High entomotoxicity and mechanism of the fungal GalNAc/Gal-specific Rhizoctonia solani lectin in pest insects

Journal of Insect Physiology 59 (2013) 295–305

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Journal of Insect Physiology

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High entomotoxicity and mechanism of the fungal GalNAc/Gal-specificRhizoctonia solani lectin in pest insects

Mohamad Hamshou a,b, Els J.M. Van Damme b, Silvia Caccia a,b, Kaat Cappelle a, Gianni Vandenborre a,b,Bart Ghesquière c,d, Kris Gevaert c,d, Guy Smagghe a,⇑a Department of Crop Protection, Faculty of Bioscience Engineering, Ghent University, Coupure links 653, B-9000 Ghent, Belgiumb Laboratory of Biochemistry and Glycobiology, Department of Molecular Biotechnology, Faculty of Bioscience Engineering, Ghent University,Coupure links 653, B-9000 Ghent, Belgiumc Department of Medical Protein Research, VIB, B-9000 Ghent, Belgiumd Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium

a r t i c l e i n f o

Article history:Received 2 November 2012Received in revised form 21 December 2012Accepted 24 December 2012Available online 2 January 2013

Keywords:Rhizoctonia solaniFungal lectinSpodoptera littoralisAcyrthosiphon pisumEntomotoxicityInsect midgutMicrovillar bindingCF-203 midgut cellsCarbohydrate bindingApoptosisDNA fragmentationCaspaseAffinity chromatographyMembrane proteinPest control

0022-1910/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.jinsphys.2012.12.003

⇑ Corresponding author. Tel.: +32 9 264 6150; fax:E-mail address: [email protected] (G. Smagg

a b s t r a c t

Whole insect assays where Rhizoctonia solani agglutinin (RSA) was fed to larval stages of the cotton leaf-worm Spodoptera littoralis and the pea aphid Acyrthosiphon pisum demonstrated a high concentration-dependent entomotoxicity, suggesting that this GalNAc/Gal-specific fungal lectin might be a good controlagent for different pest insects. RSA at 10 mg/g in the solid diet of 2nd-instar caterpillars caused 84%weight reduction after 8 days with none of the caterpillars reaching the 4th-instar stage. In suckingaphids, 50% mortality was achieved after 3 days with 9 lM of RSA in the liquid diet.

Feeding of FITC-labeled RSA to both insect pest species revealed strong lectin binding at the apical/luminal side of the midgut epithelium with the brush border zone, suggesting the insect midgut as a pri-mary insecticide target tissue for RSA. This was also confirmed with cell cultures in vitro, where there washigh fluorescence binding at the microvillar zone with primary cultures of larval midgut columnar cells ofS. littoralis, and also at the surface with the insect midgut CF-203 cell line without lectin uptake in themidgut cells.

In vitro assays using insect midgut CF-203 cells, revealed that RSA was highly toxic with an EC50 of0.3 lM. Preincubation with GalNAc and saponin indicated that this action of RSA was carbohydrate-bind-ing dependent and happened at the surface of the cells. Intoxicated CF-203 cells showed symptoms ofapoptosis as nuclear condensation and DNA fragmentation, and this concurred with an increase of cas-pase-3/7, -8 and -9 activities. Finally, RSA affinity chromatography of membrane extracts of CF-203 cellsfollowed by LC–MS/MS allowed the identification of 5747 unique peptides, among which four putativelyglycosylated membrane proteins that are associated with apoptosis induction, namely Fas-associated fac-tor, Apoptosis-linked gene-2, Neuroglian and CG2076, as potential binding targets for RSA. These data arediscussed in relation to the physiological effects of RSA.

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1. Introduction

Lectins or agglutinins are a very diverse group of carbohydrate-binding proteins that are widely distributed throughout livingorganisms, including plants, animals, fungi, bacteria and viruses(Vasta and Ahmed, 2008; Michiels et al., 2010; Hartmann and Lind-horst, 2011; Khan and Khan, 2011). Each lectin contains at leastone non-catalytic domain that can reversibly bind to a specific car-bohydrate (Loris, 2002). Lectins play a role in various biologicalprocesses such as cell–cell recognition, defense reactions or storage(Van Damme, 2008). In the last decade many plant lectins were

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shown to exert entomotoxic properties by affecting the survivalor development of pest insects belonging to different orders (Van-denborre et al., 2011). Furthermore, several plant lectins with dif-ferent carbohydrate specificities were found to be toxic to insectcell lines originating from lepidopteran tissues (Smagghe et al.,2005, 2009).

To our knowledge only very few fungal lectins have been re-ported to possess insecticidal activity towards pest insects. Oneof them is the Xerocomus chrysenteron lectin (XCL), a lectin isolatedfrom the edible mushroom X. chrysenteron, which showed hightoxicity towards the fruit fly Drosophila melanogaster and theaphids Acyrthosiphon pisum and Myzus persicae (Trigueros et al.,2003; Jaber et al., 2008). Recently, insecticidal activity was also re-ported for the fungal lectin isolated from the phytopathogenic fun-

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gi Sclerotinia sclerotiorum. This Sclerotinia sclerotiorum agglutinin(SSA) showed high toxicity towards the pea aphid A. pisum andthe insect midgut cell line CF-203 (Hamshou et al., 2010a).

Rhizoctonia solani agglutinin (RSA) is a lectin that was isolatedfrom the soil pathogen R. solani (Vranken et al., 1987). This fungusproduces black sclerotia which enables the fungus to survive in thesoil under harsh conditions for a long time. RSA is a homodimericprotein consisting of two non-covalently associated subunits of15.5 kDa with high affinity for N-acetylgalactosamine (GalNAc),galactose (Gal) and more complex glycoproteins (Candy et al.,2001). RSA is structurally and evolutionary related to the familyof proteins possessing a ricin-type lectin motif (Candy et al.,2001). Since RSA is abundantly present in the sclerotes, it has beenproposed that the lectin serves as a storage protein with potentialdefense function (Kellens and Peumans, 1990).

