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International Biodeterioration & Biodegradation 75 (2012) 158e166

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International Biodeterioration & Biodegradation

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Effect of Microgramma vaccinifolia rhizome lectin on survival and digestiveenzymes of Nasutitermes corniger (Isoptera, Termitidae)

Lidiane Pereira de Albuquerque, Giselly Maria de Sá Santana, Emmanuel Viana Pontual,Thiago Henrique Napoleão, Luana Cassandra Breitenbach Barroso Coelho, Patrícia Maria Guedes Paiva*

Departamento de Bioquímica - CCB, Universidade Federal de Pernambuco, Recife, PE 50670-420, Brazil

a r t i c l e i n f o

Article history:Received 12 April 2012Received in revised form7 June 2012Accepted 8 June 2012Available online 30 October 2012

Keywords:Microgramma vaccinifoliaLectinTermiticidal activityTrypsin inhibitionCellulasesPhosphatase

* Corresponding author. Tel.: þ55 8121268540; faxE-mail address: ppaivaufpe@yahoo.com.br (P.M.G.

0964-8305/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.ibiod.2012.06.030

a b s t r a c t

Nasutitermes corniger is a termite responsible for biodeterioration of buildings, paintings, books, andmonuments. Plant lectins can be environmentally friendly agents for termite control. This work describesthe termiticidal activity of Microgramma vaccinifolia rhizome lectin (MvRL) against N. corniger workersand soldiers. Also, it evaluates the possibility that MvRL changes trypsin-like activity as well as activity ofa-amylase, phosphatase, and cellulase in termite gut. MvRL was isolated by chromatography on a chitincolumn. MvRL, a thermo-stable 17-kDa glycosylated lectin, was inhibited by mannose and glycoproteins.MvRL showed high toxicity against N. corniger (LC50 of 0.130 and 0.085 mg ml�1 for workers and soldiers,respectively, for 3 days), and did not show repellent and feeding-rejection effects. MvRL inhibitedtrypsin-like activity from worker gut extract (Ki of 2.0 mM), although it did not affect a-amylase activitiesand stimulated acid phosphatase activity from extracts of workers and soldiers by factors of 1.6 and 3.1,respectively. The lectin also stimulated worker endoglucanase activity, and neutralized this activity insoldier gut extract. The b-glucosidase activity from both castes was inhibited by MvRL. In conclusion,MvRL killed N. corniger workers and soldiers through termiticidal mechanisms that may include theirchitin-binding and enzyme-modulating properties.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Nasutitermes (Termitidae family) constitutes the largest genus ofwood-feeding termites, whose distribution stretches mainly overtropical regions. Nasutitermes species have been reported to invadeurban environments in the Brazilian semi-arid region, attackingwood in roofs, linings, and structural spansof buildings (Meyer, 2005;Paes et al., 2007). Due to these negative impacts, Nasutitermes cor-niger is considered an insectpest, andnewmethods for its control andmonitoring have become the object of intensive scientific research.

Synthetic pesticides have been the predominantmethod used toprevent termite attack. However, the toxicity to non-target organ-isms has brought about the need to search for alternative andbiodegradable compounds. From this perspective, phytoinsecticideshave been considered an effective and environmentally friendlyalternative, because they generally present lower persistence andresidual action, being rapidly degraded (Verma et al., 2009).

Lectins are proteins that bind to carbohydrates present ondifferent cell surfaces (Correia et al., 2008). Several kinds of

: þ55 8121268576.Paiva).

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biological activity have been attributed to these proteins; anexample is insecticide effect (Paiva et al., 2011a). The interactionbetween chitin-binding lectins and N-acetylglucosamine residuesof chitin and glycosylated proteins in the peritrophic matrix mayaffect membrane integrity. It has also been suggested that lectinscan destabilize insect metabolism by interfering in enzyme func-tions (Macedo et al., 2007; Paiva et al., 2011a; Napoleão et al., 2012).

Lectins from a lichen species, Cladonia verticillaris (Silva et al.,2009), as well as from the bark, heartwood, and leaf of Myracro-druon urundeuva (Sá et al., 2008; Napoleão et al., 2011), Bauhiniamonandra secondary roots (Souza et al., 2011), Opuntia ficus indicacladodes (Paiva et al., 2011b), and Crataeva tapia bark (Araújo et al.,2012) have been reported to exert termiticidal activity againstN. corniger. Since these reports are recent, the mechanisms of ter-miticidal activity of lectins should bemore thoroughly investigated.Napoleão et al. (2011) reported that the mechanisms of termiticidallectins from M. urundeuva can involve a chitin-binding property,resistance to degradation by termite proteases, and antibacterialactivity against symbiotic bacteria found in N. corniger gut.

