Journal of Insect Physiology · 2016. 8. 22. · the peritrophic membrane of larval sugar cane...

Journal of Insect Physiology xxx (2010) xxx–xxx

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IP-2445; No. of Pages 9

The fate of vicilins, 7S storage globulins, in larvae and adult Callosobruchusmaculatus (Coleoptera: Chrysomelidae: Bruchinae)

Sheila M. Souza a, Adriana F. Uchôa b, José R. Silva c, Richard I. Samuels d, Antônia E.A. Oliveira a,Eliana M. Oliveira e, Ricardo T. Linhares f, Daniel Alexandre f, Carlos P. Silva f,*a Laboratório de Quı́mica e Função de Proteı́nas e Peptı́deos, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro, 28013-600 Campos dos

Goytacazes, RJ, Brazilb Departamento de Biologia, Centro de Biociências, Universidade Federal do Rio Grande do Norte, 59072-970 Natal, RN, Brazilc Departamento de Bioquı́mica, Instituto de Quı́mica, Universidade Federal do Rio de Janeiro, 27930-560 Macaé, RJ, Brazild Laboratório de Entomologia e Fitopatologia, Centro de Ciências e Tecnologia Agropecuárias, Universidade Estadual do Norte Fluminense Darcy Ribeiro, 28013-602 Campos dos

Goytacazes, RJ, Brazile Laboratório Central de Microscopia Eletrônica, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, SC, Brazilf Departamento de Bioquı́mica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, 88040-900 Florianópolis, SC, Brazil

A R T I C L E I N F O

Article history:

Received 12 January 2010

Received in revised form 4 March 2010

Accepted 5 March 2010

Keywords:

Protein absorption

Fat body

Antimicrobial peptides

Vigna unguiculata

A B S T R A C T

The fate of vicilins ingested by Callosobruchus maculatus and the physiological importance of these

proteins in larvae and adults were investigated. Vicilins were quantified by ELISA in the haemolymph

and fat body during larval development (2nd to 4th instars), in pupae and adults, as well as in ovaries and

eggs. Western blot analysis demonstrated that the majority of absorbed vicilins were degraded in the fat

body. Tracing the fate of vicilins using FITC revealed that the FITC–vicilin complex was present inside

cells of the fat body of the larvae and in the fat bodies of both male and female adult C. maculatus.

Labelled vicilin was also detected in ovocytes and eggs. Based on the results presented here, we propose

that following absorption, vicilins accumulate in the fat body, where they are partially degraded. These

peptides are retained throughout the development of the insects and eventually are sequestered by the

eggs. It is possible that accumulation in the eggs is a defensive strategy against pathogen attack as these

peptides are known to have antimicrobial activity. Quantifications performed on internal organs from

larvae of C. maculatus exposed to extremely dry seeds demonstrated that the vicilin concentration in the

haemolymph and fat body was significantly higher when compared to larvae fed on control seeds. These

results suggest that absorbed vicilins may also be involved in the survival of larvae in dry environments.

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1. Introduction

Seeds of legumes (Fabaceae) such as cowpea (Vigna unguiculata)and the common bean (Phaseolus vulgaris) are an important staplesource of dietary proteins, particularly in developing countries, butcultivated beans are subject to postharvest losses by larvae of bean-beetles (Coleoptera: Chrysomelidae: Bruchinae), also known asbruchids or inappropriately as weevils. The most important speciesare the cowpea weevil (Callosobruchus maculatus), the common beanweevil (Acanthoscelides obtectus) and the Mexican bean weevil(Zabrotes subfasciatus). Due to adaptations developed by thesestored-bean pests, seed destruction is often so high that the pulsegrains become unsuitable for human consumption and unviable forreplanting or commercialization. These adaptations involve thecapacity to develop in a hostile environment (low humidity and in

* Corresponding author. Fax: +55 48 3234 4069.

E-mail address: [email protected] (C.P. Silva).

Please cite this article in press as: Souza, S.M., et al., The fate of vicilins,(Coleoptera: Chrysomelidae: Bruchinae). J. Insect Physiol. (2010), do

0022-1910/$ – see front matter � 2010 Elsevier Ltd. All rights reserved.doi:10.1016/j.jinsphys.2010.03.009

the presence of insecticidal plant secondary compounds), a multi-voltine life cycle and following emergence, adults mate and ovipositimmediately without the need for water or food.