In the present study, the toxicity of RSA was investigated to-wards the larval stages of the cotton leafworm Spodoptera littoralisand A. pisum pea aphids. Both insect species are important pestinsects causing high damage in agriculture worldwide and arerepresentatives for two important groups of pest insects with bit-ing–chewing and piercing–sucking mouthparts, respectively. Tostudy the target tissue for the lectin in both caterpillars and aphids,feeding experiments were performed with FITC-labeled lectin. Fur-thermore, analyses were done with primary cultures of matureepithelial cells of the midgut from S. littoralis larvae that showedhigh sensitivity for RSA. FITC-labeled RSA showed binding to thecell surface of these cells without internalization of the lectin.Interestingly, similar observations were made with the insectCF-203 cell line that was originated from the midgut of anotherlepidopteran, the spruce budworm (Choristoneura fumiferana).Subsequently, cell death induction in midgut CF-203 cells uponexposure to RSA was investigated for DNA fragmentation, nuclearcondensation and induction of caspase activities. Finally, potentialtarget proteins in the cell membrane of these midgut CF-203 cellswere identified using RSA affinity chromatography followed byLC–MS/MS.

2. Materials and methods

2.1. Insects

A continuous colony of the cotton leafworm S. littoralis was kepton an agar-based artificial diet (Iga and Smagghe, 2011). Pea aphids(A. pisum) were kept on young broad bean (Vicia faba) plants(Nachman et al., 2011). Both insects were maintained under stan-dardized conditions of 23–25 �C, 60–70% relative humidity and a16:8 (light:dark) photoperiod.

2.2. Purification of RSA and labeling with FITC

RSA was purified from sclerotes of the R. solani strain AG 1-1Busing a combination of affinity chromatography on Gal-Sepharose4B and ion exchange chromatography on a Q Sepharose Fast Flowcolumn (GE Healthcare, Uppsala, Sweden) as described previously(Hamshou et al., 2010b). The purity of the lectin was analyzed bySDS–PAGE. After purification, RSA was labeled with FITC usingthe method described in Hamshou et al. (2010a).

2.3. Treatment of S. littoralis with RSA via artificial diet

Newborn (0–6 h old) 1st-instar larvae of S. littoralis were fed onStonefly Heliothis artificial diet containing different concentrationsof purified RSA (1, 5 and 10 mg/g fresh weight) in the diet for8 days as described previously (Caccia et al., 2012). At the end ofthis experiment, the individual larval weight was determined

and total larval mortality in each treatment was recorded. Pertreatment, three replications of 10 insects each were performed,and the experiment was repeated twice. A total of 60 insects wereanalyzed for each lectin concentration.

2.4. Treatment of A. pisum with RSA via artificial diet

In the aphid bioassay, 15 neonates (0–12 h old) of A. pisum werefed on an artificial liquid diet supplemented with purified RSA atdifferent concentrations (1.5–35 lM), essentially as described pre-viously (Smagghe et al., 2010). In the control treatment, the dietwas supplemented with an equivalent volume of phosphate buf-fered saline (PBS; 135 mM NaCl, 3 mM KCl, 1.5 mM KH2PO4 and8 mM Na2HPO4; pH 7.5). In these experiments, survival of A. pisumnymphs was scored daily during 3 days. Data were expressed asmeans ± SE based on three repeats and the experiment was re-peated five times. After Abbott’s correction for mortality in thecontrols (<15%), the toxicity results were analyzed and the mediantoxicity concentration (LC50) and the 95% confidence limits (95%CL)calculated in Prism v4 (GraphPad, La Jolla, CA) as described before(Nachman et al., 2011).

2.5. Histofluorescence procedures

Second-instars of the cotton leafworm and 4th-instars of thepea aphid were fed for 24 h on an artificial diet containing FITC-la-beled RSA at concentrations of 2 mg/g and 30 lM, respectively. Inthe controls insects were treated as in the treatments but withoutaddition of FITC-labeled RSA. Afterwards the insects were fixed inCarnoy solution (ethanol: chloroform:acetic acid, 6:3:1) for 24 hand then dehydrated in a graded series of ethanol (70%, 95% and100% for 30 min each) essentially as described before (Smaggheand Degheele, 1994). After dehydration, insect bodies were placedin butanol for 24 h and subsequently infiltrated using butanol:par-affin (1:1) for few hours. Finally, specimens were embedded inpure paraffin overnight and serial sections of 10 lm thickness werecut using a microtome (Jung AG, Heidelberg, Germany). Afterdewaxing and mounting, the location of FITC-labeled RSA in the in-sect tissues was analyzed using an Olympus BX51 fluorescencemicroscope (Olympus, Aartselaar, Belgium).

2.6. Bioassay with insect midgut cell cultures

The cytotoxic effect of RSA was investigated towards the midgutCF-203 cells, a cell line initially established from the midgut of thespruce budworm (C. fumiferana) (Sohi et al., 1993). The CF-203 cellswere cultured in Insect-Xpress medium (Bio-Whittaker-CambrexBioscience, Walkersville, MD) supplemented with 2.5% FBS (Sig-ma–Aldrich, Bornem, Belgium) as described before (Shahidi-Nog-habi et al., 2010). Therefore wells of a 96-well microtiter platewere loaded with 100 ll of a cell suspension, containing 2 � 105

cells per ml, and exposed to different concentrations of RSA(0.03, 0.3 and 0.7 lM) or PBS in the control treatment. The plateswere incubated for 4 days at 27 �C. For each concentration, 4replicates were performed, and each experiment was repeatedtwice. After incubation, the cell numbers were counted using the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide(MTT) (Sigma–Aldrich) method as described by Decombel et al.(2005).

