002, 2004). During thetransition, genes associated with vitellogenesis are turnedoff and a different set of chorion genes is turned on. Thisleads to the synthesis of proteins which will constitute theeggshell, the vitelline membrane (VM) and the chorioniclayers of the egg as described for several insects (Kafatoset al., 1977; Margaritis et al., 1980; Orr-Weaver, 1991).Secretion of eggshell by follicle cells has already beenstudied in insects such as R. prolixus, Schistocerca gregaria,Drosophila melanogaster, Scaptomyza sp., Bombyx mori

and Leptinotarsa decemlineata (Beament, 1946b; King,1970; Blau and Kafatos, 1978; Kimber, 1980; Margaritiset al., 1980; Kambysellis, 1993; Leclerc and Regier, 1993;Regier et al., 1993; Pascucci et al., 1996; Papassideri et al.,2003). Insect eggshells are normally composed of threelayers, the VM, endochorion and exochorion (passing fromthe oocyte outwards). The endochorion and exochoriontogether are known as the chorion. The eggshell isassembled with distinct proteins specialized in protectingthe oocyte by apposition of newly synthesized protein uponexisting layers (Giorgi, 1977). In R. prolixus when the

Please cite this article as: Bouts, D.M.D., et al., Biochemical properties of th

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oocyte has reached its full size the eggshell formation starts(Beament, 1946b). The oocyte membrane, which has beentransporting material from the follicle cells to the yolkcavity, becomes the VM and is the base for the depositionof the chorion (Beament, 1946b). Rhodnius chorion isconstituted of the endochorion with five membranes: (1)inner polyphenol layer, (2) resistant protein layer, (3) outerpolyphenol layer, (4) amber layer and (5) soft proteinlayer; and the exochorion with two membranes: (1) softexochorion and (2) resistant exochorion (Beament, 1946b).The eggshell synthesis in Reduviidae has been studiedless than the Drosophila system. Eggshell formation inRhodnius begins with deposition of choriogenic proteinsonto the oocyte membrane during stage 9 of oogenesis,when the T oocyte length is around 1500–2000 mm (Prattand Davey, 1972; Bjornsson and Huebner, 2004). InDrosophila the VM proteins are also synthesized duringthe early stages of eggshell formation (stages 8–10),while endochorion and exochorion proteins are synthesizedlater, during stages 11–14 (Margaritis, 1985; Pascucci et al.,1996). The numbers of proteins that constitute the egg-shell are very variable for different insect groups. TheSDS–PAGE of eggshell proteins of several species ofDrosophila, shows six major bands with comparableelectrophoretic mobility (Thireos et al., 1980). While, thesilkmoth’s eggshell is considerably more complex, wheremore than 100 proteins have been identified in Antheraea

polyphemus (Regier et al., 1982).The eggshell is designed to facilitate fertilization and to

allow respiration of the developing embryo (Beament,1946a; King and Aggarwal, 1965; Telfer and Anderson,1968; Mazur et al., 1982; Berg, 2005). At the same time, itmust protect the embryo against microorganisms possiblyusing antimicrobial agents associated with the eggshell. Thepresence of antimicrobial agents associated with eggs hasbeen described in two insects. Marchini et al. (1997)described a peptide produced by accessory glands ofCeratitis capitata that have an antimicrobial activity.Lamberty et al. (2001) purified a peptide from femalesalivary glands of the termite Pseudacanthotermes spiniger

and suggested a possible antifungal role of this peptide inthe eggshell, since this female insect smears its eggs withsaliva during egg development.The present study describes for the first time that the

follicle cell of the Reduviidae bug R. prolixus synthesizetwo proteins that end up associated with the eggshell.These proteins are synthesized by follicle cells in aperiod that coincides with the end of vitellogenesis andbeginning of choriogenesis and are deposited onto theoocytes where they associate with the oocyte microvilli.Following the end of the choriogenesis fertilization occursand embryogenesis is started. The presence of theseproteins was monitored from oocyte up to all embryogen-esis and they remained associated with the eggshell. It issuggested that these proteins are part of the VM. Thepresence of antifungal activity associated with eggshellproteins is discussed.

e major proteins from Rhodnius prolixus eggshell. Insect Biochem. Mol.

ARTICLE IN PRESSD.M.D. Bouts et al. / Insect Biochemistry and Molecular Biology ] (]]]]) ]]]–]]] 3

2. Materials and methods

2.1. Rhodnius prolixus rearing

Insects were taken from a colony of R. prolixus

maintained at 28 1C and 70–80% relative humidity. Theinsects were adult mated females fed on rabbit blood at 3-week intervals following guidelines set by the UniversidadeFederal do Rio de Janeiro/UFRJ Institutional AnimalCare Committee.

2.2. Polyacrylamide gel electrophoresis

Electrophoresis was performed in the presence of sodiumdodecyl sulfate (SDS) (Laemmli, 1970) in a 10% poly-acrylamide gel, followed by staining with coomassiebrilliant blue G. The gels were destained using a mixtureof 7% acetic acid and 40% methanol. The molecular massof purified Rp30 and Rp45 proteins was estimated bySDS–PAGE separating gel using the following proteins:bovine serum albumin (BSA-66 kDa), ovalbumin (45 kDa),glyceraldehyde-3-phosphate dehydrogenase (36 kDa), car-bonic anhydrase (29 kDa) and cytochrome c (12 kDa)(Sigma, St. Louis, MO, USA).