In nature, the relationship between bruchids and legumes isunique (Johnson, 1981; Jermy and Szentesi, 2003; Kergoat et al.,2007; Tuda, 2007). Approximately 80% of bruchid species onlydevelop inside leguminous seeds (Southgate, 1979; Johnson,1981). Interactions between bruchids and their seed hosts arecomplex and led to the appearance of adaptive mechanismsenabling the insects to reproduce and develop despite the plantdefenses. The effective seed defenses that reduce losses andretention of their reproductive potential are highly varied amongspecies of Fabaceae, including a variety of defensive proteins(Janzen, 1981; Carlini and Grossi-de-Sá, 2002; Ng, 2004).

Following oviposition within the seeds, larval development willonly be successful if the larvae are capable of ingesting,assimilating, or detoxifying the defensive compounds present inthe seeds. Among the protein-based defense arsenal of bean seeds(Tribe Phaseoleae) and other species of Fabaceae are the lectin

7S storage globulins, in larvae and adult Callosobruchus maculatusi:10.1016/j.jinsphys.2010.03.009

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S.M. Souza et al. / Journal of Insect Physiology xxx (2010) xxx–xxx2

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super-family that includes arcelins, the lectin phytohemagglutininA (PHA), a-amylase inhibitors (a-AI) (Huesing et al., 1991;Nishizawa et al., 2007), several types of peptidase inhibitors(Ryan, 1990) and variant vicilins (Macedo et al., 1993; Sales et al.,2001).

Vicilins are traditionally known as storage seed proteins of theglobulin 7S type present in leguminous seeds and in non-legumeplants (Shutov et al., 1995). However, since the early 1990s,additional functions have been inferred from their phylogeneticrelationships or chemical properties. Experimentally confirmedproperties include: insecticidal activity (Macedo et al., 1993; Motaet al., 2003; Paes et al., 2008), involvement in desiccation/hydration process (Bäumlein et al., 1995; Shutov et al., 1998),sucrose-binding activity (Braun et al., 1996) and antimicrobialactivity (Gomes et al., 1997; Marcus et al., 1999; Ng, 2004;Manners, 2007).

Studies of antifungal plant defenses have revealed that peptidesliberated from the partial proteolysis of vicilins are toxic todifferent species of fungal pathogens (Chung et al., 1997; Marcuset al., 1999, 2008; Wang et al., 2001). A growing body of evidencesuggests that during seed germination, a finely regulatedproteolysis generates vicilin-derived antifungal peptides that arereleased to the surrounding fluid to protect the plantula in earlystages of establishment (Marcus et al., 2008).

The defensive role of vicilins against bean-beetles was firstreported from the African cowpea genotype IT81D-1045,resistant to C. maculatus (Macedo et al., 1993). However, themechanism of action of the vicilins on this bruchid is not yetunderstood, although evidence suggests that chitin-bindingactivity of these proteins is important (Sales et al., 2001). Ithas been demonstrated that vicilins from V. unguiculata bind tothe peritrophic membrane of larval sugar cane stalk borer(Diatraea saccharalis) and yellow meal worm (Tenebrio molitor),significantly reducing the survival rate of these insects (Motaet al., 2003; Paes et al., 2008).

In addition to the evidence that vicilins may exert their effectsfollowing interaction with constituents on the surface of C.maculatus midgut epithelium, the systemic distribution of vicilinsin larvae has also been investigated. Uchôa et al. (2006)demonstrated that vicilins from cowpea were absorbed intactby larval C. maculatus, and subsequently detected in thehaemolymph and internal organs such as fat body and malphigiantubules. Curiously, vicilins were also detected in C. maculatusadults, which do not feed on the seeds.

Aiming to further understand the physiological significance ofvicilin absorption by C. maculatus larvae and why they areconserved in adults, quantitative and qualitative approaches wereused to trace the fate of the absorbed vicilins. The resultsdemonstrated that vicilins are partially hydrolysed in the fat bodyof larvae and adults and subsequently deposited in eggs.Experiments also showed that vicilin concentration in thehaemolymph and fat body was significantly higher when larvaedeveloped inside extremely dry seeds, indicating a possible role inadaptation to this hostile environment.

2. Materials and methods

2.1. Rearing of insects

The colony of the cowpea weevil C. maculatus used in this workwas initiated with animals supplied originally by Dr. J.H.R. Santos,Centro de Ciências Agrárias, Universidade Federal do Ceará,Fortaleza, CE, Brazil. Stock cultures of this species have beenmaintained continuously since 1984. Insects were reared on V.unguiculata seeds in natural photoperiod and maintained at29 � 1 8C and relative humidity of 65 � 5%.