The data on cell toxicity were analyzed by one-way analysis ofvariance (ANOVA) using a post hoc Tukey–Kramer test to detectsignificant differences between treatments. Statistical analyseswere performed using SPSS v15.0 (SPSS Inc., Chicago, IL). In addi-tion, a concentration–response curve and a median response(50%) concentration (EC50) with the corresponding 95%CL wereestimated with Prism v4 as described above.

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2.7. Effect of sugars on cell toxicity of RSA in midgut CF-203 cells

The effects of sugars on the cellular toxicity of RSA in midgutCF-203 cells were investigated. Preliminary finding range tests,using the same methodology as described above, demonstratedthat RSA at 0.03, 0.15, 0.3 and 0.7 lM caused a respective cytotox-icity in CF-203 cells of 23 ± 2%, 55 ± 2%, 56 ± 2% and 73 ± 1%, ascompared to the untreated control cells upon incubation during24 h. Subsequently, the effect of different carbohydrates or glyco-proteins was tested in assays containing 0.15 lM RSA, since thisconcentration was shown to yield a good response. Therefore,0.15 lM RSA was incubated for 1 h with GalNAc (100 mM) andasialomucin (10 mg/ml), while the non-specific sugar mannose(100 mM) was used as a negative control. These carbohydrate/gly-coprotein concentrations have been used before with Schneider 2cells of Drosophila without loss of cell viability (Hamshou et al.,2012). Afterwards, the mixture was added to the CF-203 cellsand incubated for 24 h at 27 �C. The MTT assay was used to calcu-late the cell toxicity as described above.

2.8. Binding of RSA to CF-203 cells

For microscopic quantification of cellular binding of FITC-la-beled RSA, the midgut CF-203 cells were grown on poly-L-lysinecoated slides overnight at 27 �C. The next day, cells were washedwith serum-free medium (EX-CELL 420, SAFC Biosciences) andincubated for 1 h at 27 �C with 0.7 lM FITC-labeled RSA (preparedin PBS). After three washes with PBS, cells were fixed with 4% para-formaldehyde for 15 min, followed by another three washes withPBS. Slides were mounted with Vectashield (Vector Labs), coveredwith a cover glass and sealed with nail polish. In the control series,cells were treated as in the treatments but without addition ofFITC-labeled RSA.

Cells were inspected with a Nikon eclipse TE2000-e epifluores-cence microscope (Nikon, France) using a 40� Plan Fluor (NA 1.30)oil immersion lens and appropriate fluorescence filters. Quantita-tive visualization of stained cells was performed on a BioRad Radi-ance 2000 confocal microscope mounted on a TE300epifluorescence body (Nikon Instruments, Paris, France) as de-scribed by Staljanssens et al. (2012).

2.9. Primary cell cultures of last instars of S. littoralis

Primary midgut cell cultures were prepared from actively feed-ing 4th instars of S. littoralis. Briefly, after midgut dissection, themidgut cells were dissociated from the basal lamina by incubationfor 1.5 h with 2 mg/ml of collagenase (Type I-AS, Sigma) in insectphysiological solution as described in Cermenati et al. (2007). Cellswere recovered and re-suspended in the same solution and incu-bated for 1 h with 0.9 lM of FITC-labeled RSA (as prepared above).In the control series, cells were treated as in the treatments with anequal amount of PBS but without addition of FITC-labeled RSA.After incubation, for microscopic analysis, cells were fixed for15 min with 4% paraformaldehyde in PBS. After three rinses withPBS, samples were mounted in Vectashield Mounting Medium(Vector Laboratories) and examined under a confocal laser scan-ning microscope (Nikon A1R; Nikon Instruments Inc., Paris, France)as described in De Geyter et al. (2012).

2.10. Effect of saponin on the cytotoxicity and binding of RSA to CF-203cells

The effect of RSA in combination with Quillaja saponaria barksaponin (Sigma, St. Louis, MO) was tested in CF-203 cells. In brief,0.3 lM of RSA and 0.001% of saponin were tested in combinationand compared to CF-203 cells in the controls (0.3 lM of RSA or

0.001% of saponin alone). As reported by De Geyter et al. (2012),CF-203 cells were incubated, under experimental conditions as de-scribed above, during 30 min with 0.001% of Q. saponaria barksaponin in the culture medium to obtain a higher cell membranepermeation. After that, cells were incubated with FITC-labeledRSA as described above.

2.11. DNA fragmentation and nuclear staining with Hoechst in themidgut cells

DNA fragmentation was analyzed as described previously(Shahidi-Noghabi et al., 2010). The extracted DNA was analyzedby gel electrophoresis on a 2% agarose gel at 100 V. DNA was visu-alized by staining with ethidium bromide.

For nuclear staining with Hoechst, CF-203 cells grown on poly-L-lysine coated glass were incubated with 0.7 lM of RSA for 24 h at27 �C. The cells were washed with PBS and fixed with 2% parafor-maldehyde for 20 min. After washing with PBS, the cell nuclei werestained with Hoechst/PBS (1:1000, v/v) for 15 min. After washingwith PBS, slides were mounted with Vectashield (Vector Labs), cov-ered with a cover glass. The cells were visualized under a Nikon Tiflorescence microscope (Nikon Benelux) using a 40� oil immersionlens and the appropriate filters to visualize Hoechst.

