Purified Cladonia verticillaris lichen lectin: Insecticidal activity on Nasutitermes corniger (Isoptera: Termitidae)

International Biodeterioration & Biodegradation 63 (2009) 334–340

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Purified Cladonia verticillaris lichen lectin: Insecticidal activity on Nasutitermes corniger (Isoptera: Termitidae)q Michele D.C. Silva a, Roberto A. Sa´ a, Thiago H. Napolea˜o a, Francis S. Gomes a, Nataly D.L. Santos a, Auristela C. Albuquerque b, Haroudo S. Xavier c, Patrı´cia M.G. Paiva a, Maria T.S. Correia a, Luana C.B.B. Coelho a, * a b c

´ ria, 50670-420 Recife, Pernambuco, Brazil ´rio de Glicoproteı´nas, Departamento de Bioquı´mica-CCB, Universidade Federal de Pernambuco, Cidade Universita Laborato ˜os, 52171-030 Recife, Pernambuco, Brazil Departamento de Biologia, Universidade Federal Rural de Pernambuco, Dois Irma ´ria, 50740-521 Recife, Pernambuco, Brazil ´ rio de Farmacognosia, Departamento de Cieˆncias Farmaceˆuticas-CCS, Universidade Federal de Pernambuco, Cidade Universita Laborato

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 August 2008 Received in revised form 15 October 2008 Accepted 3 November 2008 Available online 5 December 2008

Cladonia verticillaris lichen lectin (ClaveLL) was isolated through Sephadex G-100 gel filtration chromatography and characterized as a pure lectin through A¨KTA-FPLC and HPLC systems. The lichen extract (LE), protein fraction (F1) and ClaveLL were assayed to evaluate their potential insecticidal and/or repellence activities on termite Nasutitermes corniger. LE, F1 and ClaveLL were evaluated for hemagglutinating activity (HA), protein concentration and presence of secondary metabolites; preparations and active ClaveLL, free of secondary metabolites, were able to induce termite mortality. ClaveLL LC50 values after 10 days for workers and soldiers were 0.196 and 0.5 mg ml1, respectively. C. verticillaris preparations are potential tools for researches involving control of termites (or other insects) of economic interest to wooden industry or agriculture as well as preservation of plant species that are targets of termites or other plagues. Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: Cladonia verticillaris Lectin Lichen Nasutitermes corniger Termite

1. Introduction Termites constitute a great group of social insects that are ecologically beneficial since they are the major responsible for decomposition of cellulose, the most abundant carbohydrate polymer in the world, acting in biorecycling promoting carbon turnover in the environment. Termites have the ability to digest cellulose due to the presence of three types of cellulases (endoglucanases, exoglucanases and b-glucosidases) with endogenous and/or symbiotic origin in their gut (Breznak and Brune, 1994). Endogenous enzymes are encoded by genes in the termite genome and symbiotic enzymes are produced by hindgut symbionts (Zhou et al., 2008). The order Isoptera is constituted of seven families that have (except Termitidae) cellulose-fermenting protozoa in the hindguts of termites (Tokuda et al., 1999). The flagellates in Termitidae have been lost and the primary digestive role is displayed

q Scientific relevance: Cladonia verticillaris lichen lectin (ClaveLL) is a novel lectin easily purified in milligram quantities that may be developed as a biotechnical tool in termite management strategies. The effect of ClaveLL on Nasutitermes corniger highlights the potential use of this bioactive protein to enhance termite resistance. * Corresponding author. Tel.: þ55 8121268540; fax: þ55 8121268576. E-mail address: [email protected] (L.C.B.B. Coelho). 0964-8305/$ – see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2008.11.002