2.2. Preparation of samples from insects

Haemolymph and fat body samples were extracted from larvaeand pupae at different days after oviposition (10 animals for eachpreparation). Fat body samples were obtained from adults duringdifferent periods following emergence (10 animals each prepara-tion) and ovaries obtained from mated females 2 days followingemergence. Only actively feeding larvae with food filled gut tractswere chosen for dissection. Pre-chilled larvae were washed withcold saline (150 mM NaCl), dried with filter paper and haemolymphsamples were obtained by piercing the cuticle with a fine needlefollowed by collection of the exuded liquid directly into pre-chilled1.5 mL tubes containing phenylthiocarbamide (0.1 mg per tube).

After collection of the haemolymph and removal of organs suchas the midgut and malphigian tubules, most of the fat body wascollected with fine forceps and transferred directly to pre-chilled1.5 mL tubes. Peripheral fat bodies attached to epidermis were alsocollected, although it was difficult to remove all of them.

Following collection, pooled haemolymph, fat body tissues andovaries were homogenized using a hand-held Potter-Elvehjemhomogenizer immersed in ice. Tissue homogenates were centri-fuged at 20,000 � g for 30 min at 4 8C and the supernatants wereused for protein and vicilin determinations.

2.3. Vicilin purification and antibody production

Vicilins were purified from C. maculatus susceptible (Epace-10)seeds employing the procedure of Macedo et al. (1993). Groundmeal extracted with 50 mM borate buffer, pH 8.0, for 30 min atroom temperature was centrifuged (30 min at 8000 � g, 5 8C) andsoluble proteins were fractionated by ammonium sulphateprecipitation. The 70–90% saturation fraction was dialysed againstdistilled water, freeze-dried and chromatographed on a DEAE-Sepharose column (2 cm � 20 cm) equilibrated with 50 mM Tris–HCl, pH 8.0, and eluted with a NaCl gradient (0–1 M) in the samebuffer. The vicilin-rich fractions were then loaded onto a SephacrylS-400 column (2.5 cm � 70 cm) in 0.1 M Tris–HCl, 0.25 M NaCl, pH8.0. Fractions containing vicilins were dialysed against distilledwater and freeze-dried.

Antisera against purified vicilins were prepared by immuniza-tion of rabbits according to established methods. Enriched IgGswere obtained by affinity chromatography of the crude immuneserum using a protein A column (protein A bound to Sepharose CL-4B). Pre-immune sera were collected before immunization andused for control experiments.

2.4. Protein determination

Protein concentration was determined according to the methodof Smith et al. (1985), as modified by Morton and Evans (1992),using bovine serum albumin as a standard. In some experiments,protein concentration was determined according to the method ofBradford (1976), using ovalbumin as a standard.

2.5. Electrophoresis and Western blotting

Proteins were separated by SDS polyacrylamide gel electro-phoresis (Laemmli, 1970). Samples (20 mg of proteins) wereprepared by adding 4� SDS sample buffer (containing 10%mercaptoethanol) and boiled for 5 min prior to loading. Gels wererun at a constant voltage of 150 V and either stained usingCoomassie blue dye (0.05% [w/v] Coomassie blue in 7% [v/v] glacialacetic acid; 40% [v/v] methanol) followed by de-staining (19% [v/v]glacial acetic acid, 40% [v/v] methanol), or transferred tonitrocellulose membranes using a BioRad Trans-blot semi-drytransfer cell, according to the manufacturer’s recommendations.


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Immunoblotting was performed using the polyclonal antibodyagainst purified vicilin as described above. Both the primary andthe secondary antibodies were used at a dilution of 1:2000. Bindingwas visualised using a horseradish peroxidase-conjugated sec-ondary antibody (Bio-Rad Laboratories, Richmond, CA, USA)according to manufacturer’s instructions.

2.6. Enzyme-linked immunosorbent assay (ELISA)

ELISA assays were performed in microtitration polystyreneplates (Nunc, Denmark). The solubilised vicilin was bound to eachwell by incubating 50 mL (1–10 mg final vicilin concentration) of0.1 M carbonate buffer (pH 9.5) for 1 h at 37 8C and overnight at4 8C. The plates were washed four times with 0.01% Tween 20 inPBS (PBS–T). Unoccupied binding sites were blocked by incubatingthe plates for 90 min at 37 8C with 280 mL of 1% gelatin in PBS, andthen washed with PBS–T. Subsequently, 50 mL of anti-vicilinantibody at a 1:2000 dilution was added and incubated for 90 minat 37 8C. After six washes with PBS–T, peroxidase-conjugated goatanti-rabbit immunoglobulin (1:2000) was added and incubated for90 min at 37 8C. The plates were washed with PBS–T and reactionwas revealed by addition of 50 mL of 0.04% o-phenylenediamineand 0.004% H2O2 in 100 mM sodium phosphate buffer, pH 5.0. Thereaction was stopped by adding 50 mL of 3N H2SO4 and sampleswere read at 492 nm in an ELISA reader (Cainelle-Gebara et al.,1995). The results are the means of at least three independentdeterminations.