2.12. Caspase activity assay in midgut cells

The activity of 3 caspases in midgut CF-203 cells upon exposureto RSA was investigated with use of commercially available sub-strates, namely the Caspase-Glo�3/7, 8 and 9 reagents (Promega),containing DEVD for caspase-3/7, LETD for caspase-8, and LEHDfor caspase-9, respectively. Hereto, 100 ll of a CF-203 cell culturecontaining 2 � 105 cells per ml was loaded to a white-walled 96-well luminometer plate. The cells were treated with 0.3 lM ofRSA. In the positive control series, we used 50 lM of hydrogen per-oxide (H2O2) (Sigma–Aldrich) for the caspase-8 and -9 assays and1% DMSO for the caspase-3/7 assay. Untreated cells were used asa negative control. The plates were incubated for 24 h at 27 �C.Three replicates for each treatment were performed. Then 100 llof the Caspase-Glo�3/7, 8 and 9 reagents was added to each well.The contents of the wells were mixed gently using a plate shakerat 500 rpm for 0.5–2 min, and the plate was subsequently incu-bated at room temperature for 1–2 h. Finally, the luminescenceof each sample was measured using a luminometer (TECAN, Infi-nite M200; Männedorf, Switzerland).

2.13. Isolation of binding partners of RSA from the membrane ofmidgut cells

Midgut CF-203 cells were collected from the culture medium bycentrifugation at 500g for 10 min at 4 �C. The supernatant was re-moved carefully and PBS containing 2 mM of phenylmethylsulpho-nyl fluoride (PMSF) was added to the pellet. The sample wasvortexed and frozen at �80 �C overnight. Next day, the extractwas thawed at 4 �C and the solution was homogenized in anEppendorf tube with a pestle. After vortexing for 1–2 min, the ex-tract was centrifuged at 16,000g for 2 h at 4 �C and the supernatantcontaining the soluble proteins was collected. The extract with thesoluble protein fraction was stored at �80 �C. The pellet was re-suspended in 10 mM of Hepes buffer (pH 7.4), containing 1.5% Tri-ton X-100 and incubated for 1 h at 4 �C. In between the sample wasvortexed every 15 min. After the incubation period, the sample wascentrifuged at 20,000g for 30 min at 4 �C and the supernatant con-taining the membrane proteins was collected and frozen at �80 �C.

An RSA column was prepared and equilibrated with 0.2 M NaClbefore loading the soluble or membrane protein fraction as de-scribed by Vandenborre et al. (2010). After washing the RSA col-

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umn with 0.2 M NaCl, the proteins captured by RSA were elutedusing 20 mM unbuffered 1,3-diaminopropane. To ensure properbinding of the proteins with RSA the most concentrated proteinfractions from the first chromatography were pooled, adjusted to0.2 M NaCl and pH 7.6, and re-chromatographed on the RSA col-umn. Only the proteins that were bound in this second chromatog-raphy were sent for MS analysis. For the sample containing themembrane proteins, a similar RSA-affinity chromatography strat-egy was used. The membrane protein fraction was loaded on thecolumn in the presence of detergent. Column washing and elutionof bound proteins was performed as described above. This chroma-tography was also repeated to ensure proper binding of the pro-teins to RSA.

Both soluble and membrane glycoproteins captured by RSAwere analyzed by LC–MS/MS as described in Schouppe et al.(2011) using an Ultimate 3000 HPLC system (Dionex) in-line con-nected to a LTQ OrbiTRAP XL mass spectrometer (ThermoElectron).

The identified protein sequences were annotated by performinga BLAST search (EMBL-EBI) against Genbank (http://blas-t.ncbi.nlm.nih.gov). For each amino acid sequence, parameterswere set on the silkworm Bombyx mori as reference organismand only matches with an e-value < 0.5 were withheld. To subdi-vide the identified proteins into functionally related subfamilies,the PANTHER database (http://www.pantherdb.org) was used. Inaddition, the number of predicted N-glycosylation sites presenton the polypeptide backbone was calculated using the NetNGlyc1.0 server (http://www.cbs.dtu.dk/services/NetNGlyc). Only Asn-X-Ser/Thr sequences (where X is any amino acid except proline)with a prediction score > 0.5 were withheld as potential N-glyco-sylation sites. Potential O-glycosylation sites in protein sequenceswere predicted using NetOGlyc 3.1 Server (http://www.cbs.dtu.dk/services/NetOGlyc-3.1/). Only sequences with aprediction score > 0.5 were withheld as potential O-glycosylationsites. Finally, the location or orientation of the predicted N- and/or O-glycosylation sites on the cell membrane was determinedusing the TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/). An enrichment analysis was performed on the glyco-protein dataset for annotation terms using the Database for Anno-tation, Visualization and Integrated Discovery (DAVID) (http://www.david.abcc.ncifcrf.gov). Statistically overrepresented annota-tion terms were detected using the Benjamini statistics for multi-ple comparison corrections to calculate the false discovery rate(FDR).

The MS data are publicly available in the PRIDE database http://tinyurl.com/82ghdjv (Accession Nos.: 22420; Username: re-view39840; Password: Qwzs!⁄..).

3. Results

3.1. Insecticidal effects of RSA on cotton leafworm caterpillars and peaaphids

RSA has high toxicity towards larvae of S. littoralis. There was astrong effect on the larval weight gain and this effect was concen-tration-dependent. At day 8, the weight of larvae fed on a solid dietcontaining RSA at 10 and 5 mg/g was dramatically (p < 0.0001) re-duced with 84 ± 2% and 72 ± 5%, respectively, compared to the con-trols (Fig. 1A) without significant differences between these twotreatments (p = 0.293). The lower lectin concentrations of 1 mg/greduced larval weight by 12 ± 5% which was not significant com-pared to the controls (p = 0.312).

This reduction in larval weight for S. littoralis was accompaniedwith effects on larval development. The majority of the control lar-vae developed into the 4th instar (78%), while only 38 ± 2%, 15 ± 5%

and 5 ± 5% of the larvae entered the 4th instar after treatment withRSA at 1, 5 and 10 mg/g, respectively (Fig. 1B). In addition, many ofthese intoxicated larvae died. At the highest RSA concentration(10 mg/g), 48 ± 5% of the treated larvae were killed, while in treat-ments with RSA at 5 and 1 mg/g the mortality was only 15 ± 2% and5 ± 2%, respectively (Fig. 1C).