Microgramma vaccinifolia is an epiphytic plant with far-reachingdistribution worldwide, though it is found preferentially in thetropics. In Brazil, M. vaccinifolia rhizomes are widely used in folk

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medicine to treat diarrhea and as a balsamic in infections in therespiratory tract (Barros and Andrade, 1997). Santana et al. (2012)reported that a protein fraction from the rhizome extract containsa lectin called MvRL, whose hemagglutinating activity depends onthe concentration of divalent cations (Ca2þ and Mg2þ) in themedium, and reaches peak values with human type O erythrocytes.

This work reports on the isolation of MvRL and the evaluationof its termiticidal and repellent activities against N. cornigerworkers and soldiers. In addition, the effects of MvRL on termitedigestive enzymes (trypsin-like, a-amylase, phosphatases, andthree cellulases: endoglucanase, exoglucanase, and b-glucosidase)were determined.

2. Materials and methods

2.1. Plant material

M. vaccinifolia (Langsd. & Fisch) was collected in Recife City, Stateof Pernambuco, northeastern Brazil. A voucher specimen isarchived under number 63,291 at the herbarium Dárdano deAndrade Lima (Instituto Agronômico de Pernambuco, Recife, Brazil).The rhizomes were washed in tap water, air-dried, powdered, andstored at �20 �C.

2.2. Insects

Colonies of N. corniger were collected from an Atlantic Forestarea located at the campus of the Universidade Federal Rural dePernambuco, Brazil. The termite colony was selected according tooverall integrity criteria. The nest was carefully removed from thetrunk of a tree using a machete and then transferred to the labo-ratory. The colony was maintained at 28 � 2 �C (70 � 5% relativehumidity) in the dark for 6 h and subsequently used in bioassays.

2.3. Chemicals

The following chemicals were purchased from SigmaeAldrich (St.Louis, MO, USA): N-acetyl-D-glucosamine, acrylamide, a-amylasefrom hog pancreas, (þ)-arabinose, Avicel, N-benzoyl-DL-arginyl-r-nitroanilide (BAPNA), bovine serum albumin, bovine trypsin,carboxymethylcellulose, casein from bovinemilk, chitinpowder fromshrimp shells, Coomassie Brilliant Blue R-250, 3,5-dinitrosalicylic acid(DNS), D(�)-fructose, D(þ)-galactose, D(þ)-glucose, glutaraldehyde,D(þ)-mannose, methyl-a-D-glucopyranoside, methyl-a-D-mannopyr-anoside, N,N0-methylenebis(acrylamide), r-nitrophenyl-b-D-gluco-pyranoside, r-nitrophenyl phosphate, r-nitrophenol, ovalbulmin,D(þ)-raffinose, L(þ)-rhamnose, sodium bicarbonate, trehalose, trish-ydroximethylaminomethane (Tris), and D(þ)-xylose). Acetic acid,agar, ammonium sulfate, chloridric acid, dibasic sodium phosphate,monobasic sodium phosphate, and sodium chloride were purchasedfrom Vetec (Rio de Janeiro, Brazil). Ammonium persulphate, calciumchloride, sodium acetate, sodium hydroxide, Schiff’s reagent, solublestarch, and N,N,N0,N0-tetramethylethylenediamine (TEMED) werepurchased from Merck (Darmstadt, Germany). All reagents were ofanalytical grade.

2.4. Isolation of MvRL

Protein fraction containing MvRL was obtained fromM. vaccinifolia rhizome extract according to Santana et al. (2012).Rhizome crude extract (10% w/v) was treated with 60% (w/v)ammonium sulfate and the 0e60% fraction, containing theprecipitated proteins, was collected by centrifugation (3000 g,15 min, 4 �C) using a Sorvall� RC 6� centrifuge (Thermo Scientific,MA, USA). The 0e60% fraction was resuspended in 0.15 M NaCl,

dialyzed in a 10 kDa cutoff membrane (SigmaeAldrich, MO, USA)against 0.15 M NaCl (8 h at 4 �C), and loaded (7.8 mg of proteins)onto a chitin column (7.5 � 1.5 cm) equilibrated (20 ml min�1

flowrate) with 0.15 M NaCl. MvRL was eluted with 1.0 M acetic acid anddialyzed against distilled water (4 h) and 0.15 M NaCl (4 h) foreluent elimination.

The MvRL was submitted to polyacrylamide gel electrophoresiscontaining sodiumdodecyl sulfate (SDS-PAGE) according to Laemmli(1970). MvRL and the molecular mass markers (SigmaMarker� kit,SigmaeAldrich, MO, USA)phosphorylase B (97,000 Da), bovineserumalbumin (66,000Da), ovalbumin (45,000Da), glyceraldehyde-3-phosphate dehydrogenase (36,000 Da), carbonic anhydrase(29,000 Da), trypsinogen (24,000 Da), a-lactalbumin (14,200 Da),and aprotinin (6500 Da) were stained with 0.02% (v/v) CoomassieBrilliant Blue in 10% acetic acid. Glycoprotein staining was also per-formed with Schiff’s reagent according to Pharmacia Fine Chemicals(1980).

