InsectBiochemistry

andMolecularBiology

Insect Biochemistry and Molecular Biology ] (]]]]) ]]]–]]]

Biochemical properties of the major proteins from

Rhodnius 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

America. The number of people infected with Trypanosoma

cruzi, the etiological agent of Chagas Disease, was

estimated at between 16 and 18 million, with a further

100 million considered at risk (TDR report, 2002).

Consequently all research concerning R. prolixus is

considered an opportunity in the direction of finding

solutions for disease control. One very important aspect of

the life cycle of this insect involves a period of embryo

development in the eggs that are deposited in the

environment. At oviposition, the eggs contain all the

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�Corresponding author. Instituto de Quımica, Departamento de

Bioquımica, Universidade Federal do Rio de Janeiro, 21941-909 Rio de

Janeiro/RJ, Brazil. Tel.: +5521 2556 6867; fax: +5521 2562 7266.

E-mail address: anamelo@iq.ufrj.br (A.C.A. Melo).1These authors contributed equally to this work.

Please cite this article as: Bouts, D.M.D., et al., Biochemical properties of the major proteins from Rhodnius prolixus eggshell. Insect Biochem. Mol.

Biol. (2007), doi:10.1016/j.ibmb.2007.07.010

nutrients and energy necessary for embryonic growth and

have to be able to protect themselves of all natural dangers.

The evolutionary success of this group undoubtedly

involves the acquisition of this ability. R. prolixus eggs

are formed in a telotrophic meroistic ovary that consists of

two semi-ovaries connected by a common oviduct. Each

hemi-ovary contains seven ovarioles and each ovariole is

composed of the vitellarium and of the terminal filament, a

lanceolate structure (the trophary), which contains the

germarium, the oocytes and pre-follicular tissues. The

vitellarium is composed of oocytes in different stages of

development that are surrounded by follicle cells (Vander-

berg, 1963; Huebner and Anderson, 1972a–c; Lutz and

Huebner, 1980, Atella et al., 2005) and each oocyte is

connected 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 growth

rate when the oocytes receive nutrients primarily from the

nurse cells. Following pre-vitellogenesis the oocytes initiate

a rapid growing phase (vitellogenesis) by the uptake of

proteins synthesized by fat body and ovary to form the

yolk 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 synthesis

of the protective eggshell (Beament, 1946b; King and

Aggarwal, 1965; Telfer and Anderson, 1968; Mazur et al.,

1982; Berg, 2005). Some evidences have showed that the

transition between the vitellogenesis to choriogenesis

depends on the involvement of cyclic nucleotides (Wang

and Telfer, 1996; Medeiros et al., 2002, 2004). During the

transition, genes associated with vitellogenesis are turned

off and a different set of chorion genes is turned on. This

leads to the synthesis of proteins which will constitute the

eggshell, the vitelline membrane (VM) and the chorionic

layers of the egg as described for several insects (Kafatos

et al., 1977; Margaritis et al., 1980; Orr-Weaver, 1991).

Secretion of eggshell by follicle cells has already been

studied 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; Margaritis

et 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 three

layers, the VM, endochorion and exochorion (passing from

the oocyte outwards). The endochorion and exochorion

together are known as the chorion. The eggshell is

assembled with distinct proteins specialized in protecting

the oocyte by apposition of newly synthesized protein upon

existing layers (Giorgi, 1977). In R. prolixus when the

oocyte has reached its full size the eggshell formation starts

(Beament, 1946b). The oocyte membrane, which has been

transporting material from the follicle cells to the yolk

cavity, becomes the VM and is the base for the deposition

of the chorion (Beament, 1946b). Rhodnius chorion is

constituted of the endochorion with five membranes: (1)

inner polyphenol layer, (2) resistant protein layer, (3) outer

polyphenol layer, (4) amber layer and (5) soft protein

layer; and the exochorion with two membranes: (1) soft

exochorion and (2) resistant exochorion (Beament, 1946b).

The eggshell synthesis in Reduviidae has been studied

less than the Drosophila system. Eggshell formation in

Rhodnius begins with deposition of choriogenic proteins

onto the oocyte membrane during stage 9 of oogenesis,

when the T oocyte length is around 1500–2000 mm (Pratt

and Davey, 1972; Bjornsson and Huebner, 2004). In

Drosophila the VM proteins are also synthesized during

the early stages of eggshell formation (stages 8–10),

while endochorion and exochorion proteins are synthesized

later, 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. The

SDS–PAGE of eggshell proteins of several species of

Drosophila, shows six major bands with comparable

electrophoretic mobility (Thireos et al., 1980). While, the

silkmoth’s eggshell is considerably more complex, where

more 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, it

must protect the embryo against microorganisms possibly

using antimicrobial agents associated with the eggshell. The

presence of antimicrobial agents associated with eggs has

been described in two insects. Marchini et al. (1997)

described a peptide produced by accessory glands of

Ceratitis capitata that have an antimicrobial activity.

Lamberty et al. (2001) purified a peptide from female

salivary glands of the termite Pseudacanthotermes spiniger

and suggested a possible antifungal role of this peptide in

the eggshell, since this female insect smears its eggs with

saliva during egg development.