Similarly, feeding of pea aphids on a liquid artificial diet con-taining increasing concentrations (1.5–35 lM) of RSA, resulted ina clear mortality compared to a control diet. Typically, high nym-phal mortality was observed at lectin concentrations of 7 lM andhigher. As depicted in Fig. 2A, the toxicity was concentration-dependent and followed a sigmoid curve; the median LC50 toxicityvalue was 9 lM (95%CL: 7–12 lM; R2 = 0.81).

3.2. Localization of RSA in the insect body of caterpillars and aphids

In a feeding experiment with 2nd instars of S. littoralis fed for24 h on a diet containing FITC-labeled RSA, there was strong fluo-rescence binding to the luminal/apical side of the midgut epithe-lium comprising the brush border microvillar zone, and typicallythere was no internalization of the FITC-labeled protein in thecytoplasm of the cells of the midgut epithelium (Fig. 1D). In thenegative controls, there was no binding (Fig. 1E).

Similarly, pea aphid A. pisum 4th instars fed for 24 h on a dietcontaining FITC-RSA also demonstrated an intense fluorescenceat the luminal/apical side of the epithelial midgut cells, containingthe brush border microvillar zone (Fig. 2B and C). In the negativecontrols, no binding was observed (Fig. 2D).

3.3. Cellular toxicity of RSA in midgut cells

Exposure of CF-203 cells to purified RSA for 4 days revealedtoxic effects, resulting in cell debris, compared to untreated cells(Fig. 3A and B). Treatment with the lowest lectin concentrationof 0.03 lM yielded low cell toxicity of 5 ± 1% without significantdifferences (p = 0.8) (Fig. 3C). This cytotoxicity increased signifi-cantly (p < 0.0001) with a 10-fold higher concentration of RSA(0.3 lM), resulting in 41 ± 8%. At the highest lectin concentrationtested (0.7 lM), there was a dramatic increase in the cellular ef-fects (p < 0.0001) towards the midgut CF-203 cells (86 ± 2%)(Fig. 3C).

In addition, the median effect concentration EC50 (with 95% CL)that kills 50% of the midgut CF-203 cells exposed to RSA, was0.3 lM (0.25–0.35 lM). The quality of the fitting to a sigmoid curvewas high with an R2 = 0.90.

3.4. Effect of carbohydrates on RSA toxicity in midgut CF-203 cells

Incubation of 0.15 lM RSA with its specific and non-specificsugars for 1 h prior to treatment of the midgut CF-203 cells showedthat 100 mM GalNAc reduced the toxicity of RSA towards CF-203cells significantly (p < 0.0001) with 55 ± 2% (Fig. 4) compared toRSA treatment in the absence of sugar. A lower inhibition of RSAcytotoxicity was observed when RSA was incubated with 10 mg/mlasialomucin (i.e., glycoprotein with complex glycans) which ex-erted 23 ± 1% reduction in cytotoxicity (p = 0.003). No inhibitoryeffects on cell toxicity of RSA towards CF-203 cells were observedafter incubation of the cells with mannose which is a non-specificsugar for RSA (Fig. 4).

3.5. Binding of RSA to the midgut cells

Confocal microscopy analysis of midgut CF-203 cells exposed toFITC-labeled RSA demonstrated that the fungal lectin RSA was onlybound to the cell surface, but was not internalized by the cells(Fig. 5A; Fig. S1).

Fig. 1. Entomotoxic effects of the fungal lectin RSA on larval growth and development of the cotton leafworm Spodoptera littoralis fed on a solid artificial diet supplementedwith different concentrations of RSA for 8 days. (A) Effect of RSA on larval weight as compared to controls. Data are presented as mean fresh larval weight ± SEM. (B) Effect ofRSA on the larval developmental stage. Data are presented as percentage of the larvae that reach the L4 stage after 8 days. (C) Effect of RSA on larval survival. Data arepresented as the percentage of mortality after 8 days. The number of insects for each treatment is indicated. Values per graphic followed by a different letter (a–b) aresignificantly different (post hoc Tukey–Kramer test with p = 0.05). (D) Cross section of a 2nd instar of cotton leafworm fed for 24 h on an artificial diet containing 60 lM ofFITC-labeled RSA. The white arrows indicate that the lectin was clearly bound to the luminal/apical side of the insect midgut epithelium, but was not internalized in themidgut cells. (E) Negative control. Gut lumen = Lum.

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Moreover, incubation of midgut CF-203 cells with 0.001% of Q.saponaria bark saponin prior to addition of RSA, revealed that sapo-nin treatment resulted in a partial internalization of RSA in thecells while most of the lectin was still present on the cell surface(Fig. 5B). However, the partial uptake of RSA in the cells was notaccompanied by a significant increase in toxicity of RSA towardsCF-203 cells. The combination of 0.3 lM RSA with 0.001% saponincaused 49 ± 2% cytotoxicity, while the respective cytotoxic effectwas 51 ± 3% and 6 ± 3% when RSA and saponin were dosed alone.

In a separate experiment with primary midgut cell culturesfrom S. littoralis larvae, incubation of these midgut cells withFITC-labeled RSA demonstrated the clear apparent binding of thefungal lectin to the tips of the microvilli of the brush border zone

of the columnar cells, but no internalization was observed in themidgut cells (Fig. 5C and D).

3.6. DNA fragmentation analysis and nuclear condensation in midgutcells by RSA

Analysis of DNA extracted from CF-203 midgut cells after 24 hincubation with 0.7 lM of RSA showed a clear DNA fragmentedpattern when analyzed on a 2% agarose gel. In contrast no DNAfragmentation was observed for DNA extracted from the controltreatment (Fig. 6A).