2.5. Protein content

The protein concentration was estimated according to Lowryet al. (1951) using bovine serum albumin (31e500 mg ml�1) asstandard.

2.6. Hemagglutinating activity

The hemagglutinating assay was carried out in microtiter plates(TPP-Techno Plastic Products, Trasadingen, Switzerland) by incu-bating 50-ml samples with 2.5% (v/v) suspension of human O-typeerythrocytes treated with glutaraldehyde for 30 min (Bing et al.,1967). One hemagglutinating unit (titer�1) was defined as thereciprocal of the highest dilution of the sample promoting fullagglutination of erythrocytes (Napoleão et al., 2011). Specifichemagglutinating activity (unit mg�1) was defined as the ratio titerto protein concentration.

Hemagglutinating activity was also evaluated after incubation(30 min) of MvRL (50 mL, 40 mg) with 200 mM monosaccharide orglycoprotein solutions (0.5 mg ml�1) before adding erythrocytes.Hemagglutinating activity assays were also performed using MvRLheated (30 min) to 30e100 �C.

2.7. Termiticidal assay

Termiticidal activity was evaluated by a no-choice bioassaybased on the method described by Kang et al. (1990). Each exper-imental unit consisted of a petri dish (90 � 15 mm, TPP-TechnoPlastic Products, Trasadingen, Switzerland) whose bottom dishwas covered with filter paper. A filter paper disk (4 cm in diameter)impregnated with 200 ml of sample in 0.15 M NaCl was put in eachdish. Termiticidal activity was evaluated for rhizome crude extract,0e60% fraction, and MvRL (0.0625, 0.125, 0.25, and 0.5 mg ml�1 ofprotein). In negative controls, papers were impregnated with0.15 M NaCl. A total of 20 active termites (at a worker-to-soldierratio of 4:1) were transferred to each dish; these were main-tained at 28 �C in the dark. Insect survival was evaluated daily, untilall insects were dead. Bioassays were carried out in quintuplicatefor each concentration, and survival rates (as percentages) wereobtained for each treatment.

2.8. Repellence and feeding-rejection assays

The repellence assay was based on Su et al. (1982). Petri dishes(90 � 15 mm, TPP-Techno Plastic Products, Trasadingen,Switzerland) were filled up with 2% agar solution. After solidifica-tion, wells were pierced in the agar by removing a central core

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25 mm in diameter and 10 peripheral cores (6 mm in diameter).Next, a punch of filter paper soaked in 15 ml of 0.15MNaCl (negativecontrol) or samples (crude extract, 0e60% fraction and MvRL) wasplaced in each peripheral well. Termites (16 workers and 4 soldiers)were then transferred to the central well and the dishes weremaintained at 28 �C in the dark. Assays were conducted in tripli-cate. The absence or presence of termites in peripheral wells, tunnelconstruction patterns in agar, and the closing of constructedgalleries by insects were observed for 15 days.

The mass loss of filter paper disks treated or untreated withMvRL was also evaluated, with the aim of determining if the lectininduced a feeding-rejection effect. For this, a termiticidal assaycontaining filter papers treated with MvRL at 0.130 mg ml�1 or0.15 M NaCl (negative control) was performed as described inSection 2.7. The mass of filter papers was determined at thebeginning of experiment and after 3 days. Bioassays were carriedout in quintuplicate.

2.9. Termite gut extracts

Groups of 50 workers of N. corniger were collected andimmobilized by placing them in a freezer at �20 �C for 10e15 min.Each termite was decapitated using an 8-mm-long, 0.3-mm needle(BD Ultra-Fine II from Becton, Dickinson and Co., NJ, USA). Then,guts were removed intact, pulling the last abdominal segments.Next, guts were stored on ice in 0.15 M NaCl. Guts of workers werethen placed in a 2-ml glass tissue grinder and manually homog-enized with 1 ml of Tris buffer (0.1 M TriseHCl pH 8.0 containing0.02 M CaCl2 and 0.15 M NaCl), acetate buffer (0.1 M sodiumacetate pH 5.5 containing 0.02 M CaCl2 and 0.15 M NaCl), orsodium phosphate buffer (0.02 M sodium phosphate pH 7.0 con-taining 0.15 M NaCl). The homogenates were then centrifuged at9000 g at 4 �C for 15 min and the collected supernatant (workergut extract) was used for evaluation of digestive enzymes. Soldiergut extract was prepared according to the same procedure, using50 soldiers.