by a consortia of prokaryotes. According to morphological and molecular features, the Termitidae family is accepted as the most recently evolved and derived from Isoptera order (Miura et al., 1998). Biodeterioration of wood by termites is a serious problem for wood utilization which has economical and environmental impacts. According to Korb (2007), an estimate from 2005 put the annual damage by termites at about US$ 50 billion worldwide. Replacement of deteriorated wood also increases the number of trees cut and the impact of deforestation (Clausen and Yang, 2007). In addition to wood damage, termites are also responsible for biodeterioration of paintings, ancient books, monuments and buildings of historical importance. Due to these negative impacts, termites are considered as plague-insects and new effective methods for termite control and monitoring have been searched (Koestler et al., 2000). The genus Nasutitermes belongs to the family Termitidae, which contains around 60% of all termite cataloged species and includes wood, grass or soil feeders (Tokuda et al., 1999; Inward et al., 2007). The Nasutitermes constitutes the largest genus of wood-feeding in the order Isoptera. The presence of bacteria able to promote cellulose and xylan hydrolysis was detected in the gut of Nasutitermes species (Warnecke et al., 2007). Representatives of these

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insects can be found mainly distributed over tropical regions (Bergamaschi et al., 2007) and Nasutitermes species can invade urban environment in Brazilian semi-arid region attacking wood employed in structures of buildings (Paes et al., 2007). The kingdom Plantae contains secondary metabolites and macromolecules resultant of the primary metabolism that can act as defensive substances against microorganisms, insects and herbivorous or in response to abiotic stress. The main role of plant secondary metabolites is believed to protect plants from attack by pathogens or predators (Zhao et al., 2005). There are many proteins in plants, as reviewed by Carlini and Grossi-de-Sa´ (2002), involved in defensive mechanisms such as lectins, ribosome-inactivating proteins, inhibitors of proteolytic enzymes and glycohydrolases, arcelins, chitinases, hemilectins, canatoxin-like protein as well as modified forms of storage proteins and ureases. Lectins are proteins of wide distribution in nature and nonimmune origin with the ability to recognize a carbohydrate or a glycosylated molecule through their reversible binding sites (Kennedy et al., 1995; Correia et al., 2008). Lectins stand out in biotechnology due to many applications and other potential utilities that are daily evaluated and studied by researchers in lectinology. Several lectins have showed effects in different life stages of many insect orders such as Coleoptera (Leite et al., 2005; Sadeghi et al., 2006; Macedo et al., 2007), Diptera (Sa´ et al., 2008b), Hemiptera (Sauvion et al., 2004), Homoptera (Bandyopadhyay et al., 2001), Hymenoptera (Couty et al., 2001) and Lepidoptera (Coelho et al., 2007; Macedo et al., 2007). The unique report of lectin toxicity on an insect of Isoptera order is shown by the lethal effect of lectin from the heartwood of a timber tree (Myracrodruon urundeuva) on N. corniger (Sa´ et al., 2008a). This lectin induced mortality of workers and soldiers with LC50 values of 0.248 mg ml1 and 0.199 mg ml1, respectively. The authors concluded that this lectin may play a role in the natural resistance of M. urundeuva. Lectins can be isolated from lichens. The involvement of these lectins in the symbiotic establishment of lichens and their possible physiological role has been studied and suggested (Elifio et al., 2000; Molina and Vicente, 2000). ClaveLL is a new lectin from C. verticillaris lichen that has been purified in our laboratory. Potential antimicrobial effects and possible use of ClaveLL as a histochemical marker are currently evaluated. In this work, we evaluated the insecticidal and repellent properties of ClaveLL, a pure lectin sample (free of secondary metabolites that can interfere in insecticidal assay), against N. corniger, a wood-damaging termite species. Successful development of alternative insecticides and repellents will preserve existing wood structures as well as lower the extinction danger of overexploited species. Additionally, a lichen extract and a protein fraction from C. verticillaris used in ClaveLL purification protocol were also investigated for insecticidal and repellent effects.