Fluorescence microscopy of CF-203 cells incubated for 24 hwith 0.7 lM of RSA revealed clear characteristic changes in nuclear

0 -0.5 0.5 1.5 2.5Logarithm concentration RSA (µM)

% m

orta

lity

100

50

0

A

Fig. 2. Interaction of RSA in pea aphids (Acyrthosiphon pisum). (A) Dose response curve of mortality of pea aphids challenged for 3 days with a liquid artificial diet containingdifferent concentrations of RSA. Data are corrected for mortality in the controls (0–20%) using Abbott’s formula. (B) Cross section of 4th instar of pea aphid A. pisum fed for24 h on an artificial diet containing FITC-labeled RSA at 30 lM, showing binding of the lectin to the luminal/apical side of the epithelium of the insect midgut (MG).Cut = outer cuticle. (C) Magnification of the midgut where the white arrows show that RSA was clearly bound to the brush border microvillar zone of the midgut cells, but wasnot internalized in the midgut cells. (D) Negative control. Gut lumen = Lum.

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morphology after RSA treatment with condensed and fragmentednuclei and apoptotic bodies. In contrast, the control cells showeda normal nucleus (Fig. 6B and C).

3.7. Caspase activity in midgut cells upon exposure to RSA

Caspase activity (-3/7, -8, and -9) towards three commercialsubstrates was investigated in CF-203 cells after 24 h of incubationwith RSA. There was an induction (p < 0.001) of about 2.0- to 2.3-fold for caspase-3/7, -8 and -9 activities when the midgut CF-203cells were treated with 0.3 lM RSA (Fig. 7).

3.8. Proteomic analysis of membrane proteins of midgut cells capturedby RSA

Extracts containing membrane proteins from the midgutCF-203 cells were analyzed by SDS–PAGE (Fig. S2), affinity chroma-tography on immobilized RSA followed by LC–MS/MS. Afterwardsproteins were identified using the silkworm B. mori as a referenceorganism. Analyses were performed in three replicates, and onaverage 5747 unique peptides were identified in the membranefractions of CF-203 cells. From these peptides, 968 unique proteinswere identified and found in all three replicates (Table S1). Of thesemembrane proteins approximately 78% were found to possessputative N-glycosylation site(s). A search for proteins known tobe involved in apoptosis yielded 40 proteins. Among the RSA-bind-ing proteins identified from the membrane fraction, only four pro-teins are known to be located in the plasma membrane and arepredicted to have one or more N- or O-glycosylation site(s). These

proteins are Fas-associated factor, Apoptosis-linked gene-2, Neu-roglian and CG2076 (Table S2).

4. Discussion

Many reports have shown that lectins, especially lectins fromplant origin, can be used as tools to control insects belonging to dif-ferent orders (Vasconcelos and Oliveira, 2004; Hussain et al., 2008;Lagarda-Diaz et al., 2009). So far, the focus of the research was onmannose-binding plant lectins such as the snowdrop lectin GNA.However, the exact mechanism of action of lectins with insecti-cidal properties still remains enigmatic and needs more in-depthstudies. Here in this project, we demonstrated that a lectin fromfungal origin and with carbohydrate specificity for GalNAc/Gal,has a significant effect on Lepidoptera and Hemiptera with bit-ing-chewing and piercing-sucking mouthparts, respectively. Itwas clear that RSA had detrimental effects on the weight, develop-ment and survival of S. littoralis larvae. Similarly, Machuka et al.(1999) investigated the activity of eight GalNAc/Gal-specific plantlectins towards the legume pod borer (Maruca vitrata) and five ofthese lectins showed insecticidal activity. Interestingly, the activityof RSA towards S. littoralis was considerably higher than for theselectins. Subsequently, we expanded the toxicity assays with RSAto the pea aphid A. pisum, an important sap-sucking insect. Someproteins have been reported to be toxic towards some insect orderswithout any toxicity effects for other orders, as is the case for Bttoxin which has high toxicity against Lepidoptera but no effecton the performance of aphids (Lawo et al., 2009). Interestingly,high mortality rates were also detected in A. pisum aphids fed on

25 µm

A B

25 µm

0

50

100

Control 0.03 0.3 0.7RSA concentration (µM)

aa

b

c

% c

ytot

oxic

ity a

fter 4

day

s c

Fig. 3. Effect of RSA on the midgut CF-203 cell line. (A) Control cells and (B) cells treated with 0.7 lM of RSA for 4 days at 27 �C. (C) Cytotoxic effect of different concentrationsof RSA (0.03, 0.3 and 0.7 lM) on CF-203 cells. The loss of viability was determined using of an MTT assay after 4 days of lectin exposure. Data are presented as meanpercentages of cytotoxicity ± SEM compared to the control. Values are followed by a different letter (a–c) are significantly different (post hoc Tukey–Kramer test with p = 0.05).

0

10

20

30

40

50

60

% c

ell t

oxic

ity

a a

b

c

0.15 µM RSA+

100 µM GalNAc

+10 mg/ml

asialomucin

+100 µM mannose

-

Fig. 4. Inhibitory effect of sugars on the cell toxicity of RSA for midgut CF-203 cells.The lectin RSA (at 0.15 lM) was preincubated with a specific sugar (100 mMGalNAc), a glycoprotein (10 mg/ml asialomucin), a non-specific sugar (100 mMmannose) and PBS in the control treatments for 1 h. Then the mixtures of RSA andsugar/glycoprotein were added to CF-203 cells and incubated for 24 h at 27 �C. Celltoxicity was measured using an MTT assay. Data are presented as mean percentagesof toxicity ± SE based compared to the control, and based on four repeats. Values arefollowed by a different letter (a–c) are significantly different (post hoc Tukey–Kramer test with p = 0.05).