2.3. Protein purification

The eggshell proteins were obtained from two differentsources: chorionated oocytes, dissected from ovaries, andeggshell collected soon after hatching. (A) Chorionated

oocytes: to obtain chorionated oocyte, ovaries weredissected under the stereomicroscope in 0.15M NaCl onthe 3rd day after adult blood meal. The chorionatedoocytes were removed from the ovary and extensivelywashed in saline. After that the chorionated oocytes weredisrupted and their yolk contents removed. The remainingeggshells were washed several times in 0.01M Tris/HCl pH8.4 in order to remove contaminating yolk proteins. (B)Eggshell: Soon after hatching the eggshells were collectedfrom the breeding cage and the embryonic cuticle carefullyremoved under the stereomicroscope. The eggshells werewashed in 0.01M Tris/HCl pH 8.4 several times. Then, theeggshells from both sources were homogenized separatelyand solubilized at room temperature (RT) as described byRegier et al. (1978) with some modifications. The eggshellswere homogenized strongly in a Potter in the presence of8M urea, 0.36M Tris/HCl (pH 8.4), 0.03M dithiothreitoland 0.1M PMSF and centrifuged at 12,000g for 10min.The supernatant was collected and stored at �20 1C forfurther use; the small precipitate obtained during centrifu-gation was discarded.

Urea-extracted proteins were applied to a 10%SDS–PAGE. After separation, the proteins were faststained using a saturated solution of KCl. The bandcorresponding to the Rp30 and Rp45 proteins was cut offand eluted by simple diffusion using 50mM ammoniumbicarbonate pH 7.8 with 0.01% SDS. After 1 h at 37 1C, the

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samples were centrifuged at 12,000g for 5min and thesupernatant was collected. The degree of purification wasmonitored using a second 10% SDS–PAGE. The resultingprotein concentration was determined using the method ofLowry et al. (1951).

2.4. Amino terminal sequencing

The Rp30 and Rp45 proteins were subjected toSDS–PAGE and then transferred to a PVDF membrane(Matsudaira, 1987). The amino terminal of these proteinswas determined by Edman degradation (Edman and Berg,1967) using Porton PI 2090 coupled to an HPLC HP-1090.The purified proteins transferred to the PVDF membranewere directly applied to the sequencer cartridge. Suchexperiments were carried out at the amino terminalsequencing facility at the Instituto de Bioquımica Medica,UFRJ.

2.5. Isolation of Rp30 and Rp45 genes

Degenerate primers (Rp30—50-TTYGCNGCNCCNT-TYTAYGG-30—Rp30 protein and Rp45—50-GGNCC-NGCNTAYTAYGA-30—Rp45 protein) were synthesizedbased on the amino terminal sequence of each proteinobtained by Edman degradation. Total RNA fromfollicular epithelium was purified using TRIzol reagent(Invitrogen). Five micrograms of total RNA were reverse-transcribed using the ‘Superscript pre-amplification system’(Invitrogen) and NotI-(dt)18 primer (Amersham-Pharma-cia). PCR reactions were performed with the respectivedegenerate and the NotI-(dt)18 primers. Amplificationconditions included 40 cycles of 94 1C—30 s, 51 1C—60 sand 68 1C—180 s. On the final cycle, 68 1C was maintainedfor an additional 6min. The PCR products were gel-purified, cloned using Perfect BluntTM cloning kit (Novagen)and sequenced at the Molecular Genetics InstrumentationFacility of the University of Georgia, Georgia, USA. Thetheoretical molecular weight of each cloned protein wasestimated using computer pI/MW for Swiss-Prot/TrEMBL (Gasteiger et al., 2005).

2.6. Alignment

The search for sequence similarities was performed bythe software FASTA and BlastP 2.2.2 using defaultparameters (Pearson and Lipman, 1988; Pearson, 1990;Altschul et al., 1997). The primary amino acid sequence ofthe two proteins was aligned using the ClustalW softwarepackage (Thompson et al., 1994). GenBank accessionnumbers are indicated in parentheses: R. prolixus Rp30(EF187283) and R. prolixus Rp45 (EF187284).

2.7. Northern-blot hybridizations

Total RNA was isolated from different tissues. For thenorthern-blot assays different follicle sizes were dissected

e major proteins from Rhodnius prolixus eggshell. Insect Biochem. Mol.

ARTICLE IN PRESSD.M.D. Bouts et al. / Insect Biochemistry and Molecular Biology ] (]]]]) ]]]–]]]4

and stored in RNAlater (Ambion) at 4 1C. Ovarioles werestaged based in terminal (T) follicle length (Pratt andDavey, 1972; Bjornsson and Huebner, 2004) following:500–600mm length follicle (early vitellogenesis), 900–1000mmlength follicle (late vitellogenesis) and 1500–2000mm lengthfollicle (choriogenesis). The apical trophic tissue (tropharies)was identified by morphology as described by Vanderberg(1963) and Huebner and Anderson (1972a). The RNAsamples (30 mg/lane) were separated by electrophoresisin 1.2% formaldehyde-agarose gels, transferred tonylon membranes (Sambrook et al., 1989) and probedwith 32P-labeled Rp30 or Rp45 full cDNA. The nylonmembrane was washed under high stringency conditions:three times with 0.6M sodium citrate, 0.6M NaCl and0.5% SDS-15min at RT, and once with 0.3M sodiumcitrate, 0.3M NaCl and 0.5% SDS-15min at 60 1C.Membranes were exposed to Kodak X-OMAT filmat �70 1C with an intensifying screen. Exposition timevaried from 1 to 7 days according to the radioactiveintensity. RNA sizes were calculated using an RNA ladder(Invitrogen).

2.8. Antiserum

Purified Rp30 (1mg) or Rp45 (1mg) proteins wereemulsified in complete Freund’s adjuvant and injectedsubcutaneously in the back of different 1.5 kg rabbits. Twoweeks after injection, a booster was given and 30 days laterblood was taken from an ear vein and the serum examinedby immunoblotting using total proteins (Towbin et al.,1979).