The present study describes for the first time that the

follicle cell of the Reduviidae bug R. prolixus synthesize

two proteins that end up associated with the eggshell.

These proteins are synthesized by follicle cells in a

period that coincides with the end of vitellogenesis and

beginning of choriogenesis and are deposited onto the

oocytes where they associate with the oocyte microvilli.

Following the end of the choriogenesis fertilization occurs

and embryogenesis is started. The presence of these

proteins was monitored from oocyte up to all embryogen-

esis and they remained associated with the eggshell. It is

suggested that these proteins are part of the VM. The

presence of antifungal activity associated with eggshell

proteins is discussed.

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Please cite this article as: Bouts, D.M.D., et al., Biochemical properties of the major proteins from Rhodnius prolixus eggshell. Insect Biochem. Mol.

Biol. (2007), doi:10.1016/j.ibmb.2007.07.010

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. The

insects were adult mated females fed on rabbit blood at 3-

week intervals following guidelines set by the Universidade

Federal do Rio de Janeiro/UFRJ Institutional Animal

Care Committee.

2.2. Polyacrylamide gel electrophoresis

Electrophoresis was performed in the presence of sodium

dodecyl sulfate (SDS) (Laemmli, 1970) in a 10% poly-

acrylamide gel, followed by staining with coomassie

brilliant blue G. The gels were destained using a mixture

of 7% acetic acid and 40% methanol. The molecular mass

of purified Rp30 and Rp45 proteins was estimated by

SDS–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 different

sources: chorionated oocytes, dissected from ovaries, and

eggshell collected soon after hatching. (A) Chorionated

oocytes: to obtain chorionated oocyte, ovaries were

dissected under the stereomicroscope in 0.15M NaCl on

the 3rd day after adult blood meal. The chorionated

oocytes were removed from the ovary and extensively

washed in saline. After that the chorionated oocytes were

disrupted and their yolk contents removed. The remaining

eggshells were washed several times in 0.01M Tris/HCl pH

8.4 in order to remove contaminating yolk proteins. (B)

Eggshell: Soon after hatching the eggshells were collected

from the breeding cage and the embryonic cuticle carefully

removed under the stereomicroscope. The eggshells were

washed in 0.01M Tris/HCl pH 8.4 several times. Then, the

eggshells from both sources were homogenized separately

and solubilized at room temperature (RT) as described by

Regier et al. (1978) with some modifications. The eggshells

were homogenized strongly in a Potter in the presence of

8M urea, 0.36M Tris/HCl (pH 8.4), 0.03M dithiothreitol

and 0.1M PMSF and centrifuged at 12,000g for 10min.

The supernatant was collected and stored at �20 1C for

further 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 fast

stained using a saturated solution of KCl. The band

corresponding to the Rp30 and Rp45 proteins was cut off

and eluted by simple diffusion using 50mM ammonium

bicarbonate pH 7.8 with 0.01% SDS. After 1 h at 37 1C, the

samples were centrifuged at 12,000g for 5min and the

supernatant was collected. The degree of purification was

monitored using a second 10% SDS–PAGE. The resulting

protein concentration was determined using the method of

Lowry et al. (1951).

2.4. Amino terminal sequencing

The Rp30 and Rp45 proteins were subjected to

SDS–PAGE and then transferred to a PVDF membrane

(Matsudaira, 1987). The amino terminal of these proteins

was 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 membrane

were directly applied to the sequencer cartridge. Such

experiments were carried out at the amino terminal

sequencing 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 synthesized

based on the amino terminal sequence of each protein

obtained by Edman degradation. Total RNA from

follicular 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 respective

degenerate and the NotI-(dt)18 primers. Amplification

conditions included 40 cycles of 94 1C—30 s, 51 1C—60 s

and 68 1C—180 s. On the final cycle, 68 1C was maintained

for an additional 6min. The PCR products were gel-

purified, cloned using Perfect BluntTM cloning kit (Novagen)

and sequenced at the Molecular Genetics Instrumentation

Facility of the University of Georgia, Georgia, USA. The

theoretical molecular weight of each cloned protein was

estimated using computer pI/MW for Swiss-Prot/Tr

EMBL (Gasteiger et al., 2005).

2.6. Alignment

The search for sequence similarities was performed by

the software FASTA and BlastP 2.2.2 using default

parameters (Pearson and Lipman, 1988; Pearson, 1990;

Altschul et al., 1997). The primary amino acid sequence of

the two proteins was aligned using the ClustalW software

package (Thompson et al., 1994). GenBank accession

numbers 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 the

northern-blot assays different follicle sizes were dissected

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Please cite this article as: Bouts, D.M.D., et al., Biochemical properties of the major proteins from Rhodnius prolixus eggshell. Insect Biochem. Mol.