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Lichen was dried at 28  C and an overnight extraction (16 h; 28  C) was performed in 0.15 M sodium phosphate buffer pH 7.0 containing 0.15 M NaCl (PBS), followed by filtration, centrifugation (3000 g, 4  C, 15 min) and protein precipitation with 30% ammonium sulphate for 4 h at 28  C. The 0–30% precipitate fraction (F1) was obtained by centrifugation (3000 g, 4  C, 15 min), submitted to protein quantification and loaded (18 mg of protein) onto a gel filtration Sephadex G-100 (1.4  63 cm) column aiming to obtain pure and active ClaveLL. The lichen extract (LE), protein fraction (F1) and ClaveLL samples obtained in the purification process had their hemagglutinating activity (HA) evaluated with 2.5% (v/v) glutaraldehyde-treated erythrocyte suspension in 0.15 M NaCl (Bing et al., 1967) according to Correia and Coelho (1995). HA (titer) was defined as the lowest dilution of the sample that showed hemagglutination. Specific HA was defined as the ratio between the titer and protein concentration (mg ml1). HA inhibition assays were performed with 200 mM N-acetylglucosamine and 0.5 mg ml1 solution of glycoproteins from rabbit and bovine fetal serum. 2.3. Phytochemical analysis Phytochemical evaluation of LE, F1 and ClaveLL samples in PBS and of a lichen methanolic extract (an infusion of 3 g of triturated lichen in methanol prepared under constant agitation for 30 min) was performed. Samples were analyzed by thin layer chromatography (TLC) on silica sheet (Merck, Germany). The analyses used several systems of development as mobile phase, reagents for adequate revelation and chromatographic standards. Each sample was investigated for the presence of: (I) alkaloids (EtOAc–HCOOH–AcOH–H2O [100:11:11:26 v/v] as mobile phase and Dragendorff’s reagent for revelation; Wagner and Bladt, 1996); (II) terpenoids and steroids (EtOAc–HCOOH–AcOH–H2O [100:0.5:0.5:0.5 v/v] and Liebermann–Burchard’s reagent; Harborne, 1998); (III) saponins (EtOAc–HCOOH–AcOH–H2O [100:11:11:26 v/v] and anisaldehyde for revelation; Wagner and Bladt, 1996); (IV) iridoids (EtOAc–HCOOH–AcOH–H2O [100:11:11:26 v/v] and vanillinsulphuric acid for revelation; Wagner and Bladt, 1996); (V) sugars (n-BuOH–Me2CO–phosphate buffer pH 5.0 [40:50:10 v/v] and 2,3,5triphenyltetrazolium chloride to reveal; Wallenfels, 1950); (VI) coumarins (Et2O–toluene–AcOH 10% [50:50:50 v/v] and UV 365 nm to detect; Wagner and Bladt, 1996); (VII) cinnamic derivatives, phenylpropanoglucosides, flavonoids and phenolic acids (EtOAc– HCOOH–AcOH–H2O [100:11:11:26 v/v] and Neu’s reagent to reveal; Wagner and Bladt, 1996; Markhan, 1982; Neu, 1956); (VIII) condensed proanthocyanidins and leucoanthocyanidins (EtOAc–HCOOH– AcOH–H2O [100:11:11:26 v/v] and vanillin-chloridric acid to reveal; Roberts et al.,1956); and (IX) hydrolysable tannins (n-BuOH–Me2CO– phosphate buffer pH 5.0 [40:50:10 v/v] and 1% iron alum to reveal; Stiasny, 1912). 2.4. Insecticidal assay

2. Material and methods 2.1. Protein content The protein concentration was estimated in all samples according to Lowry et al. (1951) using bovine serum albumin (31– 500 mg/mL) as standard. 2.2. Lectin purification C. verticillaris lichen was collected at Alhandra City, State of Paraı´ba, northeastern Brazil and taxonomic identification was kindly performed by Dr. Eugeˆnia Cristina Gonçalves Pereira (Universidade Federal de Pernambuco). C. verticillaris lichen lectin (ClaveLL) was obtained through a sequential purification protocol.