2.9. Immunoblotting

The Rp30 and Rp45 proteins were separated by agradient 6.5–22% SDS–PAGE for 180min at 2mA/cm andthen electrotransferred to a nitrocellulose membrane in25mM Tris, 192mM glycine, 20% methanol (pH 8.3) for120min at 150mA, followed by staining with PonceauRed, or prepared for immunostaining as follows: themembrane was incubated with antiserum raised againstpurified Rp30 or Rp45 proteins followed by secondaryanti-rabbit antibody conjugated with alkaline phosphataseand developed with NBT/BCIP (Towbin et al., 1979). Afterimmunostaining the membrane was washed several timeswith water and dried at RT. As a control of molecular massa pre-stained protein mix composed of myosin (205 kDa),b-galactosidase (116 kDa), phosphorylase b (97 kDa), BSA(66 kDa), ovalbumin (45 kDa), glyceraldehyde-3-phosphatedehydrogenase (36 kDa), carbonic anhydrase (29 kDa),trypsinogen (24 kDa), soybean trypsin inhibitor (20 kDa)and a-lactoalbumin (14 kDa) (Sigma, St Louis, MO, USA)was used. For the extraction of Rp30 and Rp45 proteinsduring embryogenesis, chorionated oocytes or eggs colle-cted at different days after oviposition were dissolvedin 8M urea as described in Section 2.3 and subjected toimmunoblotting as describe above.

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2.10. Aspergillus niger cultures and antifungal activity

A. niger strain (EK 0197) was collected by spontaneousspore decantation suspended in air at Petri plates with solidSabouraud medium (dextrose 40 g, peptone 10 g, agar 15 gper liter) at RT. The antifungal activity was assayed asdescribed by Broekaert et al. (1990) with some modifications.The fungal strain was grown at RT in liquid Sabouraudmedium (LSM). The fungal cells were seeded in a 96-micro-titer plate in LSM at a density of 3� 102 conidia/mL (100mLper well). Twenty microliters of the diluted-protein solution([Rp30] ¼ 1.0 mM; [Rp45] ¼ 0.1; 0.2; 0.5; 0.8 and 1.0 mM;[ALB] ¼ 1.0 mM) were added in different wells and the cellsuspension was incubated for 48 h at RT. The turbidity ofeach well was measured at 540 nm using a VERSAmax

microplate reader (Molecular Devices). Images werecaptured using a Zeiss NC-80 camera attached to a Zeiss-Stemi 2000-C microscope. In the electron microscopyassay, fungal colonies were grown in the presence of 10and 80 mM of Rp45 protein and 10 mM of BSA was used asa control.

2.11. Ovary preparation

Ovaries were dissected 3 days after blood meal. Thefollicles were examined under a Zeiss stereomicroscope andthe ovarioles separated for morphological analysis andimmunolocalization as described below. To obtain a layerof follicle cells, each follicle was opened up usingiridectomy scissors. Then the cytoplasm of the oocyteswas discarded so that the final preparation was a layer offollicle cells attached to the oocyte membrane. Thispreparation was also used for immunolocalization. Isolatedovarioles or a layer of dissected follicle cells attached to theoocyte membrane were fixed using 4% paraformaldehydein PBS. The fixed preparation was mounted onto coverglasses coated with poly-L-lysine, washed with PBS, andtreated with 150mM NH4Cl for 20min. Permeation wasobtained by treatment with 0.1% Triton X-100 in PBS for5min at RT. Non-specific staining was avoided bytreatment with PBS containing 1.5% BSA and 0.5% fishgelatin (blocking buffer-BB) for 30min. After incubationwith antiserum raised against Rp30 or Rp45 proteins(diluted 1:5000) for 60min, the preparations were washedwith BB and finally incubated with goat anti-rabbitsecondary antibody associated with fluorescein (Gibco,Grand Island, NY, USA) diluted 1:100 in BB, for 60minin the dark. The preparation was mounted with 0.2Mn-propyl gallate in 9:1 glycerol-PBS and analyzed usingZeiss laser scanning microscope (LSM 310). The imagesobtained were all processed using Adobe Photoshop.

2.12. Transmission electron microscopy

A. niger cultures were treated with Rp45 protein (10 mMor 80 mM) or BSA (10 mM) and then fixed for 2 h at RTwith 2.5% glutaraldehyde in 0.1M cacodylate buffer (CB),

e major proteins from Rhodnius prolixus eggshell. Insect Biochem. Mol.

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Fig. 1. Purification of urea-extracted proteins (Rp30 and Rp45) from

Rhodnius eggshells. Urea-extracted proteins from eggshells were applied

on the top of a 10% SDS–PAGE (Lane 1). After the run, proteins were

stained using a solution of 1M KCl. The corresponding bands to Rp30

and Rp45 proteins were cut off and eluted by simple diffusing with 50mM

ammonium bicarbonate pH 7.8 plus 0.01% SDS. The purification degree

was monitored using a second 10% SDS/PAGE stained with Coomassie

blue. Lane 1: urea-extracted proteins; Lane 2: purified Rp30 protein; Lane

3: purified Rp45 protein. Arrows indicate the positions of proteins named

Rp30 and Rp45. The numbers on the left are indicating molecular mass

standards.

D.M.D. Bouts et al. / Insect Biochemistry and Molecular Biology ] (]]]]) ]]]–]]] 5

pH 7.2. Post-fixation was carried out in 1% osmiumtetroxide in CB containing 0.8% potassium ferrocyanideand 5mM CaCl2. Thereafter, the cells were dehydrated inacetone and embedded in Epon. Ultrathin sections werestained with uranyl acetate and lead citrate and observedunder a Zeiss EM-900 electron microscope. As a controlnon-treated A. niger cells were processed by the sameprocedure and analyzed.

2.13. Immunoelectron microscopy localization of Rp30 and

Rp45 proteins

Follicles were fixed in a mixture of 0.1% glutaraldehydetype I and 4% paraformaldehyde in PBS (pH 7.2) for120min at RT. After fixation, oocytes were washed in PBSand dehydrated in a series of methanol solutions (30–90%),and finally embedded in Unicryl (British Biocell) at �20 1Cunder UV illumination. Ultra thin sections were collectedon 300 mesh nickel grids. The sections were subsequentlyincubated in PBS (pH 7.4) containing 150mM NH4Clfor 30min, PBS containing 1.5% BSA, 0.5% fish gelatinand 0.1% Tween 20 (blocking buffer Tween-BBT) for30min. Subsequently, samples were incubated in BBTcontaining antibodies raised against Rp30 or Rp45proteins for 60min (dilution 1:500). Afterwards, sectionswere washed in BBT, incubated with 10 nm gold-labeledgoat anti-rabbit IgG (1:100) (Sigma, St Louis, MO, USA)for 60min, and thoroughly washed in PBS. Grids wereexamined in a Zeiss EM-900 electron microscope, afterstaining with uranyl acetate and lead citrate. Controlexperiments were performed using nonimmune serumfollowed by incubation with gold-labeled goat–anti-rabbit IgG.