2.7. Covalent conjugation of FITC (fluorescein isothiocyanate) to

vicilin

FITC was covalently coupled to vicilins from V. unguiculata(genotype Epace-10). FITC (50 mg in 1 mL anhydrous DMSO) wasimmediately diluted in 0.75 M bicarbonate buffer, pH 9.5 before use.Following addition of FITC to give a ratio of 1 mg per mg of vicilin, thetube was wrapped in foil; incubated and rotated at roomtemperature for 1 h. The un-reacted FITC was removed by dialysisagainst distilled water. The resulting solution was freeze-dried.

2.8. Feeding C. maculatus larvae using artificial seeds

In order to verify the fate of the labelled vicilins in larvae of C.maculatus, the FITC–vicilin complex was mixed with cowpea flourat the concentration of 2.0% (w/w). C. maculatus larvae weretransferred at the beginning of the fourth instar (when larvae areactively consuming their diet) to gelatin capsules containingmixtures of the seed flour of V. unguiculata and the FITC–vicilincomplex. The flour was inserted and compacted inside gelatincapsules with the aid of spatulas and glass rods. Larvae removedfrom seeds of V. unguiculata were transferred to a cavity producedin the compacted mass of flour in one half of the gelatin capsule, ata ratio of three larvae per capsule. Following this, the two halves ofthe capsules were carefully joined together in order to permit thefeeding movements of the larvae and maintained in the dark.

Controls were used in which only FITC was mixed with seedflour at the concentration of 2.0% (w/w) in order to assess the levelof FITC absorption. Capsules containing only cowpea flour wereused as controls to evaluate auto-fluorescence of the internalorgans.

After the feeding period, larvae were removed and dissected forcollection of the midgut and fat body. Some larvae were left tocomplete their metamorphosis until emergence of adults. In orderto visualise and document the presence of labelled vicilins bymicroscopy from larvae, adults and eggs, fresh portions weremounted on glass slides and visualised using a laser Confocalmicroscope (Leica DMI6000 B Microscope).


In order to obtain low humidity seeds, drier than the controlseeds used to maintain the insect colonies, seeds were maintainedin a hermetically sealed glass container in the presence of silica gelfor 1 month. The low humidity seeds were exposed to females for24 h and maintained with silica gel until the larvae reached thefourth instar, when they were dissected and samples from theirhaemolymph and fat body were used for vicilin quantification byELISA as previously described.

2.9. Extraction and quantification of vicilins from C. maculatus eggs

C. maculatus females 3 days after emergence were transferred toglass vials and maintained during 24 h inside an incubator at 28 8Cand 70% RH. After this time, the eggs laid on cover glass slides wereremoved with a fine needle, placed in a 1.5 mL tube andhomogenized (50 eggs/150 mL) in 100 mM phosphate buffer with500 mM NaCl, pH 7.6, for 30 min at 4 8C. The homogenate wascentrifuged at 10,000 � g for 30 min at 4 8C and the proteinconcentration in supernatant was determined by the Bradfordmethod (Bradford, 1976). Supernatant proteins were diluted with100 mM carbonate buffer (pH 9.5) to a final protein concentrationof 20 mg/100 mL and vicilin determinations were carried out byELISA as previously described.

2.10. Statistical analysis

The one-way analysis of variance (ANOVA) was used to analysethe data throughout the experiments. The Student t-test was usedwhen the ANOVA indicated a difference between sample means.

3. Results

3.1. Vicilin quantification during the larval development and in pupae

of C. maculatus

Vicilin polypeptide subunits extracted from haemolymph andfat body of larvae fed on cowpea seeds were detected byelectrophoresis followed by Western blotting and quantified byELISA. The same polyclonal antibody was used here as in Uchôaet al. (2006), therefore control experiments for the specificity ofthis antibody were omitted from this paper. However, in allWestern blots control lanes, purified vicilin molecules were used.

In order to quantify vicilins in larvae of C. maculatus 14, 16, 18,20 days after oviposition and in pupae (22 and 24 days afteroviposition), the concentrations of these proteins in the haemo-lymph and fat body were determined by ELISA. Results expressedin ng of vicilin per animal showed that higher quantities of vicilinswere found in the fat body than in the haemolymph (Fig. 1).

Fig. 1 also showed that an increase in vicilin concentration wasobserved up to the 16th day of development, followed by adecrease during the 4 days before pupation.