2.10. Effect of MvRL on enzyme activities from N. corniger gut

2.10.1. Trypsin-like activityTrypsin activity exerted by termite gut extracts was determined

in 96-well microtiter plates (TPP-Techno Plastic Products, Trasa-dingen, Switzerland) using the synthetic substrate BAPNA asdescribed by Kakade et al. (1969). Termite gut extract in Tris buffer(10 ml, 81 mg of protein) was incubated (60 min, 37 �C) with 8 mM

Fig. 1. Isolation of MvRL by chromatography on chitin column. (A) Chromatography of 0e60Arrows demonstrate eluents added. Fractions of 2.0 ml were collected and evaluated for hemto 58 were pooled and named MvRL. (B) SDS-PAGE (12%, w/v) of MvRL stained with 0.02%standards in electrophoresis. (C) SDS-PAGE (12%, w/v) of MvRL stained with Schiff’s reagen

BApNA (5 ml) in Tris buffer (185 ml). Enzyme activity was evaluatedmeasuring absorbance at 405 nm. One unit of trypsin-like activitywas defined as the amount of enzyme that hydrolyzes 1 mmol ofBApNA per minute under the established conditions. Control ofsubstrate hydrolysis was performed by incubation (60 min, 37 �C)of bovine trypsin (5 ml, 0.5 mg) with 8mMBApNA (5 ml). Assayswereperformed in quadruplicate.

Trypsin-like activity was evaluated after incubation (30 min,37 �C) of MvRL (0.875e5.0 mM) with termite gut extract in Trisbuffer (10 ml, 81 mg of protein). Next, 4 or 8 mM BApNA (5 ml) wasadded and the mixture was then incubated for 60 min at 37 �C.Reaction blanks containing only substrate or MvRL were also per-formed. Inhibition curves were plotted and a Dixon plot analysiswas employed to determine the constant of inhibition (Ki). Dixonplots were generated using the reciprocal velocity (1/v) versuslectin concentration. Intersection of the regression lines for each[BApNA] yielded the Ki (Segel, 1975). Reaction blanks containingonly substrate or MvRL were also performed. Assays were per-formed in triplicate.

2.10.2. a-Amylase activityThe assay was carried out based on the method described by

Bernfeld (1955). Termite gut extract in acetate buffer pH 5.5 (100 ml;530 mg of protein) was incubated at 50 �C for 10 min with 400 ml ofa 1% (w/v) soluble starch (Merck, Germany) solution in 0.1 Msodium acetate pH 5.5 containing 0.02 M CaCl2 and 0.15 M NaCl.The reaction was stopped by adding 500 ml of 3,5-dinitrosalicylicacid (DNS). Next, the assays were heated at 100 �C in boilingwater for 6 min and immediately cooled in ice for 15 min. Then,absorbance was measured at 540 nm. The amount of reducingsugars was determined using a standard reaction curve constructedbased in the different glucose concentrations with DNS(Y ¼ 0.1183X e 0.0704, where Y is absorbance at 540 nm and X isglucose concentration in mg ml�1). One unit of a-amylase activitywas defined as the amount of enzyme required to generate 1 mmolof glucose per minute. As a positive control, the same procedurewas carried out with a-amylase (400 ml, 400 mg) from hog pancreas.Reaction blanks were performed without starch. Assays were per-formed in triplicate.

The effect of lectin on a-amylase activity was evaluated byincubating (15 min at 27 �C) termite gut extract in acetate buffer(100 ml; 530 mg of protein) with MvRL (62.5e250 mg) beforedetermination of enzyme activity. Reaction blanks containing onlystarch or MvRL were also performed. Assays were performed intriplicate.

% fraction from rhizome extract on chitin column. The washing step used 0.15 M NaCl.agglutinating activity (HA). ABS 280 nm (,,A,D), log specific HA (SHA, x). Fractions 43(v/v) Coomassie Blue in 10% (v/v) acetic acid. Molecular mass markers were used ast. Arrows indicate MvRL polypeptide.

Table 1Purification of Microgramma vaccinifolia rhizome lectin (MvRL).

Sample Protein(mg ml�1)

Specific hemagglutinatingactivity (units mg�1)

Purification(times)

Rhizome extract 2.0 16 1.00e60% fraction 1.8 116 7.3MvRL 0.3 426 26.6

Hemagglutinating activity (titer�1) was determined with human type-O erythro-cytes. Specific activity corresponds to the ratio hemagglutinating activity to proteinconcentration (mg ml�1). Purification corresponds to the ratio specific HA of thepurification step to specific HA of the rhizome extract.

Table 2Hemagglutinating activity of MvRL in presence of monosaccharides andglycoproteins.