M. Hamshou et al. / Journal of Insect Physiology 59 (2013) 295–305 301

a liquid artificial diet containing different concentrations of RSA.Recently, we also reported the entomotoxicity of another fungallectin, namely SSA from S. sclerotiorum, which also possesses astrong toxicity towards aphids (Hamshou et al., 2010a). The LC50

of SSA against A. pisum was 2 (1–3) lM which is close to theLC50 of 9 (7–12) lM for RSA. Moreover, Trigueros et al. (2003) re-ported also toxicity by XCL against A. pisum with an LC50 of15 lM. Taken together, these data all confirm the potency of fungallectins with GalNAc/Gal-specificity in controlling leaf-eating andsap-sucking pest insects. However, further testing with pest insectsunder more practice-related conditions and risk assessment testsfor hazards in beneficial organisms are required before implemen-tation of these insecticidal lectins in IPM strategies.

In histology experiments, the two insects of this study, S. litto-ralis and A. pisum were fed on a diet containing FITC-labeled RSA.It was clear that the GalNAc/Gal-specific lectin bound to the brushborder zone at the luminal/apical side of the columnar cells of themidgut of both insects, without internalization in these cells. Sim-ilar results were also observed when RSA binding was analyzedwith primary midgut cell cultures from S. littoralis, confirmingthe importance of lectin binding at the tips of the microvilli onthe surface of the epithelial cells. Subsequently, further studieswere done using the insect CF-203 cell line originating from themidgut of another lepidopteran, the spruce budworm. Also here,RSA bound specifically to the cell membrane. To better understandthe mechanism of action of RSA at the insect midgut epithelium,midgut CF-203 cells were exposed to the fungal lectin. It was clearthat RSA induced a significant toxicity. In addition it was shownthat binding of RSA to the cell surface was carbohydrate-mediatedas proven by the fact that lectin cytotoxicity was inhibited only byspecific carbohydrates or glycoproteins. This fungal lectin alsoshowed a high cellular activity against the embryonic Schneider2 cells from the dipteran D. melanogaster and this concurred with

Fig. 5. Confocal fluorescence microscopy of midgut CF-203 cells. (A) An optical section of the cells shows binding of RSA at the cell surface of cells treated with 0.7 lM of RSAfor 1 h (Supplementary Fig. 3 presents the Z-stacks). (B) Midgut CF-203 cells were incubated with 0.001% saponin for 30 min prior to RSA treatment as in (A). Saponintreatment allowed partial internalization of RSA in CF-203 cells. (C) Bright field and (D) confocal laser scanning images of a primary midgut columnar cell culture from S.littoralis. The white arrow indicates that there is a clear apparent binding of FITC-labeled RSA to the tips of the microvilli of the brush border zone of the columnar epithelialmidgut cells. Scale bar = 10 lm.

A

Marker control RSA

10000 bp --

1500 bp ---

500 bp ---

200 bp ---

B

C

Fig. 6. (A) DNA fragmentation in midgut CF-203 cells treated with 0.7 lM of RSAcompared to control cells. Ten micrograms of DNA was loaded on the 2% agarosegel. The first lane shows the Mass Ruler DNA Ladder Mix (Fermentas). Nuclearcondensation/fragmentation in CF-203 (B) control cells and (C) cells treated with0.7 lM RSA. Scale bar = 10 lm.

302 M. Hamshou et al. / Journal of Insect Physiology 59 (2013) 295–305

a carbohydrate-mediated binding of RSA on the surface of the cells(Hamshou et al., 2012). In addition, Hamshou et al. (2010a) also re-ported significant toxicity of SSA to midgut CF-203 cells with spe-cific binding to the cell surface. These findings together with theresults of a histofluorescence study on A. pisum aphids and S. litto-ralis caterpillars demonstrated the insect midgut as a primary

insecticide target for RSA. Indeed in the past, several studies haveshown that lectins (can) interact with specific receptors in the in-sect midgut. For instance, ferritin can act as a target site for thesnowdrop lectin (GNA) in the midgut of the cotton leafworm S. lit-toralis (Sadeghi et al., 2009). Harper et al. (1995) also demonstratedbinding of some lectins to Ostrinia nubilalis brush border mem-brane proteins. Furthermore, Fitches and Gatehouse (1998) ob-served that GNA and ConA can bind to soluble and brush bordermembrane enzymes in the midgut of Lacanobia oleracea, which af-fected the activities of soluble and brush border membrane en-zymes. Finally, we also believe that other tissues and cell typesmay bind GalNAc/Gal-specific lectins. However, future analysis isrequired to confirm the presence of these carbohydrate decora-tions in different tissues/cells in the insect body.

Apoptosis, also known as programmed cell death (PCD), is amechanism by which cells undergo death to control cell prolifera-tion or in response to DNA damage. Apoptotic cells can be charac-terized by typical morphological changes such as blebbing orbudding, cell shrinkage, nuclear fragmentation, chromatin conden-sation and chromosomal DNA fragmentation (Lawen, 2003; Maet al., 2005). Interestingly, evidence of apoptosis was seen in mid-gut CF-203 cells intoxicated with RSA as judged especially by DNAfragmentation in addition to the presence of apoptotic bodies. Acti-vation for some caspases (-3/7, -8, and -9) was observed, albeit atlow levels. Here it should be mentioned that we made use of thecommercially available substrates (namely the Caspase-Glo�3/7,8 and 9 reagents from Promega, containing DEVD for caspase-3/7,LETD for caspase-8, and LEHD for caspase-9) that are mainly usedin mammalian research to measure caspase activities which mayexplain the relatively lower responses with a 2-fold induction.

Fig. 7. Caspase activities in midgut CF-203 cells treated with RSA. (A–C) Caspase-3/7, -8 and -9 activities in midgut CF-203 cells: cells were treated during 24 h with 0.3 lM ofRSA, 50 lM of H2O2 in the caspase-8 and -9 assays, and 0.01% of DMSO in the caspase-3/7 assay as positive control; untreated cells were used as negative control. Thecommercial substrates of Caspase-Glo�3/7, -8 and -9 kits from Promega were used to measure caspase activities. Data are presented as mean relative luminescence units(RLU) ± SD after 1 h reaction with caspase-3/7, -8 and -9 reagents. Values are followed by a different letter (a–c) are significantly different (post hoc Tukey–Kramer test withp = 0.05).