3.2. Vicilin quantification in the fat body of adults and in eggs of C.

maculatus

Quantitative determinations of vicilins in the fat body of adultmale and female C. maculatus showed that immunoreactivepeptides are present during the first 10 days of adult life(Fig. 2). Vicilin concentration was highest during the first 2 daysof adult life in both males and females. In female insects, vicilinconcentration was significantly higher during days 1 and 2 thanthe following 7 days of development.

ELISA determinations showed that the vicilin-derived peptideswere also present in the ovaries of mated C. maculatus females(0.33 � 0.015 ng vicilin equivalent/mg protein, n = 3, cohort of 15animals). Vicilins were also detected in eggs laid by C. maculatus



Fig. 2. Vicilin concentration in the fat body of male and female Callosobruchusmaculatus determined by ELISA during different periods following emergence.

Results are expressed as vicilin concentration (mg per insect). The histograms showthe mean of three independent experiments containing 10 animals in each cohort,

while the bars show the standard deviations. Significant differences (*) in vicilin

concentration in the fat bodies of female insects were seen on the 1st and 2nd days

following emergence (P < 0.001), according to Student’s t-test, when compared to

all other periods tested.

Fig. 4. Western blot analysis of vicilins from fat body of adult Callosobruchusmaculatus during different days following emergence. (A) Females and (B) males.

Lane V, vicilin standard. Numbers represent days following emergence; arrow

heads indicate vicilin subunits and the asterisks indicate the position of vicilin-

derived peptides.

Fig. 1. Vicilin concentration in the haemolymph and fat body of Callosobruchusmaculatus larvae (14–20 days after oviposition) and pupae (22 and 24 days after

oviposition) determined by ELISA. Error bars represent standard deviations

calculated from three independent samples containing 10 animals each.


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females (49.45 � 7.28 ng vicilin equivalent/mg protein, n = 3, cohortof 50 eggs).

3.3. Western blot analysis

The presence of vicilins in the haemolymph and in the fat bodyof C. maculatus larvae and pupae during different periods of

Fig. 3. Western blot analysis of vicilin proteins from haemolymph and fat body of CallosHaemolymph and (B) fat body. Lane V, vicilin standard. Numbers represent days after ov

vicilin-derived peptides.


development was confirmed by Western blotting (Fig. 3). Positiverecognition in both larvae and pupae was seen in the haemolymph(Fig. 3A) and fat body (Fig. 3B). The immuno-recognition of threevicilin subunits in the haemolymph suggests that the trimeric formof the vicilin molecule was maintained following absorption fromthe midgut. However, in pupae and in the period up to pupation, arange of peptides with higher electrophoretic motilities than thethree original vicilin subunits were positively recognised by theantibody (Fig. 3B). The low intensity of the peptide bands wasprobably due to their low concentrations. In order to help visualisethe position of these bands on the gels, asterisks were used inFigs. 3 and 4. The recognition pattern by the antibody in the fatbody was similar to that observed in the pupae suggesting that thevicilin subunits were hydrolysed in the fat body.

obruchus maculatus larvae and pupae during different periods after oviposition. (A)

iposition. Arrows indicate vicilin subunits and the asterisks indicate the position of



Fig. 5. Vicilin concentration (ng per insect) determined by ELISA in the haemolymphand fat body of Callosobruchus maculatus larvae developing inside control seeds and

in seeds with extremely low humidity. The histograms are the mean values and the

bars represent standard deviations calculated from three independent samples

from 10 animals in each cohort. Different letters represent statistical differences

(P < 0.001) according to Student’s t-test.


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Fig. 4 confirms that the immunoreactive vicilin-derivedpeptides are present in the fat body of both females (Fig. 4A)and males (Fig. 4B).

3.4. Differences in vicilin uptake when larvae developed on extremely

low humidity seeds

In order to verify if the absorption of vicilin is correlated withenvironmental humidity, control seeds (approx. 15% RH) were

Fig. 6. Fluorescence microscopy localisation of FITC labelled vicilin in the internal organsFITC–vicilin for 24 h. Following the feeding period, the animals were dissected and sec

visualised using a laser confocal microscope. Control animals were fed with unlabelled vi

as fluorescence was minimal. Plate A: confocal laser scanning micrograph (single optica

bright-field and fluorescence images of the midgut section. Note fluorescence in the mi

midgut epithelium (Ep). Plate D: confocal laser scanning micrograph of fat body section (s

superimposition of bright-field image and fluorescence microscopy of the fat body sec


compared to seeds with extremely low humidity (approx. 5% RH)infested by C. maculatus. Fig. 5 shows that the vicilin concentrationin both the haemolymph and fat body of larvae aged 16 days afteroviposition significantly increased when the larvae were main-tained on low humidity seeds.