Carbohydrate Specific hemagglutinatingactivity of MvRL

Monosaccharide (200 mM)(þ)-Arabinose 426D(�)-Fructose 426D(þ)-Galactose 426D(þ)-Glucose 426D(þ)-Mannose 54Methyl-a-D-glucopyranoside 426Methyl-a-D-mannopyranoside 426N-acetyl-D-glucosamine 426D(þ)-Raffinose 426L(þ)-Rhamnose 426Trehalose 426D(þ)-Xylose 426

Glycoprotein (500 mg mL�1)Bovine serum albumin 426Casein 54Ovalbumin 54

Hemagglutinating activity was determined using human O-type erythrocytes.Specific hemagglutinating activity was calculated from the ratio of titer�1 to proteinconcentration (mg ml�1). Specific hemagglutinating activity of MvRL in absence ofglycosylated molecules was 426.

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2.10.3. Acid and alkaline phosphatase activitiesThe activity of phosphatases was determined according to an

adaptation of the method developed by Asakura (1978) describedby Koodalingam et al. (2011). Termite gut extract in sodium phos-phate buffer (50 ml; 180 mg of protein) was mixed with 450 ml of0.05 M sodium acetate buffer, pH 4.0 (for determination of acidphosphatase activity) or 0.05 M TriseHCl, pH 8.0 (for alkalinephosphatase activity). Next, 500 ml of 12.5 mM r-nitrophenylphosphate prepared in acetate or Tris buffer was added to themixtures. After incubation for 15 min at 37 �C in a water bath, theenzyme reaction was stopped by adding 100 ml of 0.5 N sodiumhydroxide and centrifugation (4000 g; 5 min) ensued. The absor-bance at 440 nm of the supernatants was then recorded. Theamount of r-nitrophenol released by hydrolysis of r-nitrophenyl-phosphate was determined using the calibration curveY ¼ 0.0013X þ 0.0648 (Y is the absorbance at 410 nm; X is the r-nitrophenol concentration in milligrams per milliliter). One unit ofacid or alkaline phosphatase activity was defined as the amount ofenzyme required to generate 1 mmol of r-nitrophenol per minute.Assays were performed in triplicate.

The activities of acid and alkaline phosphates from termite gutextract in sodium phosphate buffer (50 ml; 180 mg of protein) werealso determined after incubation (15 min, 37 �C) with MvRL (25e200 mg). Reaction blanks containing only substrate or MvRL werealso performed. Assays were performed in triplicate.

2.10.4. Cellulase activitiesAssays for endoglucanase and exoglucanase were carried out

according to the methods described by Li et al. (2009) and Woodand Bhat (1988), respectively, with adaptations. The reactionsstarted by incubating (50 �C, 10 min) worker (100 ml; 330 mg ofprotein) or soldier (100 ml; 270 mg of protein) gut extracts with400 ml of a solution containing 1% (w/v) carboxymethylcellulose(for endoglucanase activity) or 1% (w/v) Avicel (for exoglucanaseactivity) in sodium acetate pH 5.5 containing 0.15 M NaCl. Afterincubation, 500 ml of 3,5-dinitrosalicylic acid was added to stop thereaction and the mixtures were heated (100 �C, 6 min) andimmediately cooled in ice (15 min). Then, absorbance at 540 nmwas measured. The amount of reducing sugars was determinedusing glucose as standard (Y ¼ 0.1183X e 0.0704, where Y is theabsorbance at 540 nm and X is the glucose concentration inmgml�1). One unit of enzyme activity was defined as the amount ofenzyme required to generate 1 mmol of glucose per minute. Blankswere performed submitting worker and soldiers gut extracts to thesame reaction steps in the absence of substrate.

An adaptation of the method described by Tan et al. (1987) wasused to assess b-glucosidase activity. Worker (100 ml; 330 mg ofprotein) or soldier (100 ml; 270 mg of protein) gut extract wasincubated (50 �C; 10 min) with 400 mL of 0.1% (w/v) r-nitrophenyl-b-D-glucopyranoside solution in sodium acetate pH 5.5 containing0.15 M NaCl. After incubation, 500 ml of 10% (w/v) sodium bicar-bonate was added to stop the reaction, and absorbance at 410 nmwas measured. The amount of r-nitrophenol released by hydrolysisof r-nitrophenyl-b-D-glucopyranoside was determined using thecalibration curve Y ¼ 32.224X þ 0.0783 (Y is the absorbance at410 nm; X is the r-nitrophenol concentration in mgml�1). One unitof activity was defined as the amount of enzyme required togenerate 1 mmol of r-nitrophenol per minute. Reaction blanks wereperformed without substrate.

The effects of MvRL on cellulase activities were determined byincubating (15 min at 27 �C) worker (100 ml; 530 mg of protein) orsoldier (100 ml; 270 mg of protein) extracts with MvRL (25e250 mg)before determination of enzyme activity. Reaction blanks contain-ing only substrate or MvRL were also performed. Assays wereperformed in triplicate.