3. Results

3.1. Purification of Rp30 and Rp45 proteins

Eggshell protein profiles were analyzed by SDS–PAGE.The protein profile of eggshells revealed six major bands(Fig. 1, Lane 1) and two of the most abundant proteinswere purified for further use (Fig. 1, Lanes 2 and 3).The molecular mass of each protein was determined basedon the mobility of standard proteins. Due to theirmolecular masses they were named R. prolixus 30 kDaprotein (Rp30) and R. prolixus 45 kDa protein (Rp45)(Fig. 1—arrows).

3.2. Immunolocalization of Rp30 and Rp45 proteins in the

follicles

Antibodies against Rp30 and Rp45 proteins wereobtained in rabbits and used for immunoblotting. Fig. 2shows that the polyclonal antibodies are specific and theydo not recognize either hemolymph or oocyte proteins thatcould potentially contaminate the preparations. Theantibodies against both proteins were clearly associated

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with follicle cells (Figs. 3B and D). The inset in Figs. 3Band D presents a panoramic view of follicle cells suggestingthat both proteins are associated with them; however thetechnique used did not have enough resolution to show uswhether the labeling was inside or outside the cells. Inorder to obtain more information at cellular level a detailedmorphological analysis of follicle cells was performed usingelectron microscopy.The immunogold labeling technique was used in order to

detail the association of Rp30 and Rp45 proteins with thesestructures. Fig. 4 shows that these proteins co-localizeinside the follicle cells (Figs. 4A(inset) and B(inset)) andalso between follicles, suggesting that they have beensecreted to the space between cells. Interestingly bothproteins strongly associate with the microvilli (Figs. 4Aand C). Detail of this association is shown in the insetof Figs. 4A and C. In order to follow the fate of theseproteins we monitored their presence by immunoassayover different stages of oogenesis and embryogenesis,from oocyte up to the point of the hatching of the firstinstar larvae. Fig. 5 shows that the amount of theRp45 protein remains unchanged, from chorionatedoocyte to the point of first instar nymph hatching. In thesame way as Rp45, the quantity of Rp30 protein remainedunchanged during the whole embryogenesis (data notshown).

e major proteins from Rhodnius prolixus eggshell. Insect Biochem. Mol.

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Fig. 2. Western blotting of Rp30 and Rp45 proteins. A gradient 6.5–22% SDS–PAGE was run and then the gel was electrotransferred to a nitrocellulose

membrane. The membrane was challenged with primary antibody against each protein, washed and then challenged with a secondary antibody conjugated

with alkaline phosphatase and developed with BCIP/NBT. Lane 1 ¼ hemolymph proteins; Lane 2 ¼ egg homogenate; and Lane 3 ¼ eggshell homogenate

was used to check antibody specificity. (A) Coomassie blue stained gel. (B) Nitrocellulose membrane after electrotransference of the gel in (A) challenged

with antibody against Rp30 protein. (C) Nitrocellulose membrane after electrotransference of a similar gel in (A) challenged with antibody against Rp45

protein. Arrows show the position of Rp30 and Rp45 proteins.

Fig. 3. Immunofluorescence of follicles challenged with antibodies against Rp45 and Rp30 proteins. The preparation was challenged with antibody

against Rp45 protein (A and B) and against Rp30 protein (C and D). The fluorescence was visualized in a confocal laser scanning microscope (fluorescence

mode) (B) and (D) using goat anti-rabbit secondary antibody associated with fluorescein. Phase contrast (A and C). INSET: follicle cells free of oocyte

challenged with antibodies against Rp45 and Rp30 proteins. FC ¼ follicle cells; Y ¼ yolk. Bar ¼ 50 mm.

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3.3. Cloning, sequencing and analysis of Rp30 and Rp45

expression

The first 23 amino acid residues of each protein werededuced by Edman degradation. The sequence VXPNAG-

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XFPGFAAPFYGXYGVXP was obtained for the Rp30protein and the sequence XGPXGLVGDAGYLTG-PAYYDXFH was obtained for the Rp45 protein. Degen-erate oligonucleotides were designed based upon thesequences obtained from Edman degradation (underlined

e major proteins from Rhodnius prolixus eggshell. Insect Biochem. Mol.

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Fig. 4. Immunolocalization of Rp45 and Rp30 proteins in sectioned follicles embedded in Unicryl. Sections were treated with (A) Anti-Rp45 protein

antibody and (B-C) with anti-Rp30 protein antibody. (A) Follicle cell and microvilli, (B) a view of follicle cells and (C) oocyte microvilli. After incubation

with primary antibody, sections were incubated with 10 nm gold-labeled goat anti-rabbit IgG. MV ¼ microvilli; OO ¼ oocyte; FC ¼ follicle cells;

Y ¼ yolk; (*) intercellular space. Arrows indicate representative gold particles. INSETS show expanded view of follicle cells (A and B) and microvilli (A

and C) together with gold particles. Bar ¼ 50 mm.

Fig. 5. Western blotting of Rp45 protein during embryogenesis. Chorionated oocyte homogenate or egg homogenate collected on different days of

embryogenesis, as indicated in the figure, were dissolved in 8M urea and used to separate the proteins in a gradient 6.5–22% SDS–PAGE. The samples

were electrotransferred to a nitrocellulose membrane and challenged with antibody against Rp45 protein. The membrane was revealed with a secondary

antibody conjugated with alkaline phosphatase and developed with NBT/BCIP. Day 0 corresponds to chorionated oocyte; days 2–11 represents the

number of days after the eggs were laid; day 15 corresponds to the eggshell left behind after hatching.