Biol. (2007), doi:10.1016/j.ibmb.2007.07.010

and stored in RNAlater (Ambion) at 4 1C. Ovarioles were

staged based in terminal (T) follicle length (Pratt and

Davey, 1972; Bjornsson and Huebner, 2004) following:

500–600mm length follicle (early vitellogenesis), 900–1000mm

length follicle (late vitellogenesis) and 1500–2000mm length

follicle (choriogenesis). The apical trophic tissue (tropharies)

was identified by morphology as described by Vanderberg

(1963) and Huebner and Anderson (1972a). The RNA

samples (30 mg/lane) were separated by electrophoresis

in 1.2% formaldehyde-agarose gels, transferred to

nylon membranes (Sambrook et al., 1989) and probed

with 32P-labeled Rp30 or Rp45 full cDNA. The nylon

membrane was washed under high stringency conditions:

three times with 0.6M sodium citrate, 0.6M NaCl and

0.5% SDS-15min at RT, and once with 0.3M sodium

citrate, 0.3M NaCl and 0.5% SDS-15min at 60 1C.

Membranes were exposed to Kodak X-OMAT film

at �70 1C with an intensifying screen. Exposition time

varied from 1 to 7 days according to the radioactive

intensity. RNA sizes were calculated using an RNA ladder

(Invitrogen).

2.8. Antiserum

Purified Rp30 (1mg) or Rp45 (1mg) proteins were

emulsified in complete Freund’s adjuvant and injected

subcutaneously in the back of different 1.5 kg rabbits. Two

weeks after injection, a booster was given and 30 days later

blood was taken from an ear vein and the serum examined

by immunoblotting using total proteins (Towbin et al.,

1979).

2.9. Immunoblotting

The Rp30 and Rp45 proteins were separated by a

gradient 6.5–22% SDS–PAGE for 180min at 2mA/cm and

then electrotransferred to a nitrocellulose membrane in

25mM Tris, 192mM glycine, 20% methanol (pH 8.3) for

120min at 150mA, followed by staining with Ponceau

Red, or prepared for immunostaining as follows: the

membrane was incubated with antiserum raised against

purified Rp30 or Rp45 proteins followed by secondary

anti-rabbit antibody conjugated with alkaline phosphatase

and developed with NBT/BCIP (Towbin et al., 1979). After

immunostaining the membrane was washed several times

with water and dried at RT. As a control of molecular mass

a 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-phosphate

dehydrogenase (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 proteins

during embryogenesis, chorionated oocytes or eggs colle-

cted at different days after oviposition were dissolved

in 8M urea as described in Section 2.3 and subjected to

immunoblotting as describe above.

2.10. Aspergillus niger cultures and antifungal activity

A. niger strain (EK 0197) was collected by spontaneous

spore decantation suspended in air at Petri plates with solid

Sabouraud medium (dextrose 40 g, peptone 10 g, agar 15 g

per liter) at RT. The antifungal activity was assayed as

described by Broekaert et al. (1990) with some modifications.

The fungal strain was grown at RT in liquid Sabouraud

medium (LSM). The fungal cells were seeded in a 96-micro-

titer plate in LSM at a density of 3� 102 conidia/mL (100mL

per 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 cell

suspension was incubated for 48 h at RT. The turbidity of

each well was measured at 540 nm using a VERSAmax

microplate reader (Molecular Devices). Images were

captured using a Zeiss NC-80 camera attached to a Zeiss-

Stemi 2000-C microscope. In the electron microscopy

assay, fungal colonies were grown in the presence of 10

and 80 mM of Rp45 protein and 10 mM of BSA was used as

a control.

2.11. Ovary preparation

Ovaries were dissected 3 days after blood meal. The

follicles were examined under a Zeiss stereomicroscope and

the ovarioles separated for morphological analysis and

immunolocalization as described below. To obtain a layer

of follicle cells, each follicle was opened up using

iridectomy scissors. Then the cytoplasm of the oocytes

was discarded so that the final preparation was a layer of

follicle cells attached to the oocyte membrane. This

preparation was also used for immunolocalization. Isolated

ovarioles or a layer of dissected follicle cells attached to the

oocyte membrane were fixed using 4% paraformaldehyde

in PBS. The fixed preparation was mounted onto cover

glasses coated with poly-L-lysine, washed with PBS, and

treated with 150mM NH4Cl for 20min. Permeation was

obtained by treatment with 0.1% Triton X-100 in PBS for

5min at RT. Non-specific staining was avoided by

treatment with PBS containing 1.5% BSA and 0.5% fish

gelatin (blocking buffer-BB) for 30min. After incubation

with antiserum raised against Rp30 or Rp45 proteins

(diluted 1:5000) for 60min, the preparations were washed

with BB and finally incubated with goat anti-rabbit

secondary antibody associated with fluorescein (Gibco,

Grand Island, NY, USA) diluted 1:100 in BB, for 60min

in the dark. The preparation was mounted with 0.2M

n-propyl gallate in 9:1 glycerol-PBS and analyzed using

Zeiss laser scanning microscope (LSM 310). The images

obtained were all processed using Adobe Photoshop.

2.12. Transmission electron microscopy

A. niger cultures were treated with Rp45 protein (10 mM

or 80 mM) or BSA (10 mM) and then fixed for 2 h at RT

with 2.5% glutaraldehyde in 0.1M cacodylate buffer (CB),

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Please cite this article as: Bouts, D.M.D., et al., Biochemical properties of the major proteins from Rhodnius prolixus eggshell. Insect Biochem. Mol.