3.5. Detection of FITC–vicilin in internal organs and eggs of C.

maculatus

In order to detect the fate of vicilin molecules in alldevelopmental stages of C. maculatus, fourth instar larvae werefed with FITC–vicilin. Two days after feeding (larvae) and 2 daysafter emergence of adults, whole-mounts of larval midgutepithelium and fat body of larvae and adults, as well as of ovariesand eggs were observed using confocal microscopy (Figs. 6–8).FITC–vicilin was detected dispersed in the cytoplasm of midgutepithelial cells, indicating that the protein crossed the epitheliumthrough a transcellular pathway (Fig. 6). Confocal images of whole-mounts of fat body from larvae and adults revealed that the FITC–vicilin complex entered the cells and was localised inside vesicles(Figs. 6 and 7A–C). Positive fluorescence was also unequivocallyobserved in ovocytes (Fig. 7D–F) and in eggs (Fig. 8).

4. Discussion

The absorption of intact proteins across the midgut epitheliumof insects has received limited attention until recently, however agrowing number of papers have confirmed that this phenomenonis more common than previously thought (review by Jeffers and

of larval Callosobruchus maculatus. Fourth instar larvae were fed with a mixture of 2%

tions of the midgut tissue and fat body were freshly mounted on glass slides and

cilin or with FITC incorporated in the cowpea flour, but results were not shown here

l section). Plate B: bright-field image of midgut section. Plate C: superimposition of

dgut lumen (L) and association with the basal lamina (arrows) that surrounds the

ingle optical section). Plate E: bright-field image of the same fat body section. Plate F:

tion. Note florescence was confined to vesicles inside cells of fat body lobules.



Fig. 7. Fluorescence microscopy detection of FITC–vicilin in adult Callosobruchus maculatus fat body and ovocyte. Fourth instar larvae were fed with a mixture of 2% FITC–vicilin and left to complete their metamorphosis until adult emergence. Females were dissected and fat body and ovocytes were freshly mounted on glass slides and

visualised using laser confocal microscopy. (A) Confocal laser scanning micrograph (single optical section); (B) bright-field and (C) superimposition of bright-field and

fluorescence images of the fat body section; (D–F) micrographs from ovocytes; (D) confocal laser scanning micrograph (single optical section); (E) bright-field micrograph; (F)

superimposition of bright-field and fluorescence images of the ovocyte.


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Roe, 2008). Initially observed in blood-feeding insects and morerecently protein absorption has been reported in a range ofphytophagous insect species (Powell et al., 1998; Hirayama et al.,2000; Sugimura et al., 2001; Fitches et al., 2001a,b; Habibi et al.,2002; Jeffers et al., 2005; Kurahashi et al., 2005; Casartelli et al.,2005, 2007; Uchôa et al., 2006). In several cases, xenobioticproteins were used to show the movement of macromoleculesacross the midgut epithelium, in addition to the normal dietaryproteins that are specifically absorbed. The capacity of someproteins and peptides to reach the internal environment via the guttissue is being considered as a promising method for deliveringinsecticides into the haemocoel (Casartelli et al., 2005; Jeffers andRoe, 2008; Fiandra et al., 2009). In spite of the potential as aninnovative technology to deliver pesticides, the details of howdietary proteins are absorbed are not yet fully understood(Modespacher et al., 1986; Allingham et al., 1992; Audsley et al.,2008; Casartelli et al., 2008; Fiandra et al., 2009).

As pointed out by Casartelli et al. (2005) and Jeffers and Roe(2008), knowledge of absorption of intact proteins by the midgutepithelium of insects is in its infancy. One of the less discussedthemes is the adaptive value of this process, or in other words, whydo some insect species absorb proteins from their diets and whatare the functions exercised by the absorbed proteins? Concerningthis issue, the function exerted by urease absorbed in larval B. moriis the best characterized to date (Hirayama et al., 1997, 1999, 2000;Sugimura et al., 2001; Kurahashi et al., 2005). The urease molecule


is specifically absorbed in an intact form, preserving its enzymeactivity. Their studies have led the authors to postulate theinvolvement of the dietary urease in the release of ammonia fromurea in the insect haemocoel. The released ammonia could beincorporated via the glutamine synthetase–glutamate synthasepathway into amino acids necessary for silk-protein synthesis,used in the production of the cocoon (Hirayama et al., 1997, 1999;Kurahashi et al., 2005).