M. Hamshou et al. / Journal of Insect Physiology 59 (2013) 295–305 303

At present, apoptosis has been extensively studied in mammals butcomparatively less tools are available for insects. In general, casp-ases are a family of cysteine proteases and are known as the mainexecutors of the apoptotic process that is activated in proteolyticcascades during cell death (Kumar and Harvey, 1995; Thornberryand Lazebnik, 1998; Taylor et al., 2008). They exist in an inactiveform in the cells and are activated upon exposure to apoptoticstimuli. Upstream initiator caspases (caspase-8, -9, -10) are firstactivated, and then as a consequence the downstream effectorcaspases (caspase-3, -6, -7) are activated which in turn inducesapoptosis (Alenzi et al., 2010).

In a last part of this work we performed a proteomics study ofthe proteins bound to RSA in an affinity chromatography experi-

ment. We focused on a subfraction of proteins consisting of puta-tively glycosylated proteins and being involved in apoptosis. Thisanalysis revealed four interesting proteins, namely Fas-associatedfactor, Apoptosis-linked gene-2, Neuroglian and CG2076, as puta-tive binding partners for RSA. This raises now the question: howis the signaling pathway of these four proteins and their involve-ment in relation to apoptosis induction? To explain the mechanismof apoptosis-induction by RSA in midgut CF-203 cells, we believethat the Fas signaling pathway is involved. As known from themammalian literature, Fas (also called Apo1 or CD95) is a deathreceptor on the cell surface and is considered as the major cellsurface receptor involved in the induction of apoptosis (Li et al.,2007). Fas triggers apoptosis by recruiting the apoptosis initiator

304 M. Hamshou et al. / Journal of Insect Physiology 59 (2013) 295–305

caspase-8 through the adaptor Fas-associated death domain (FADD)which binds to the death domain of Fas and the death effector do-main of caspase-8 (Krammer, 2000). After activation of caspase-8by Fas, apoptosis could be induced via activation effector caspasessuch as caspase-7 directly or indirectly by releasing cytochrome cfrom mitochondria into the cytosol. The release of cytochrome c re-sults in an activation of caspase-9 which induces apoptosis via acti-vation of downstream effector caspases (Scaffidi et al., 1998;Shatnyeva et al., 2011). In the current study, we believe that a sim-ilar pathway, as reported for the Fas signaling pathway, could beresponsible for the induction of apoptosis in midgut CF-203 cellsupon exposure to RSA, especially also given the fact that Fas wasreported to have two N-glycosylation sites as well as a Thr-rich re-gion that can be O-glycosylated (Li et al., 2007). Indeed these datasuggest that Fas could be a suitable target for RSA in the inductionof apoptosis in insect midgut cells via activation of caspases.Although the Fas protein itself was not found among the proteinseluted from the RSA affinity column, we identified two proteinsthat are linked with Fas, namely Fas-associated factor and Apopto-sis-linked gene-2. For the first protein, the Fas-associated factor,Chu et al. (1995) reported that the Fas-associated factor-1 bindsto FADD, which in turn induces the activation of caspase-8 (Ryuand Kim, 2001; Ryu et al., 2003). For the second protein, the Apop-tosis-linked gene-2, Jung et al. (2001) reported that this proteinalso binds to FADD, which protein translocates then from the plas-ma membrane to the cytosol (Maki and Shibata, 2007). For the twoother proteins, Neuroglian and CG2076, there exists unfortunatelyno information on their signaling pathway in apoptosis. Based onthese data we have set out a working model. We hypothesize thatthe binding of RSA to the carbohydrate decoration of the Fas-asso-ciated factor could lead to activation of caspase-8 which in turncould activate caspase-7 directly or indirectly via activation of cas-pase-9. Finally, activation of caspase-7 could then induce apopto-sis. Indeed in insect midgut CF-203 cells we measured anactivated caspase-8, -9 and -3/7 upon exposure to RSA. The respec-tive homolog of caspase-8 and -9 in insects (D. melanogaster) isDredd and Dronc, and that of caspase-7 is Drice, Dcp-1, Decay or/and Damm. The involvement of Dronc and Drice in apoptosis-induction has been reported recently (Cooper et al., 2009; Cooperand Mitchell-Foster, 2011).

In conclusion, we believe that RSA is inducing apoptosis in in-sect midgut cells by activation of the Fas signaling pathway induc-ing caspases. This then helps to explain the strong entomotoxicaction of this fungal GalNAc/Gal-specific lectin in insects as ob-served for the first time in this project with caterpillars and aphids.However, further work to investigate the function and importanceof the two other membrane proteins, Neuroglian and CG2076, maybe useful to further complement our current understanding ofapoptosis-induction by RSA. If involved, then RNAi of the mem-brane protein expression should rescue the midgut CF-203 cellsfrom the toxic effects of RSA.

Acknowledgements

M. Hamshou is recipient of a Ph.D. scholarship from the Minis-try of Higher Education and General Commission for ScientificAgricultural Research in Syria. This research was supported bythe Special Research Council of Ghent University (BOF-UGent; pro-ject BOF10/GOA/003), the Hercules Foundation, and the Fund forScientific Research-Flanders (FWO-Vlaanderen; 3G016306) to G.Smagghe and E.J.M. Van Damme. The UGent/VIB lab acknowledgessupport by research grants from the FWO-Vlaanderen (3G016306,G.0156.05, G.0077.06 and G.0042.07), the Concerted Research Ac-tions (BOF07/GOA/012) from BOF-UGent, and the InteruniversityAttraction Poles (IUAP06). B. Ghesquière is a postdoctoral researchfellow of the FWO-Vlaanderen.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.jinsphys.2012.12.003.

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