Biol. (2007), doi:10.1016/j.ibmb.2007.07.010

pH 7.2. Post-fixation was carried out in 1% osmium

tetroxide in CB containing 0.8% potassium ferrocyanide

and 5mM CaCl2. Thereafter, the cells were dehydrated in

acetone and embedded in Epon. Ultrathin sections were

stained with uranyl acetate and lead citrate and observed

under a Zeiss EM-900 electron microscope. As a control

non-treated A. niger cells were processed by the same

procedure and analyzed.

2.13. Immunoelectron microscopy localization of Rp30 and

Rp45 proteins

Follicles were fixed in a mixture of 0.1% glutaraldehyde

type I and 4% paraformaldehyde in PBS (pH 7.2) for

120min at RT. After fixation, oocytes were washed in PBS

and dehydrated in a series of methanol solutions (30–90%),

and finally embedded in Unicryl (British Biocell) at �20 1C

under UV illumination. Ultra thin sections were collected

on 300 mesh nickel grids. The sections were subsequently

incubated in PBS (pH 7.4) containing 150mM NH4Cl

for 30min, PBS containing 1.5% BSA, 0.5% fish gelatin

and 0.1% Tween 20 (blocking buffer Tween-BBT) for

30min. Subsequently, samples were incubated in BBT

containing antibodies raised against Rp30 or Rp45

proteins for 60min (dilution 1:500). Afterwards, sections

were washed in BBT, incubated with 10 nm gold-labeled

goat anti-rabbit IgG (1:100) (Sigma, St Louis, MO, USA)

for 60min, and thoroughly washed in PBS. Grids were

examined in a Zeiss EM-900 electron microscope, after

staining with uranyl acetate and lead citrate. Control

experiments were performed using nonimmune serum

followed 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 proteins

were purified for further use (Fig. 1, Lanes 2 and 3).

The molecular mass of each protein was determined based

on the mobility of standard proteins. Due to their

molecular masses they were named R. prolixus 30 kDa

protein (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 were

obtained in rabbits and used for immunoblotting. Fig. 2

shows that the polyclonal antibodies are specific and they

do not recognize either hemolymph or oocyte proteins that

could potentially contaminate the preparations. The

antibodies against both proteins were clearly associated

with follicle cells (Figs. 3B and D). The inset in Figs. 3B

and D presents a panoramic view of follicle cells suggesting

that both proteins are associated with them; however the

technique used did not have enough resolution to show us

whether the labeling was inside or outside the cells. In

order to obtain more information at cellular level a detailed

morphological analysis of follicle cells was performed using

electron microscopy.

The immunogold labeling technique was used in order to

detail the association of Rp30 and Rp45 proteins with these

structures. Fig. 4 shows that these proteins co-localize

inside the follicle cells (Figs. 4A(inset) and B(inset)) and

also between follicles, suggesting that they have been

secreted to the space between cells. Interestingly both

proteins strongly associate with the microvilli (Figs. 4A

and C). Detail of this association is shown in the inset

of Figs. 4A and C. In order to follow the fate of these

proteins we monitored their presence by immunoassay

over different stages of oogenesis and embryogenesis,

from oocyte up to the point of the hatching of the first

instar larvae. Fig. 5 shows that the amount of the

Rp45 protein remains unchanged, from chorionated

oocyte to the point of first instar nymph hatching. In the

same way as Rp45, the quantity of Rp30 protein remained

unchanged during the whole embryogenesis (data not

shown).

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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

Please cite this article as: Bouts, D.M.D., et al., Biochemical properties of the major proteins from Rhodnius prolixus eggshell. Insect Biochem. Mol.

Biol. (2007), doi:10.1016/j.ibmb.2007.07.010

3.3. Cloning, sequencing and analysis of Rp30 and Rp45

expression

The first 23 amino acid residues of each protein were

deduced by Edman degradation. The sequence VXPNAG-

XFPGFAAPFYGXYGVXP was obtained for the Rp30

protein and the sequence XGPXGLVGDAGYLTG-

PAYYDXFH was obtained for the Rp45 protein. Degen-

erate oligonucleotides were designed based upon the

sequences obtained from Edman degradation (underlined

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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.

D.M.D. Bouts et al. / Insect Biochemistry and Molecular Biology ] (]]]]) ]]]–]]]6

Please cite this article as: Bouts, D.M.D., et al., Biochemical properties of the major proteins from Rhodnius prolixus eggshell. Insect Biochem. Mol.

Biol. (2007), doi:10.1016/j.ibmb.2007.07.010

above—see Section 2.4 and 2.5). Ovaries were dissected

and RNA extracted with TRIzol reagent. The RNA was

used in a first strand cDNA synthesis reaction with

NotI(dT)18 primers. The NotI(dT)18 primer was used

together with the degenerate primers in separate polymer-

ase chain reactions (PCR) to amplify the cDNA coding for

the protein of interest. The clones encode a partial peptide

of 220 and 362 amino acids in length to Rp30 and Rp45,

respectively, which are missing the N-terminus. The

N-terminus also includes 10 amino acids of Rp30 and 14

amino acids of Rp45 from Edman degradation. The cDNA

cloned products had molecular sizes of 663 bp (Rp30) and

1089 bp (Rp45), corresponding to polypeptides with pre-

dicted mass of 24623.85Da and 38015.32Da, respectively.