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above—see Section 2.4 and 2.5). Ovaries were dissectedand RNA extracted with TRIzol reagent. The RNA wasused in a first strand cDNA synthesis reaction withNotI(dT)18 primers. The NotI(dT)18 primer was usedtogether with the degenerate primers in separate polymer-ase chain reactions (PCR) to amplify the cDNA coding forthe protein of interest. The clones encode a partial peptideof 220 and 362 amino acids in length to Rp30 and Rp45,respectively, which are missing the N-terminus. TheN-terminus also includes 10 amino acids of Rp30 and 14

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amino acids of Rp45 from Edman degradation. The cDNAcloned products had molecular sizes of 663 bp (Rp30) and1089 bp (Rp45), corresponding to polypeptides with pre-dicted mass of 24623.85Da and 38015.32Da, respectively.The theoretical molecular masses were lower than observedin SDS–PAGE, probably due to post-translational mod-ifications. An extension of 24 amino acids of N-terminus ofRp45 was also obtained using data from EST randomsequencing of cDNA library from R. prolixus follicle cellswhich confirmed a cleavage signal peptide in a deduced

e major proteins from Rhodnius prolixus eggshell. Insect Biochem. Mol.

ARTICLE IN PRESSD.M.D. Bouts et al. / Insect Biochemistry and Molecular Biology ] (]]]]) ]]]–]]]8

sequence (data not shown) (personal communication ofPaiva-Silva G. O. and Oliveira P. L., as part of R. prolixus

Genome Consortium). Since the cloning was made usingdegenerate oligonucleotides obtained from purified pro-teins the 50UTR region is still unknown this may explainwhy the size of RNA is much bigger than the protein theyencoded. The amino acid sequences deduced by thenucleotide sequence of Rp30 and Rp45 proteins arerepresented in Fig. 6A. The first 23 amino acids fromRp45 sequence obtained by Edman degradation was alsoconfirmed using the same data from EST randomsequencing of the cDNA library as mentioned above, thuswe can identify the X’s in the sequence were two cysteine(one in the first position and another one in the fourthposition) and one glycine (in the 21st position). The clone

Fig. 6. Deduced amino acid sequence of the Rp30 and Rp45 proteins. (A) A

residues are indicated by (*) and residues with similar properties by (:). Al

corresponding to 57.2% identity and 163 similar residues corresponding to 77.

are shaded and boxed. (B) Comparative analysis of C-terminal amino acid se

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sequence of Rp30 identified the two X’s in the N-terminuswhich were one serine in the 18th position and one glycinein the 22nd position.Both sequences present repetitive motifs of valine–pro-

line–valine (VPV) at every 15 amino acid in their centraldomains. Alignment of both sequences was performed andshowed great homologies in their VPV repetitive domain(Fig. 6A(boxed)), and a total of 57.2% identity and 77.48%similarity.The amino acid sequences of Rp30 and Rp45 proteins were

compared with other proteins by FASTA and BlastP 2.2.2(Pearson and Lipman, 1988; Pearson, 1990; Altschul et al.,1997). The alignment did not show similarity to any knowneggshell proteins. The Rp30 protein revealed similarity with aglycine-rich cuticle protein from B. mori (GenBank accession

lignment of amino acid sequence of Rp30 and Rp45 proteins. Identical

ignment results indicate that the proteins present 132 identical residues

48% similarity. Repetitive motives of VPV at every 15 amino acid residues

quence of Rp30 protein and R&R consensus motif.

e major proteins from Rhodnius prolixus eggshell. Insect Biochem. Mol.

ARTICLE IN PRESSD.M.D. Bouts et al. / Insect Biochemistry and Molecular Biology ] (]]]]) ]]]–]]] 9

no. AB197878—30.366% identity–47.644% similarity) whichalso presents a VPV motif; and with a cuticle protein fromAedes aegypti (GenBank accession no. EAT48061—26.257%identity–51.397% similarity). Interestingly the 25 C-terminalamino acid of Rp30 protein showed a modification of the‘‘R&R consensus sequence’’ motif proposed by Rebers andRiddiford (1988) which is present in many cuticle proteinsand is demonstrated to bind chitin (Rebers and Willis, 2001;Togawa et al., 2004) (Fig. 6B).

The Rp45 protein aligned well with glycine-rich proteinssuch as those found in Oryza sativa (GenBank accessionno. Q6ZF32—38.636% identity–61.364% similarity) andstructural proteins such as elastin precursor (GenBankaccession no. P07916—36.765% identity–58.088% similarity)and the flagelliform silk protein (GenBank accession no.Q9BIU8—34.965% identity–53.147% similarity), whereglycine residues are also abundant. The region of theRp45 protein which presents similarities with the glycine-rich proteins is the amino and carboxyl-terminal domain.The Rp30 protein does not possess this region. Thecomparison revealed that the Rp45 protein possesseshomology with a cytoskeletal protein (GenBank accessionno. Q39721—30.714% identity–53.571% similarity), that

Fig. 7. Expression of Rp30 and Rp45 genes by northern blot analysis. (A)

RNA samples were isolated from different tissues. (a) Membrane probed

with full cDNA-Rp30 gene; (b) membrane probed with full cDNA-Rp45

gene; (c) ribosomal protein gene was also amplified from each sample as a

control for RNA integrity. Tissues are indicated on the top of Fig. 8A. (B)

RNA from different tissues. Lane 1: ovary of non-blood-fed female; Lane

2: trophary; Lane 3: 500–600mm length follicle; Lane 4: 900–1000 mmlength follicle; Lane 5: 1500–2000mm length follicle; Lane 6: laid egg; Lane

7: ovary of blood-fed female. (d) Membrane probed with full cDNA-Rp30

gene; (e) membrane probed with full cDNA-Rp45 gene.