In several cases, the absorbed proteins are not useful for theinsect, but conversely, they can cause toxic effects. The mannose-binding lectin from snowdrop (Galanthus nivalis), known as GNA, iscapable of crossing the midgut tissue of the plant hopper Nilaparvatalugens and accumulates in the haemolymph, fat body and ovarioles,causing a decrease in the reproductive capacity of the insect (Powellet al., 1998). Fitches et al. (2001a) have demonstrated the potential ofP. vulgaris phytohemagglutinin (PHA) isolectins to bind to midguttissue and detected their presence in organs other than the gut inlarvae of the tomato moth Lacanobia oleracea, causing antibiosis.Seed proteins called arcelins, potentially toxic proteins from seeds ofthe common bean P. vulgaris, are also capable of crossing the midgutepithelium of larval Z. subfasciatus, being detected in the haemo-lymph and fat body (Paes et al., 2000). Due to their capacity to crossthe insect midgut epithelium, some lectins have being used todeliver biologically active compounds into the haemocoel of insects(Fitches et al., 2002, 2004; Down et al., 2006; Trung et al., 2006;Jeffers and Roe, 2008).



Fig. 8. Detection of FITC–vicilin in Callosobruchus maculatus eggs. Fourth instar larvae were fed with a mixture of 2% FITC–vicilin and left to complete their development. Eggswere freshly mounted on glass slides and visualised using laser confocal microscopy. (A) Egg produced by a FITC–vicilin fed animal as observed using confocal laser scanning

microscopy. (B) Bright-field microscopy of an egg from a FITC–vicilin fed animal. (C) Superimposition of confocal and bright-field images of the same egg. (D) Egg produced by

an animal fed on a control diet containing only cowpea flour observed using confocal laser scanning microscopy. (E) Bright-field microscopy. (F) Superimposition of

fluorescence and bright-field images of an egg from control animal.


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Vicilins are thought to be multifunctional proteins. In additionto their function as seed storage proteins, vicilin-like proteins arethought to be involved in cellular desiccation/hydration processesand are toxic to some insect species (Bäumlein et al., 1995; Shutovet al., 1998; Mota et al., 2003; Moura et al., 2007; Paes et al., 2008).As the modification of vicilin molecules by conjugation with FITCdid not interfere with their absorption, this technique was used totrack the fate of the FITC–vicilin complex in larvae and in adult C.maculatus. The transport of vicilins in the larval bruchid C.maculatus appears to be similar to that of other proteins, whichare absorbed intact from the midgut lumen into the haemocoel ofdifferent insect species.

The variant vicilins may cause toxicity to C. maculatus bybinding to midgut epithelial cell surfaces, specifically to consti-tuents of the cell glycocalix (Macedo et al., 1993; Sales et al., 2001),although there were no obvious signs of damage to the cells (Saleset al., 2001). Oral administration of V. unguiculata vicilins to larvalsugar cane stalk borer D. saccharalis (Mota et al., 2003) and tolarvae yellow meal worm T. molitor (Paes et al., 2008) showed thatthese molecules bound to the peritrophic membranes, leading toadverse effects on larval development.

Aiming to further understand the putative toxic effects ofvariant vicilins to larval C. maculatus, systemic effects, rather thanlocalised effects on the midgut tissue were investigated (Uchôaet al., 2006). Evidence suggested that the binding of vicilins to thebrush border membrane leads to their absorption throughout theepithelium in an intact form, without causing any apparent


damage to the gut cells. In the experiments described here, the fateof vicilins was tracked following ingestion by larvae of C.maculatus. Experiments confirmed the transport of vicilins fromthe midgut lumen into the haemolymph and fat body of thisbruchid species (Figs. 1–3). Furthermore, vicilin was also detectedduring the second to fourth larval instar, and subsequently vicilinswere detected following pupation (Figs. 1 and 2). Surprisingly,vicilin-derived peptides could also be detected in the fat bodies ofadult males and females until at least 10 days after emergence(Fig. 4), although the pattern in females was different to that ofmales.

Western blotting analysis confirmed that entire vicilin mole-cules were absorbed by larvae of different ages and circulated intheir trimeric conformation in the haemolymph (Fig. 3). However,analysis of samples collected from the fat bodies of larvae duringthe late fourth instar, pupae or adults showed that the vicilinmolecules were partially degraded, liberating immunoreactivevicilin-derived peptides which continued to be recognised by thepolyclonal anti-vicilin antibody (Figs. 3 and 4). The presence ofvicilin in the fat body of adult bruchids is interesting, especially asthe adults do not feed under our experimental conditions.Therefore we suggest that vicilin or vicilin-derived peptidesdetected in adults were originally incorporated during the larvalphase and conserved during the pupal and adult phases. Theadaptive significance of this finding was further investigated.Vicilin-derived peptides in the fat body were quantified during thefirst 10 days of adult development. The low concentrations seen in