The theoretical molecular masses were lower than observed

in SDS–PAGE, probably due to post-translational mod-

ifications. An extension of 24 amino acids of N-terminus of

Rp45 was also obtained using data from EST random

sequencing of cDNA library from R. prolixus follicle cells

which confirmed a cleavage signal peptide in a deduced

ARTICLE IN PRESS

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.

D.M.D. Bouts et al. / Insect Biochemistry and Molecular Biology ] (]]]]) ]]]–]]] 7

Please cite this article as: Bouts, D.M.D., et al., Biochemical properties of the major proteins from Rhodnius prolixus eggshell. Insect Biochem. Mol.

Biol. (2007), doi:10.1016/j.ibmb.2007.07.010

sequence (data not shown) (personal communication of

Paiva-Silva G. O. and Oliveira P. L., as part of R. prolixus

Genome Consortium). Since the cloning was made using

degenerate oligonucleotides obtained from purified pro-

teins the 50UTR region is still unknown this may explain

why the size of RNA is much bigger than the protein they

encoded. The amino acid sequences deduced by the

nucleotide sequence of Rp30 and Rp45 proteins are

represented in Fig. 6A. The first 23 amino acids from

Rp45 sequence obtained by Edman degradation was also

confirmed using the same data from EST random

sequencing of the cDNA library as mentioned above, thus

we can identify the X’s in the sequence were two cysteine

(one in the first position and another one in the fourth

position) and one glycine (in the 21st position). The clone

sequence of Rp30 identified the two X’s in the N-terminus

which were one serine in the 18th position and one glycine

in the 22nd position.

Both sequences present repetitive motifs of valine–pro-

line–valine (VPV) at every 15 amino acid in their central

domains. Alignment of both sequences was performed and

showed 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 known

eggshell proteins. The Rp30 protein revealed similarity with a

glycine-rich cuticle protein from B. mori (GenBank accession

ARTICLE IN PRESS

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

residues are indicated by (*) and residues with similar properties by (:). Alignment results indicate that the proteins present 132 identical residues

corresponding to 57.2% identity and 163 similar residues corresponding to 77.48% similarity. Repetitive motives of VPV at every 15 amino acid residues

are shaded and boxed. (B) Comparative analysis of C-terminal amino acid sequence of Rp30 protein and R&R consensus motif.

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

Please cite this article as: Bouts, D.M.D., et al., Biochemical properties of the major proteins from Rhodnius prolixus eggshell. Insect Biochem. Mol.

Biol. (2007), doi:10.1016/j.ibmb.2007.07.010

no. AB197878—30.366% identity–47.644% similarity) which

also presents a VPV motif; and with a cuticle protein from

Aedes aegypti (GenBank accession no. EAT48061—26.257%

identity–51.397% similarity). Interestingly the 25 C-terminal

amino acid of Rp30 protein showed a modification of the

‘‘R&R consensus sequence’’ motif proposed by Rebers and

Riddiford (1988) which is present in many cuticle proteins

and is demonstrated to bind chitin (Rebers and Willis, 2001;

Togawa et al., 2004) (Fig. 6B).

The Rp45 protein aligned well with glycine-rich proteins

such as those found in Oryza sativa (GenBank accession

no. Q6ZF32—38.636% identity–61.364% similarity) and

structural proteins such as elastin precursor (GenBank

accession no. P07916—36.765% identity–58.088% similarity)

and the flagelliform silk protein (GenBank accession no.

Q9BIU8—34.965% identity–53.147% similarity), where

glycine residues are also abundant. The region of the

Rp45 protein which presents similarities with the glycine-

rich proteins is the amino and carboxyl-terminal domain.

The Rp30 protein does not possess this region. The

comparison revealed that the Rp45 protein possesses

homology with a cytoskeletal protein (GenBank accession

no. Q39721—30.714% identity–53.571% similarity), that

also presented the VPV repetitive sequence. The amino acid

content 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%) and

arginine (7.0%). In the Rp45 protein the most frequent

amino 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 was

performed. Results demonstrated that the expression of

these genes only occurred in the ovaries (Fig. 7A). The

Rp30 and Rp45 probes hybridized with a 4.1 and 4.9 kb

band, respectively. The Rp30 probe was also observed to

cross-hybridize to 6.3 kb band and 4.9 kb Rp45 band (data

not shown). Probably these facts are due to a similarity of

sequences between these RNAs. Moreover, this suggests

the existence of another protein with a molecular weight

larger than either Rp30 or Rp45 expressed in follicle cells

with a similar sequence. To investigate the expression

pattern of the Rp30 and Rp45 genes in the ovaries another

northern-blot assay was performed with follicles in

different development stages (Fig. 7B). Ovaries were

ARTICLE IN PRESS

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 mm

length 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.

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.

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

Please cite this article as: Bouts, D.M.D., et al., Biochemical properties of the major proteins from Rhodnius prolixus eggshell. Insect Biochem. Mol.