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also presented the VPV repetitive sequence. The amino acidcontent from deduced sequence varies in both proteins.The most abundant amino acid in Rp30 protein was valine(20.0%), followed by proline (11.3%), histidine (8.3%) andarginine (7.0%). In the Rp45 protein the most frequentamino acid also was valine (21.5%), followed by glycine(13.0%), alanine (9.2%) and proline (8.2%). This propor-tion could change when total sequence will be obtained.In order to analyze the expression patterns of Rp30 and

Rp45 genes in different tissues a northern-blot assay wasperformed. Results demonstrated that the expression ofthese genes only occurred in the ovaries (Fig. 7A). TheRp30 and Rp45 probes hybridized with a 4.1 and 4.9 kbband, respectively. The Rp30 probe was also observed tocross-hybridize to 6.3 kb band and 4.9 kb Rp45 band (datanot shown). Probably these facts are due to a similarity ofsequences between these RNAs. Moreover, this suggeststhe existence of another protein with a molecular weightlarger than either Rp30 or Rp45 expressed in follicle cellswith a similar sequence. To investigate the expressionpattern of the Rp30 and Rp45 genes in the ovaries anothernorthern-blot assay was performed with follicles indifferent development stages (Fig. 7B). Ovaries were

Fig. 8. Profile of Aspergillus niger growth. (A) Fungal cell growth was

monitored for 48 h at 540 nm (turbidity) in the presence and absence of

Rp45 protein. (—E—) control; (—K—) 1mM Rp45 protein; (—m—)

1mM BSA. (B) A. niger was allowed to grow for 48 h in the presence of

different concentrations of Rp45 protein as indicated in the figure. Photos

represent the slots containing different concentrations of Rp45 protein.

e major proteins from Rhodnius prolixus eggshell. Insect Biochem. Mol.

ARTICLE IN PRESSD.M.D. Bouts et al. / Insect Biochemistry and Molecular Biology ] (]]]]) ]]]–]]]10

dissected on the third day after a blood meal and stored inRNAlater (Ambion). Ovarioles were subdivided intotropharies (Vanderberg, 1963) and follicle staged interminal (T) follicles length in accordance to Pratt andDavey (1972) and Bjornsson and Huebner (2004) asfollows: 500–600 mm (early vitellogenesis), 900–1000 mm(late vitellogenesis) and 1500–2000 mm (choriogenesis), laideggs (negative control) and total ovaries (positive control).Expression of both genes was observed only in the1500–2000 mm follicle lengths and in the positive control(Fig. 7B—panels d and e). This result reinforces the factthat the putative proteins are exclusively from chorionicfollicles and that they may constitute a novel gene family.

Fig. 9. Morphology of Aspergillus niger following treatment with Rp45 protein

medium. (B) Panoramic view of A. niger cell treated with 10mMBSA. (C) View

shows that a 5 h-treatment with 10 mM of Rp45 protein is enough to induce the

following treatment with 10mM of Rp45 protein. (E and F) View of cell wall a

Gly ¼ glycogen particles; CW ¼ cell wall; M ¼ membrane; M-L ¼ multi-lame

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3.4. Antifungal activity

The results evidencing that the Rp30 and Rp45 proteinsremain associated with the eggshell are consistent with therole of these proteins in protecting the embryo duringdevelopment. To obtain further insight into possiblefunctions, these proteins were tested for antifungal activityin part due to their close association with the embryo.Fig. 8A shows that Rp45 protein inhibits the growth ofA. niger while BSA, extracted from the gel by the sameprocedure used to purify Rp45 protein, presented no effect.In order to determine its dose dependence, A. niger wasgrown in a medium containing different concentrations of

. (A) Panoramic view of non-treated A. niger cell grown for 48 h in culture

of A. niger cell following treatment with 10mMRp45 protein for 48 h. Inset

appearance of multi-lamellar structure. (D) Detail of swollen mitochondria

nd cytoplasm alterations following treatment with 80mM of Rp45 protein.

llar structure; MT ¼ mitochondria; VAC ¼ vacuoles. Bar ¼ 1mm.

e major proteins from Rhodnius prolixus eggshell. Insect Biochem. Mol.

ARTICLE IN PRESSD.M.D. Bouts et al. / Insect Biochemistry and Molecular Biology ] (]]]]) ]]]–]]] 11

Rp45 protein (Fig. 8B). The concentration of Rp45 proteinnecessary to inhibit 50% of A. niger growth was 0.91 mM.The Rp30 protein revealed no antifungal activity, at least,against A. niger (data not shown).

3.5. Morphological alterations of A. niger promoted by the

presence of Rp45 protein

The addition of Rp45 protein in the medium, besidesinhibiting A. niger growth, also induced morphologicalalterations in the fungal cells as well as in the cell wall(Fig. 9). The control cell (Fig. 9A) or BSA-treated cell(Fig. 9B) showed normal morphology. Fungal cellspresented a large amount of glycogen, mitochondria anda well-developed cell wall (Figs. 9A and B). The fact thatBSA (purified by the same procedure used to obtain Rp45protein) did not affect the morphology of fungal cellssuggests that the procedure used to obtain Rp45 proteindid not bring contaminants from the acrylamide gel thatcould potentially affect the fungus. The treatment with10 mM of Rp45 protein for 48 h induced the appearanceof multi-lamellar structures (Fig. 9C) absent in controlcells (Fig. 9A and B). The inset in Fig. 9C shows that a5 h-treatment is enough to induce the appearance of thesestructures. Ten micromolars of Rp45 protein also affectedthe mitochondria organization (Fig. 9D), clearly showingswollen mitochondria.

An increase in the concentration of Rp45 protein from10 to 80 mM leads to more significant effects on themorphology of the cells (Figs. 9E and F). A disorganiza-tion of the cell wall is clearly seen (Inset—Fig. 9F) as wellas the presence of large vacuoles (Figs. 9E and F).