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males did not fluctuate over time, whereas, females have relativelyhigh vicilin-derived peptide concentrations during the first 2 daysof adult life, falling dramatically on the 3rd day of development andremained low until the end of the experiment (Fig. 2). Fig. 4 showsthat the positive reaction with the anti-vicilin polyclonal antibodyis associated to vicilin-derived peptides in both males and females.Vicilins detected by ELISA were quantified in the ovaries(0.33 � 0.015 ng vicilin equivalent/mg protein, n = 3, cohorts of 15animals) and in the eggs (49.45 � 7.28 ng vicilin equivalent/mgprotein, n = 3, cohorts of 50 eggs).

To address the question of the possible functions of absorbedvicilins, the hypothesis that vicilins are used as a mechanism thatpermits the bruchid larvae to survive in the relatively dryenvironment of the mature seed cotyledon was investigated.Following the rearing of C. maculatus larvae inside extremely dryseeds, vicilin concentration in the haemolymph and fat body wassignificantly higher when compared to larvae reared on controlseeds (Fig. 5). Taking into account that vicilin-like molecules areassociated to the desiccation/hydration process in other models(Bäumlein et al., 1995; Shutov et al., 1998) and that the fat body isthe main organ associated to adaptive anhydrobiosis in someinsects (Watanabe et al., 2005), this hypothesis merits furtherinvestigation. The participation of vicilins as storage proteins or asenergy sources is improbable, as the larvae are in constant contactwith in their diet. Furthermore, according to Wightman (1978),Callosobruchus analis adults, a close relative of C. maculatus, usestorage lipids instead of proteins as an energy source.

At the end of the larval phase and in adults, the trimericconformation of the vicilin molecule was not easily detected andWestern blotting experiments revealed the predominance ofimmunoreactive peptide fragments (Figs. 3 and 4). Consideringthat vicilin-derived peptides are known to have antimicrobialactivity (Chung et al., 1997; Marcus et al., 1999, 2008; Wang et al.,2001; Moura et al., 2007), it is possible that vicilins deposited in theeggs may act as protective compounds against pathogens. Thequantitative determinations (ELISA), the FITC–vicilin complexlocalisation by confocal microscopy and Western blotting ofsamples from females and in eggs confirmed that vicilin andvicilin-derived peptides are indeed passed from the ovaries to theeggs (Figs. 7 and 8). The deposition of vicilins in the internal organscould also protect the larvae and adults against entomopathogenicfungi and parasitoids.

The results presented in this paper shed light on the possiblefunctions associated to the absorption of a storage seed protein bya phytophagous insect. Very little is known about the moleculardetails of the absorptive process of entire proteins in insect gutsand even less is known of the functional significance of this process(reviewed by Jeffers and Roe, 2008). As pointed out by Uchôa et al.(2006), even if part of the dietary vicilin is digested in the midgutlumen after ingestion by the larvae, the stability of vicilinmolecules and their affinity for the putative receptor may be highenough to mediate its transport from the midgut lumen into thehaemolymph. Further investigation of the vicilin-binding mole-cule(s) in the midgut tissue, the transport of vicilins in thehaemolymph, the internalization and the controlled hydrolysis inthe fat body and their deposition in the eggs will hopefully increaseour comprehension of their roles in larvae and adult bruchids.

Acknowledgements

We thank Cristovão Barros Pinheiro for expert technicalassistance. This work was supported by the Brazilian researchagencies CNPq and FAPERJ. J.R. Silva is member of the InstitutoNacional de Ciência e Tecnologia em Entomologia Molecular(INCT-EM). A.E.A. Oliveira, R.I. Samuels and C.P. Silva are CNPqresearch fellows.


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The fate of vicilins, 7S storage globulins, in larvae and adult Callosobruchus maculatus (Coleoptera: Chrysomelidae: Bruch...IntroductionMaterials and methodsRearing of insectsPreparation of samples from insectsVicilin purification and antibody productionProtein determinationElectrophoresis and Western blottingEnzyme-linked immunosorbent assay (ELISA)Covalent conjugation of FITC (fluorescein isothiocyanate) to vicilinFeeding C. maculatus larvae using artificial seedsExtraction and quantification of vicilins from C. maculatus eggsStatistical analysis

ResultsVicilin quantification during the larval development and in pupae of C. maculatusVicilin quantification in the fat body of adults and in eggs of C. maculatusWestern blot analysisDifferences in vicilin uptake when larvae developed on extremely low humidity seedsDetection of FITC–vicilin in internal organs and eggs of C. maculatus

DiscussionAcknowledgementsReferences

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