Biol. (2007), doi:10.1016/j.ibmb.2007.07.010

dissected on the third day after a blood meal and stored in

RNAlater (Ambion). Ovarioles were subdivided into

tropharies (Vanderberg, 1963) and follicle staged in

terminal (T) follicles length in accordance to Pratt and

Davey (1972) and Bjornsson and Huebner (2004) as

follows: 500–600 mm (early vitellogenesis), 900–1000 mm

(late vitellogenesis) and 1500–2000 mm (choriogenesis), laid

eggs (negative control) and total ovaries (positive control).

Expression of both genes was observed only in the

1500–2000 mm follicle lengths and in the positive control

(Fig. 7B—panels d and e). This result reinforces the fact

that the putative proteins are exclusively from chorionic

follicles and that they may constitute a novel gene family.

3.4. Antifungal activity

The results evidencing that the Rp30 and Rp45 proteins

remain associated with the eggshell are consistent with the

role of these proteins in protecting the embryo during

development. To obtain further insight into possible

functions, these proteins were tested for antifungal activity

in part due to their close association with the embryo.

Fig. 8A shows that Rp45 protein inhibits the growth of

A. niger while BSA, extracted from the gel by the same

procedure used to purify Rp45 protein, presented no effect.

In order to determine its dose dependence, A. niger was

grown in a medium containing different concentrations of

ARTICLE IN PRESS

Fig. 9. Morphology of Aspergillus niger following treatment with Rp45 protein. (A) Panoramic view of non-treated A. niger cell grown for 48 h in culture

medium. (B) Panoramic view of A. niger cell treated with 10mMBSA. (C) View of A. niger cell following treatment with 10mMRp45 protein for 48 h. Inset

shows that a 5 h-treatment with 10 mM of Rp45 protein is enough to induce the appearance of multi-lamellar structure. (D) Detail of swollen mitochondria

following treatment with 10mM of Rp45 protein. (E and F) View of cell wall and cytoplasm alterations following treatment with 80mM of Rp45 protein.

Gly ¼ glycogen particles; CW ¼ cell wall; M ¼ membrane; M-L ¼ multi-lamellar structure; MT ¼ mitochondria; VAC ¼ vacuoles. Bar ¼ 1mm.

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

Please cite this article as: Bouts, D.M.D., et al., Biochemical properties of the major proteins from Rhodnius prolixus eggshell. Insect Biochem. Mol.

Biol. (2007), doi:10.1016/j.ibmb.2007.07.010

Rp45 protein (Fig. 8B). The concentration of Rp45 protein

necessary 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, besides

inhibiting A. niger growth, also induced morphological

alterations 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 cells

presented a large amount of glycogen, mitochondria and

a well-developed cell wall (Figs. 9A and B). The fact that

BSA (purified by the same procedure used to obtain Rp45

protein) did not affect the morphology of fungal cells

suggests that the procedure used to obtain Rp45 protein

did not bring contaminants from the acrylamide gel that

could potentially affect the fungus. The treatment with

10 mM of Rp45 protein for 48 h induced the appearance

of multi-lamellar structures (Fig. 9C) absent in control

cells (Fig. 9A and B). The inset in Fig. 9C shows that a

5 h-treatment is enough to induce the appearance of these

structures. Ten micromolars of Rp45 protein also affected

the mitochondria organization (Fig. 9D), clearly showing

swollen mitochondria.

An increase in the concentration of Rp45 protein from

10 to 80 mM leads to more significant effects on the

morphology of the cells (Figs. 9E and F). A disorganiza-

tion of the cell wall is clearly seen (Inset—Fig. 9F) as well

as the presence of large vacuoles (Figs. 9E and F).

4. Discussion

Numerous studies have been published on the secretion

and morphogenesis of chorion in different insects (Regier

et al., 1978; Kimber, 1980; Margaritis et al., 1980; Mazur

et al., 1980; Regier et al., 1982; Hamodrakas et al., 1985;

Margaritis, 1985; Papassideri and Margaritis, 1996). In

Drosophila, the chorion genes are amplified by the follicle

cells in response to developmental signals, prior to their

transcription (Orr-Weaver, 1991). The number of proteins

in chorion varies for different insects. In D. melanogaster

about 20 chorion proteins are present in the eggshell, while

about 186 proteins were resolved by two-dimension gel

electrophoresis as chorion constituents in A. polyphemus

(Regier et al., 1980, 1982). All the genes responsible for

the proteins that will be part of the eggshell are turned on,

at the same time the genes that take part in vitellogenesis

are turned off (Kafatos et al., 1977). In Hyalophora

cecropia the termination of vitellogenin uptake seems to

be associated with the increase of cAMP (Wang and

Telfer, 1996). In R. prolixus, Medeiros et al. (2002, 2004)

provided evidences that eicosanoids control the oogenesis,

through the modulation of cAMP levels. Whether or

not eicosanoids are related to the transition from

vitellogenesis to choriogenesis and which role cAMP

plays in the control of the gene expression remains to be

defined.