4. Discussion

Numerous studies have been published on the secretionand morphogenesis of chorion in different insects (Regieret al., 1978; Kimber, 1980; Margaritis et al., 1980; Mazuret al., 1980; Regier et al., 1982; Hamodrakas et al., 1985;Margaritis, 1985; Papassideri and Margaritis, 1996). InDrosophila, the chorion genes are amplified by the folliclecells in response to developmental signals, prior to theirtranscription (Orr-Weaver, 1991). The number of proteinsin chorion varies for different insects. In D. melanogaster

about 20 chorion proteins are present in the eggshell, whileabout 186 proteins were resolved by two-dimension gelelectrophoresis as chorion constituents in A. polyphemus

(Regier et al., 1980, 1982). All the genes responsible forthe proteins that will be part of the eggshell are turned on,at the same time the genes that take part in vitellogenesisare turned off (Kafatos et al., 1977). In Hyalophora

cecropia the termination of vitellogenin uptake seems tobe associated with the increase of cAMP (Wang andTelfer, 1996). In R. prolixus, Medeiros et al. (2002, 2004)provided evidences that eicosanoids control the oogenesis,through the modulation of cAMP levels. Whether ornot eicosanoids are related to the transition from

Please cite this article as: Bouts, D.M.D., et al., Biochemical properties of th

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vitellogenesis to choriogenesis and which role cAMPplays in the control of the gene expression remains to bedefined.It has been well established that in most insects the

eggshell synthesis occurs by apposition of material over apre-existing layer, such as VM (Giorgi, 1977; Margaritiset al., 1980; Margaritis, 1985). Ultrastructurally chorionconsists of fibrous layers that run in parallel to the chorionsurface (Smith et al., 1971; Kafatos et al., 1977; Mazuret al., 1982). At the end of morphogenesis the chorionstructure is finalized by the formation of disulphide bonds(Blau and Kafatos, 1978).The results presented here indicate that R. prolixus

eggshell formation might follow the general patterndescribed above. The Rp30 and Rp45 proteins isolatedfrom the eggshell are synthesized by follicle cells and areeither secreted to the space between them or onto theoocytes. Here we showed that they associate withthe oocyte membrane, especially at the microvilli, duringthe initial stage of choriogenesis. The localization of Rp30and Rp45 inside the follicle cells, their association with theoocyte membrane and also to the eggshell left behind bythe first instar nymph clearly evidence that these proteinsare important during the early stage of eggshell construc-tion. Considering that all eggshell layers are formedsequentially by apposition of proteins during the lastpart of oogenesis (Giorgi, 1977; Margaritis et al., 1980;Margaritis, 1985), it is tempting to speculate that theseproteins are used to build up the VM. Althoughcontroversy exists with respect to the use of the term VMand its origin (Clements, 1992; Bate and Arias, 1993; Valleet al., 1999) here it is used in accordance with Beament(1946b). Another evidence that reinforces our hypothesisconcerning these proteins are a component of VM is thatscraping the innermost layer of the eggshell, named VM byBeament (1946b), Rp30 and Rp45 can be obtained (datanot shown). These proteins similar to what is observed inthe Drosophila system remain insoluble throughout theembryogenesis.The R. prolixus Rp30 and Rp45 proteins have very

similar sequences, which are mainly found in their centraldomains. These data suggest that the genes that codifythese proteins may be paralogous, being originated by theduplication of an ancestral gene followed by a divergencein their sequences. The central domains of these proteins,constructed by the repetitive VPV consensus sequencesexactly eight times in tandem, probably present a peculiarthree-dimensional structure. As this very similar feature ispresent in both proteins, it must play an importantfunctional role. The fact that in both proteins there areexactly eight of these repetitive units suggest that in orderto fold correctly, this domain must be present. The VPVmotif was also found in hypothetic proteins available inthe genome project of other vector insects such asA. aegypti (PS50326—identity 36.67% that has a valinerich region and a signal peptide cleavage) and Anopheles

gambiae (ENSANGP00000022326—identity 35.66% that

e major proteins from Rhodnius prolixus eggshell. Insect Biochem. Mol.

ARTICLE IN PRESSD.M.D. Bouts et al. / Insect Biochemistry and Molecular Biology ] (]]]]) ]]]–]]]12

has proline- and valine-rich regions). The VPV motif is alsofound in articulin proteins isolated from epiplasm (struc-ture characteristic of protist) that is a proteinaceous layerorganized as a continuous sheet used to maintain the cellshape (Peck, 1977; Marrs and Bouck, 1992). Articulinspresent aberrant migration on SDS–PAGE and similar toRp30 and Rp45 proteins of Rhodnius, the predictedmolecular mass from open ready frame does not corre-spond to that observed in the SDS–PAGE. The differencesin molecular mass can be attributed either to post-transla-tional modification of proteins or to intrinsic properties ofthose polypeptides as suggested for articulins (Huttenlauchet al., 1998a, b).

An interesting result was observed in the C terminalsequence of Rp30 protein which showed a modification ofthe ‘‘R&R consensus’’ (Rebers and Riddiford, 1988), acuticle motif protein. This consensus is the most commonregion which confers the ability of cuticle proteins to bindchitin (Iconomidou et al., 2005). The presence of thisconsensus region in the Rp30 protein associated withthe observation that the proteins remained intact through-out embryogenesis suggest that this protein may beinvolved with the binding or the accumulation ofchitin in the specific region of the egg. We suggest thatRp30 may be involved in embryonic cuticle formation.Recent infra-red analysis of Rhodnius embryonic cuticleclearly showed the presence of chitin (data notshown). Considering that the embryonic cuticle is formedin close contact with VM this possibility cannot be ruledout.

On the other hand, the Rp45 protein presented identitieswith glycine-rich proteins such as elastin. These proteinsnormally perform structural tasks and are also able toretract to their initial position after being stretched(Sachetto-Martins et al., 2000).

Eggshell assembly is a complex process involvingtemporal as well as spatial regulation and depends onVM proteins. In Drosophila, it is considered that VMproteins are assembled in similar ways of elastins are toassemble extracellular matrices (Manogaran and Waring,2004). Here it is suggested that both Rp30 and RP45proteins are components of VM.