2.11. Statistical analysis

Standard deviations (SD) were calculated using GraphPad Prismversion 4.0 for Windows (GraphPad Software, San Diego, CA, USA)and data were expressed as a mean of replicates � SD. Significantdifferences between treatment groups used in the termiticidalassay were analyzed by Student’s t-test (significance at p < 0.05)using the Origin 6.0 program. The lethal concentrations required tokill 50% (LC50) of termites after 3 days were calculated by probitanalysis with a reliability interval of 95% using the computer soft-ware StatPlus� 2006 (AnalystSoft, Vancouver, BC, Canada).

3. Results

Proteins (256 mg) from rhizome crude extract precipitatedusing ammonium sulfate were chromatographed on a chitincolumn. MvRL (4.0 mg; specific hemagglutinating activity of 426)was recovered after elution with 1.0 M acetic acid (Fig. 1A). Thechitin chromatography step was efficient in removing pigmentspresent in the 0e60% fraction. SDS-PAGE revealed the presence ofa single 17-kDa polypeptide band (Fig. 1B) that was stained usingSchiff’s reagent (Fig. 1C). The purification process resulted in anincrease in hemagglutinating activity. The MvRL purificationprocedure is summarized in Table 1.

The carbohydrate-binding specificity of MvRL was determinedby evaluating the effect of monosaccharides and glycoproteins onhemagglutinating activity (Table 2). The activity of MvRL wasinhibited bymannose, casein, and ovalbumin. The stability of MvRL

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hemagglutinating activity to heating was also determined, and thespecific activity (426) was not reduced, even at 100 �C.

Termiticidal assays using rhizome extract, 0e60% fraction andMvRL showed that all preparations exerted termiticidal actionagainst N. corniger workers. Student’s t-test revealed that theextract (Fig. 2A) and 0e60% fraction (Fig. 2C) at 0.125, 0.25, and0.5 mg ml�1 significantly (p < 0.05) induced the mortality ofworkers in comparison with the control. MvRL was termiticidal forworkers at all concentrations evaluated (Fig. 2E). The LC50 value ofMvRL on workers determined for 3 days was 0.130 mg ml�1. MvRLwas more active against workers than the rhizome extract and the0e60% fraction, indicating that the lectin is the active principle incrude preparations.

Rhizome extract did not interfere significantly (p < 0.05) in thesurvival of soldiers (Fig. 2B), while 0e60% fraction at 0.25 and0.5 mg ml�1 significantly (p < 0.05) induced death of soldiers(Fig. 2D). MvRL showed a strong termiticidal effect (LC50 of0.085 mg ml�1) against soldiers, increasing the mortality rate at all

Fig. 2. Effect of rhizome extract (A and B), 0e60% fraction (C and D), and MvRL (E and F) on swere 0.0625 (�), 0.125 (A), 0.25 (>), and 0.5 (D) mg ml�1. 0.15 M NaCl was the negative

tested concentrations (Fig. 2D). Rhizome extract, 0e60% fraction,and MvRL killed all termites between 4 and 7 days, while incontrols mortality of all termites was detected only after 11e12days. The significant differences (p < 0.05) between the dataobtained in the control and treatments with M. vaccinifolia prepa-rations provide strong evidence that they induced prematuremortality of termites.

In the repellence assay (Fig. 3), the insects built galleriesrandomly, ingesting the agar, with no preference for or rejection ofwells containing samples and negative control. In addition, thetermites maintained these galleries opened and active throughoutthe experiment. These results indicate that the rhizome extract, the0e60% fraction, and MvRL did not exert repellent activity againstN. corniger.

The results from the feeding-rejection assay showed that, after 3days, the mass loss of filter paper disks treated with MvRL at LC50for workers was 3.9 � 1.7 mg. This value was higher than thatdetermined for the control disks (2.23� 0.5mg), indicating that the

urvival of N. cornigerworkers (A, C and E) and soldiers (B, D and F). Concentrations usedcontrol (,). Each point represents the mean � SD of five experiments.

Fig. 3. Aspects of termite repellence assay from a lower standpoint, after 15 days. Thesamples evaluated were the negative control 0.15 M NaCl (1), rhizome extract at0.5 mg ml�1 of protein (2), 0e60% fraction at 0.5 mg ml�1 of protein (3), and MvRL at0.5 mg ml�1 (4). The termites were removed from the plate before photography. Thepicture was taken against a black background, for better visualization.

Fig. 4. Effect of MvRL on N. corniger trypsin-like and acid phosphatase activities. (A)Trypsin-like activity from worker gut extract toward 8 mM BApNA in the presence ofMvRL. (B) Acid phosphatase activity from worker gut extract in the presence of MvRL.(C) Effect of MvRL on acid phosphatase activity from soldier gut extract.

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lectin has no feeding-rejection effect. The mass loss of filter papersfrom the repellence assay could not be determined, since diskswere spoiled by the presence of agar.