It has been well established that in most insects the

eggshell synthesis occurs by apposition of material over a

pre-existing layer, such as VM (Giorgi, 1977; Margaritis

et al., 1980; Margaritis, 1985). Ultrastructurally chorion

consists of fibrous layers that run in parallel to the chorion

surface (Smith et al., 1971; Kafatos et al., 1977; Mazur

et al., 1982). At the end of morphogenesis the chorion

structure 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 pattern

described above. The Rp30 and Rp45 proteins isolated

from the eggshell are synthesized by follicle cells and are

either secreted to the space between them or onto the

oocytes. Here we showed that they associate with

the oocyte membrane, especially at the microvilli, during

the initial stage of choriogenesis. The localization of Rp30

and Rp45 inside the follicle cells, their association with the

oocyte membrane and also to the eggshell left behind by

the first instar nymph clearly evidence that these proteins

are important during the early stage of eggshell construc-

tion. Considering that all eggshell layers are formed

sequentially by apposition of proteins during the last

part of oogenesis (Giorgi, 1977; Margaritis et al., 1980;

Margaritis, 1985), it is tempting to speculate that these

proteins are used to build up the VM. Although

controversy exists with respect to the use of the term VM

and its origin (Clements, 1992; Bate and Arias, 1993; Valle

et al., 1999) here it is used in accordance with Beament

(1946b). Another evidence that reinforces our hypothesis

concerning these proteins are a component of VM is that

scraping the innermost layer of the eggshell, named VM by

Beament (1946b), Rp30 and Rp45 can be obtained (data

not shown). These proteins similar to what is observed in

the Drosophila system remain insoluble throughout the

embryogenesis.

The R. prolixus Rp30 and Rp45 proteins have very

similar sequences, which are mainly found in their central

domains. These data suggest that the genes that codify

these proteins may be paralogous, being originated by the

duplication of an ancestral gene followed by a divergence

in their sequences. The central domains of these proteins,

constructed by the repetitive VPV consensus sequences

exactly eight times in tandem, probably present a peculiar

three-dimensional structure. As this very similar feature is

present in both proteins, it must play an important

functional role. The fact that in both proteins there are

exactly eight of these repetitive units suggest that in order

to fold correctly, this domain must be present. The VPV

motif was also found in hypothetic proteins available in

the genome project of other vector insects such as

A. aegypti (PS50326—identity 36.67% that has a valine

rich region and a signal peptide cleavage) and Anopheles

gambiae (ENSANGP00000022326—identity 35.66% that

ARTICLE IN PRESS

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

Please cite this article as: Bouts, D.M.D., et al., Biochemical properties of the major proteins from Rhodnius prolixus eggshell. Insect Biochem. Mol.

Biol. (2007), doi:10.1016/j.ibmb.2007.07.010

has proline- and valine-rich regions). The VPV motif is also

found in articulin proteins isolated from epiplasm (struc-

ture characteristic of protist) that is a proteinaceous layer

organized as a continuous sheet used to maintain the cell

shape (Peck, 1977; Marrs and Bouck, 1992). Articulins

present aberrant migration on SDS–PAGE and similar to

Rp30 and Rp45 proteins of Rhodnius, the predicted

molecular mass from open ready frame does not corre-

spond to that observed in the SDS–PAGE. The differences

in molecular mass can be attributed either to post-transla-

tional modification of proteins or to intrinsic properties of

those polypeptides as suggested for articulins (Huttenlauch

et al., 1998a, b).

An interesting result was observed in the C terminal

sequence of Rp30 protein which showed a modification of

the ‘‘R&R consensus’’ (Rebers and Riddiford, 1988), a

cuticle motif protein. This consensus is the most common

region which confers the ability of cuticle proteins to bind

chitin (Iconomidou et al., 2005). The presence of this

consensus region in the Rp30 protein associated with

the observation that the proteins remained intact through-

out embryogenesis suggest that this protein may be

involved with the binding or the accumulation of

chitin in the specific region of the egg. We suggest that

Rp30 may be involved in embryonic cuticle formation.

Recent infra-red analysis of Rhodnius embryonic cuticle

clearly showed the presence of chitin (data not

shown). Considering that the embryonic cuticle is formed

in close contact with VM this possibility cannot be ruled

out.

On the other hand, the Rp45 protein presented identities

with glycine-rich proteins such as elastin. These proteins

normally perform structural tasks and are also able to

retract to their initial position after being stretched

(Sachetto-Martins et al., 2000).

Eggshell assembly is a complex process involving

temporal as well as spatial regulation and depends on

VM proteins. In Drosophila, it is considered that VM

proteins are assembled in similar ways of elastins are to

assemble extracellular matrices (Manogaran and Waring,

2004). Here it is suggested that both Rp30 and RP45

proteins are components of VM.

Some characteristics observed in the Rp30 and Rp45

proteins, such as a close association with the embryo and

the amino acid composition (rich in glycine), linked to the

knowledge in literature that most antimicrobial peptides in

insects are glycine-rich peptides (Bulet et al., 1999; Otvos,

2000) led us to test the possibility of these proteins

presenting antimicrobial activities. Here we show that

one of these proteins, the Rp45, presents an antifungal

activity against A. niger in micromolar concentration. This

result 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 amounts

of liquid, fungus growth was never found. As far as we

know, this is the first report relating eggshell proteins

with antifungal activity. A. niger was used in our assays

because it is a member of the most common group of fungi

in the environment and it also has entomopathogenic

potential (Moraes et al., 2001), at least against mosquitoes.