Some characteristics observed in the Rp30 and Rp45proteins, such as a close association with the embryo andthe amino acid composition (rich in glycine), linked to theknowledge in literature that most antimicrobial peptides ininsects are glycine-rich peptides (Bulet et al., 1999; Otvos,2000) led us to test the possibility of these proteinspresenting antimicrobial activities. Here we show thatone of these proteins, the Rp45, presents an antifungalactivity against A. niger in micromolar concentration. Thisresult explains, in part, an intriguing observation concern-ing our colony. The insect cages are maintained at 70–80%humidity at 28 1C, a condition suitable for fungus growth.Although the cages become very humid, soon after feeding,due to the fact that R. prolixus feces contain large amountsof liquid, fungus growth was never found. As far as we

Please cite this article as: Bouts, D.M.D., et al., Biochemical properties of th

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know, this is the first report relating eggshell proteinswith antifungal activity. A. niger was used in our assaysbecause it is a member of the most common group of fungiin the environment and it also has entomopathogenicpotential (Moraes et al., 2001), at least against mosquitoes.Another fungus, Fusarium solani is also pathogenic foreggs of Panstrongylus geniculatus (Hartung and Lugo,1996). The presence of antifungal activity, associatedwith the eggshell, was possibly important during theevolution of insect species. Their need of an open spacein the eggshell, to allow fertilization and gas exchange forembryo respiration, possibly evolved in parallel withthe acquisition of antimicrobial agents that could beassociated with the eggshell. The fact that Rp30 proteindid not inhibit Aspergillus growth does not necessarilymean that this protein is not an antifungal agent. We arenow testing both proteins against a variety of other fungiand bacteria.Arthropods produce a number of different peptides to

protect them against the invasion of microorganisms asreviewed by Otvos (2000) and Bulet et al. (2004), but only afew reports have described the presence of peptides withantimicrobial activities associated with the eggshell ofinsects (Marchini et al., 1997; Lamberty et al., 2001) andnematodes (Lopez-Llorca et al., 2002). Here we haveshown for the first time that an eggshell component of animportant insect vector has an antifungal activity. In orderto benefit from the yolk, the fungus must first penetrate theeggshell. The contact of Rp45 protein of Rhodnius eggwith the invading fungus hyphae may be enough toblock the invasion. Considering that Rp45 protein is notsoluble when associated with the eggshell, its effect ispossibly elicited by contact. In terms of embryo develop-ment it is not necessary to kill the fungus; therefore afungistatic effect should be just enough. Supportingthis hypothesis, the addition of A. niger to the eggs ofR. prolixus, under a condition suitable for fungus growth,is not enough to destroy the eggs and the nymphs hatchnormally.Fungal cells develop mechanisms to secrete enzymes

(Hube, 2000; Naglik et al., 2003; Santos et al., 2006) ontothe hosts in order to invade their cells. Thus, host cells haveto counteract the effect of these enzymes in order tosurvive. Extracellular proteinases of saprophytic fungi suchas A. niger are secreted primarily to provide nutrients forthe cells, but this biochemical property can be used to fulfillspecialized functions during the infective process (Nagliket al., 2003). The authors did not investigate whether theeffect elicited by Rp45 protein was due to the effect of theentire molecule or of the peptides derived from the Rp45protein by the action of putative proteases from the fungus.In any event the biological effect was elicited protecting theembryo. The mechanism of the Rp45 protein is now underinvestigation.Antifungal agents generally inhibit enzymatic reactions

involved in fungal cellular biosynthesis, including aminoacids, nucleotides, lipids and polysaccharides, but the

e major proteins from Rhodnius prolixus eggshell. Insect Biochem. Mol.

ARTICLE IN PRESSD.M.D. Bouts et al. / Insect Biochemistry and Molecular Biology ] (]]]]) ]]]–]]] 13

fungicide effect can also be achieved by interference inintracellular transduction pathways (Kojima et al., 2004).In fungi a cross-talk between cAMP and calcium signalingpathway exists (Bencina et al., 2005), suggesting thepossibility of an antifungal agent to induce a metabolicimbalance simultaneously in several different metabolicpathways in the fungus making the study of the actionmechanism of an agent a difficult task.

A large number of antifungal proteins have beendescribed over the last two decades due to immunocom-promised hosts such as AIDS patients under treatmentwith immunosuppressive therapies and organ transplantrecipients. The target of these antifungal proteins varies butantifungal protein active on the fungal cell wall, plasmamembrane, and intracellular targets can be recognized(Theis and Sthal, 2004). The mechanisms of action are asvaried as their sources and include cell wall degradation,membrane channel inhibition, pore formation, damage tocellular ribosome, and inhibition of DNA synthesis and cellcycle (Selitrennikoff, 2001).

The A. niger morphological alterations observed afterthe addition of Rp45 protein to the culture medium includealteration of cell walls and intracellular structures leadingto the appearance of swollen mitochondria and a largeamount of vacuoles. In Saccharomyces cerevisiae vacuolesare central in much of the physiology of the organism. Thisorganelle is involved with pH and osmoregulation, proteindegradation, storage of amino acids, ions and polypho-sphates and sporulation. Thus interference in this organellemay potentially alter several metabolic pathways at thesame time to such an extent that it could end up as adefective organism (Klionsky et al., 1990). Differentantifungal agents such as echinocandin induce the appear-ance of multi-lamellar structure in Candida albicans

(Cassone et al., 1981), a signal of cell injury, but themechanism leading to this is not known. The fact thatRp45 protein is active simultaneously against the fungalcell wall and intracellular targets makes this protein apotential fungicide.

Acknowledgments

We wish to express our gratitude to Jose de Souza LimaJunior and Litiane M. Rodrigues for maintaining ourcolony of Rhodnius prolixus; to Rosane O. M. M. da Costa(in memoriam) for their technical support in the biochem-ical work and to Noemia Rodrigues and Sebastiao Cruz(in memoriam) for their assistance on electron microscopy.A special thanks to SJT and SJ. This work was supportedby grants from MCT/Conselho Nacional de Desenvolvi-mento Cientıfico e Tecnologico (CNPq), Conselho deAperfeic-oamento de Ensino Superior (CAPES), Financia-dora de Estudos e Projetos (FINEP), Programa de Apoioao Desenvolvimento Cientıfico e Tecnologico (PADCT),Programa de Nucleos de Excelencia (PRONEX) andFundac- ao de Amparo a Pesquisa Carlos Chagas Filho(FAPERJ).

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