Evaluation of digestive enzyme activities from gut of N. cornigerworkers showed the presence of trypsin-like (1.0 mU/mg), a-amylase (5.5 U/mg), acid phosphatase (216 mU/mg), and cellulase(endoglucanase: 2.18 U/mg; exoglucanase: 149 mU/mg; b-glucosi-dase: 227 mU/mg) activities. Soldier extract did not show trypsin-like activity, while a-amylase and acid phosphatase activitieswere 0.8 and 45.0 mU/mg, respectively. Endoglucanase, exogluca-nase, and b-glucosidase activities in soldier extract were 1.09 U/mg,266 mU/mg, and 320 mU/mg, respectively. Alkaline phosphatasewas not detected in extracts from guts of both castes.

Trypsin-like activity in workers was significantly (p < 0.05)reduced in the presence of MvRL, and a Ki of 2.0 mM was calculated(Fig. 4A). MvRL did not affect a-amylase activity in workers andsoldiers, although acid phophatases from workers and soldiersincreased 1.6 and 3.1 times, respectively (Fig. 4B and C). Endoglu-canase activity in workers increased in the presence of MvRL(Fig. 5A), while this activity in soldier extract was neutralized afterincubation with lectin (Fig. 5B). Exoglucanase activity in eithercaste was not affected by MvRL. On the other hand, b-glucosidaseactivity was reducedwhen the extract fromworkers or soldiers waspreviously incubated with MvRL (Fig. 5C and D).

4. Discussion

MvRL was successfully isolated by an inexpensive and rapidprotocol, consisting of treatment of rhizome extract with ammo-nium sulfate and chromatography on chitin column. The molecularmass of MvRL was similar to that reported by Santana et al. (2012),which evaluated the electrophoresis profile of MvRL-rich prepa-ration (0e60% fraction described here). The glycosylation of MvRLwas identified by SDS-PAGE and the presence of sugar moiety in theMvRL structure may be associated with its stability during heating,similarly to the M. urundeuva leaf lectin, which is glycosylated andstable to heating at 100 �C (Napoleão et al., 2011).

The binding specificity of a lectin can be determined byincubating it with a carbohydrate solution before determination ofhemagglutinating activity. The binding to monosaccharide orglycosylated moieties of glycoproteins prevents the interactionof lectin with glycoconjugates on the erythrocyte surface, and

hemagglutinating activity is reduced or neutralized (Paiva et al.,2011a). MvRL was shown to be a mannose-binding lectin, and itsactivity was also inhibited by glycoproteins. Although MvRLhemagglutinating activity was not affected by N-acetyl-D-glucos-amine, the lectin was able to bind to chitin, a polymer of thismonosaccharide. It has been suggested that the carbohydrate-binding sites of some lectins, such as that from M. urundeuva leaf,may have an extended geometry that allows it to recognizecomplex sugar molecules (glycoproteins and carbohydrate poly-mers) better than simple sugars (Rougé et al., 2003; Singh et al.,2007; Napoleão et al., 2011).

Fig. 5. Effects of MvRL on N. corniger endoglucanase and b-glucosidase activities. Worker (A) and soldier (B) endoglucanase activity in the presence of MvRL. b-glucosidase activitiesfrom workers (C) and soldiers (D) in the presence of MvRL.

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MvRL was toxic on termites, since the survival time of workersand soldiers exposed to lectin was lower, in comparison with thecontrol. MvRL was more active against N. corniger workers (LC50 of0.130 mg ml�1 for 3 days) than lectins from C. tapia bark (LC50 of0.45 mg ml�1 for 6 days) or C. verticillaris lichen (LC50 of0.196mgml�1 for 10 days); it was alsomore active than lectins fromM. urundeuva bark, heartwood, and leaf, whose LC50 ranged from0.248 to 0.974 mg ml�1 for 4 days (Sá et al., 2008; Silva et al., 2009;Napoleão et al., 2011; Araújo et al., 2012). The LC50 values for O. ficusindica cladodes and B. monandra roots (0.116 and 0.09 mg ml�1,respectively) against workers (Souza et al., 2011; Paiva et al., 2011b)were lower than those determined for MvRL. However, MvRL wasmore effective than these lectins, since it was possible to determineits LC50 after just 3 days, while the values for O. ficus indica andB. monandra lectins could only be determined after 4 and 12 days,respectively. Regarding the effect against soldiers, the LC50 of MvRL(0.085 mg ml�1) was lower than the values determined for all lec-tins that were able to kill representatives of this caste. In summary,MvRL can be considered as the best alternative among all lectinsthat have shown termiticidal activity so far.