Another fungus, Fusarium solani is also pathogenic for

eggs of Panstrongylus geniculatus (Hartung and Lugo,

1996). The presence of antifungal activity, associated

with the eggshell, was possibly important during the

evolution of insect species. Their need of an open space

in the eggshell, to allow fertilization and gas exchange for

embryo respiration, possibly evolved in parallel with

the acquisition of antimicrobial agents that could be

associated with the eggshell. The fact that Rp30 protein

did not inhibit Aspergillus growth does not necessarily

mean that this protein is not an antifungal agent. We are

now testing both proteins against a variety of other fungi

and bacteria.

Arthropods produce a number of different peptides to

protect them against the invasion of microorganisms as

reviewed by Otvos (2000) and Bulet et al. (2004), but only a

few reports have described the presence of peptides with

antimicrobial activities associated with the eggshell of

insects (Marchini et al., 1997; Lamberty et al., 2001) and

nematodes (Lopez-Llorca et al., 2002). Here we have

shown for the first time that an eggshell component of an

important insect vector has an antifungal activity. In order

to benefit from the yolk, the fungus must first penetrate the

eggshell. The contact of Rp45 protein of Rhodnius egg

with the invading fungus hyphae may be enough to

block the invasion. Considering that Rp45 protein is not

soluble when associated with the eggshell, its effect is

possibly elicited by contact. In terms of embryo develop-

ment it is not necessary to kill the fungus; therefore a

fungistatic effect should be just enough. Supporting

this hypothesis, the addition of A. niger to the eggs of

R. prolixus, under a condition suitable for fungus growth,

is not enough to destroy the eggs and the nymphs hatch

normally.

Fungal cells develop mechanisms to secrete enzymes

(Hube, 2000; Naglik et al., 2003; Santos et al., 2006) onto

the hosts in order to invade their cells. Thus, host cells have

to counteract the effect of these enzymes in order to

survive. Extracellular proteinases of saprophytic fungi such

as A. niger are secreted primarily to provide nutrients for

the cells, but this biochemical property can be used to fulfill

specialized functions during the infective process (Naglik

et al., 2003). The authors did not investigate whether the

effect elicited by Rp45 protein was due to the effect of the

entire molecule or of the peptides derived from the Rp45

protein by the action of putative proteases from the fungus.

In any event the biological effect was elicited protecting the

embryo. The mechanism of the Rp45 protein is now under

investigation.

Antifungal agents generally inhibit enzymatic reactions

involved in fungal cellular biosynthesis, including amino

acids, nucleotides, lipids and polysaccharides, but the

ARTICLE IN PRESS

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

Please cite this article as: Bouts, D.M.D., et al., Biochemical properties of the major proteins from Rhodnius prolixus eggshell. Insect Biochem. Mol.

Biol. (2007), doi:10.1016/j.ibmb.2007.07.010

fungicide effect can also be achieved by interference in

intracellular transduction pathways (Kojima et al., 2004).

In fungi a cross-talk between cAMP and calcium signaling

pathway exists (Bencina et al., 2005), suggesting the

possibility of an antifungal agent to induce a metabolic

imbalance simultaneously in several different metabolic

pathways in the fungus making the study of the action

mechanism of an agent a difficult task.

A large number of antifungal proteins have been

described over the last two decades due to immunocom-

promised hosts such as AIDS patients under treatment

with immunosuppressive therapies and organ transplant

recipients. The target of these antifungal proteins varies but

antifungal protein active on the fungal cell wall, plasma

membrane, and intracellular targets can be recognized

(Theis and Sthal, 2004). The mechanisms of action are as

varied as their sources and include cell wall degradation,

membrane channel inhibition, pore formation, damage to

cellular ribosome, and inhibition of DNA synthesis and cell

cycle (Selitrennikoff, 2001).

The A. niger morphological alterations observed after

the addition of Rp45 protein to the culture medium include

alteration of cell walls and intracellular structures leading

to the appearance of swollen mitochondria and a large

amount of vacuoles. In Saccharomyces cerevisiae vacuoles

are central in much of the physiology of the organism. This

organelle is involved with pH and osmoregulation, protein

degradation, storage of amino acids, ions and polypho-

sphates and sporulation. Thus interference in this organelle

may potentially alter several metabolic pathways at the

same time to such an extent that it could end up as a

defective organism (Klionsky et al., 1990). Different

antifungal 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 the

mechanism leading to this is not known. The fact that

Rp45 protein is active simultaneously against the fungal

cell wall and intracellular targets makes this protein a

potential fungicide.

Acknowledgments

We wish to express our gratitude to Jose de Souza Lima

Junior and Litiane M. Rodrigues for maintaining our

colony 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 supported

by grants from MCT/Conselho Nacional de Desenvolvi-

mento Cientıfico e Tecnologico (CNPq), Conselho de

Aperfeic-oamento de Ensino Superior (CAPES), Financia-

dora de Estudos e Projetos (FINEP), Programa de Apoio

ao Desenvolvimento Cientıfico e Tecnologico (PADCT),

Programa de Nucleos de Excelencia (PRONEX) and

Fundac- ao de Amparo a Pesquisa Carlos Chagas Filho

(FAPERJ).

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