N. corniger are insects that have the ability to build tunnels andgalleries. In the presence of possible toxic substances they can reactin self-defense, closing these spaces to avoid physical contact (Suet al., 1982). Using the same assay employed here, the repellentproperty of methanolic extract from M. urundeuva heartwood wasdetected by closing of the galleries constructed next to theperipheral wells containing it (Sá et al., 2009). The investigation ofrepellent activity revealed that the extract, the 0e60% fraction, andMvRL did not exert repellent activity against termites. This result issimilar to those found for all the other termiticidal lectins (Sá et al.,2008; Silva et al., 2009; Napoleão et al., 2011; Souza et al., 2011;Paiva et al., 2011b; Araújo et al., 2012). The absence of repellentactivity of plant lectins against termites can be explained by the factthat proteins are not usually detected by olfactory sensilla ofinsects, since they are not volatile.

MvRL had no feeding-rejection activity, which indicates that, inthe termiticidal assay, the insecticidal effect of lectin was due totoxic action by ingestion. In addition, it seems that the contact andingestion of lectin do not induce any rejection response by termites,which is a valuable characteristic for termite control, since theinsects will not stop ingesting the insecticide before death.

The mechanisms of termiticidal action of lectins still need to beelucidated. Napoleão et al. (2011) suggested that the chitin-bindingproperty, the bactericidal effect on gut symbionts, as well as theresistance to proteolysis in the insect gut are related to the termiti-cidal activity of M. urundeuva bark, heartwood, and leaf lectins. Inthis work, we investigated the ability of MvRL to modulate theactivity of digestive enzymes from gut of N. corniger workers andsoldiers, sincemetabolic alterations can lead to insect death (Macedoet al., 2007).

MvRL inhibited the trypsin-like activity from N. cornigerworkers, and may have led to impairment of digestion andabsorption of essential amino acids. In termites, protease activityplays an important role in the digestion of peptidic components indiets, such as hydrolyzable peptides found in soil (Ji and Brune,2005). The trypsin-like activity from the gut of Ephestia kuehniellaand Aedes aegypti larvae was also inhibited by lectins from Annonacoriacea seeds and M. urundeuva leaf, respectively (Coelho et al.,2007; Napoleão et al., 2012). Lectins are able to block the activityof enzymes by binding to the sugar moiety in glycosylatedenzymes, or by binding to sites other than the substrate binding sitein non-glycosylated enzymes (Macedo et al., 2007).

Gut extracts fromN. cornigerworkers and soldiers didnot containalkaline phosphatase activity, but acid phosphatase activity wasdetected in gut extracts from both castes. Acid phosphatases areimportant in diverse physiological processes for insects, and areconsidered marker enzymes to evaluate the interference of insecti-cides on insects’ physiological status (Koodalingam et al., 2011). Thestimulation of acidphosphatase activity byMvRL can lead to damageto termite gut structure, since this enzyme is usually involved in

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tissue autolysis in several animals, including insects (Nunes et al.,2006; Zhu et al., 2009).

Cellulases are important enzymes in the digestion of cellulose,the major nutrient of wood-eating termites. These enzymes usuallyact together in a balancedmanner and the stimulation/inhibition ofthe activity of one or more cellulases can deregulate the digestionprocess. The endoglucanase activities in workers and soldiers weredifferently modulated byMvRL, whichmay be linked to the distincteffect of lectin on survival of these castes. The inhibition of b-glucosidase activities in workers and soldiers may lead to strongimpairment of digestion, since the action of this enzyme is crucialbecause it finishes the digestion of cellulose to glucose, andhydrolyzes celobiose, an important inhibitor of cellulose digestion,when present at high concentrations.

5. Conclusions

MvRL, a heat-stable lectin with chitin-binding ability, showeda high level of toxicity against N. corniger workers and soldiers,being the most active termiticidal lectin reported so far. MvRL didnot show a repellent property, and thus its toxicity may result indisturbance of colony organization, structure, and maintenance.The mechanisms of MvRL termiticidal activity may involve imbal-ance of various physiological processes due to inhibition of trypsin-like activity at worker gut, stimulation of acid phosphatase activityat gut of both castes, as well as modulation of cellulase activities ingut of workers and soldiers, deregulating the entire process ofcellulose digestion.

Acknowledgments

The authors express their gratitude to the Conselho Nacional deDesenvolvimento Científico e Tecnológico (CNPq) for researchgrants and fellowships (L.C.B.B. Coelho and P.M.G. Paiva), Coor-denação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES),and Fundação de Amparo à Ciência e Tecnologia do Estado dePernambuco (FACEPE) for research grants. L.P. Albuquerque wouldlike to thank CAPES and the Brazilian Ministry of Education (Pro-grama de Reestruturação e Expansão das Universidades Federais -REUNI) for a graduate scholarship. G.M.S. Santana and E.V. Pontualwould like to thank FACEPE for graduate scholarships, and T.H.Napoleão would like to thank CAPES for a graduate scholarship. Theauthors are also deeply grateful to Maria Barbosa Reis da Silva fortechnical assistance and Felix Nonnemacher for English editing.

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