The termite species N. corniger was collected in the campus of the Universidade Federal Rural de Pernambuco and identified by Dr. Luiz Roberto Fontes (Superintendeˆncia de Controle de Endemias, SUCEN, Brazil). Termite colonies were kept at vegetation house from the Departamento de Agronomia from Universidade Federal Rural de Pernambuco. Termiticidal activity was evaluated by a bioassay based on the method described by Sa´ et al. (2008a). Assays were carried out in Petri plates (90  15 mm) with the lower plate covered with filter paper. Disks of filter paper (4 cm diameter) impregnated with 200 mL of LE or F1 samples at 20, 2 or 1 mg ml1 of protein (corresponding to 320, 32 or 16 mg of protein cm2, respectively) were placed in plates for feeding by the insects. Treatments with the lectin contained ClaveLL samples at 0.6, 0.4, 0.2 or 0.05 mg ml1

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corresponding to 9.6, 6.4, 3.2 or 0.8 mg of protein cm2, respectively. PBS was used as negative control. The paper disks containing the samples were dried at 28  C and then placed in the plates. Twenty insects (16 workers and 4 soldiers) from termite colonies were carefully transferred to the plates. All plates were kept in darkness at 28  C. Monitoring of assays was performed daily to detect death of insects and to guarantee the humidity of the plates by adding a water drop to the paper covering the plate until all of the insects had died. Assays were made in quintuplicate. Total experimental duration was defined as the number of days to reach 100% mortality rate in negative control. The survival rates (%) were obtained for each treatment and expressed as mean  standard deviation (SD). Statistical analysis was performed using the computer package StatplusÒ 2006 (AnalystSoft, Canada) for determination of the protein concentration required to kill 50% of insects in each treatment (LC50) for LE, F1 (after 4 days) and ClaveLL (after 10 days) by probit analysis method with a reliability interval of 95%. Significant differences between each possible pair of treatments were established performing Student’s t-test (p < 0.05), assuming equal variances (homoscedastic) and considering the accumulative mortality during all assays using OriginÔ 6.0 (Microcal, USA) software. 2.5. Repellence assay The potential repellent property from LE, F1 and ClaveLL was evaluated using an assay based on Sa´ et al. (2008a). A 2% (w/v) agar solution in distilled water was added to Petri plates until the border of the plates so that there was no space between the superior surface of agar and plate covers. After solidification wells were made in agar by the removal of 1 central cylinder (25 mm diameter) and 8 peripheral cylinders (6 mm diameter). Disks of filter paper (6 mm diameter) were impregnated with LE or F1 samples at 300, 150, 30 or 15 mg of total protein (corresponding to 1060, 530, 106 or 53 mg cm2, respectively) and ClaveLL sample at 11.5, 5.75, 2.25, 1.5 or 0.75 mg of total lectin (corresponding to 40.6, 20.3, 7.95, 5.3 or 2.65 mg lectin cm2, respectively). PBS was used as negative control. The paper disks were put to dry at 28  C and placed in the peripheral wells. Each protein concentration was present in double in plates and each plate was carried out in triplicate, performing a total of 6 replicates for each protein concentration assayed. Twenty insects (16 workers and 4 soldiers) were carefully transferred to central wells in the plates that were maintained in darkness at 28  C. Plates were observed daily to evaluate absence or presence of termites in wells, construction standards of tunnels in agar, closing by insects of constructed galleries and death of insects. 3. Results 3.1. Lectin isolation An amount of 20 g from C. verticillaris flour allowed obtaining 1162.50 mg (total protein) in LE, 510.14 mg in F1 and 18.58 mg of purified ClaveLL using a simple protocol. ClaveLL represents 0.093% of the dry weight of crude flour, and 1.59% and 3.64% of total protein present in LE and F1, respectively. LE, F1 and ClaveLL agglutinated glutaraldehyde-treated rat, chicken, quail, rabbit (mostly) and human (all ABO system) erythrocytes. LE and F1 showed high protein concentration (7.75 and 19.85 mg ml1, respectively), and specific HA (33 and 412, respectively). HA of ClaveLL (specific HA of 780.5) was reduced in the presence of N-acetylglucosamine and abolished in the presence of rabbit and bovine fetal serum. All protein samples including active ClaveLL and free of secondary metabolites were chosen to evaluate their potential insecticidal properties via lectin effect.

3.2. Phytochemical analysis Phytochemical evaluation of LE, F1 and ClaveLL samples showed none of the secondary metabolites (alkaloids, terpenoids, steroids, saponins, iridoids and polyphenols as coumarins, cinnamic derivatives, phenylpropanoglucosides, flavonoids, phenolic acids, condensed proanthocyanidins, leucoanthocyanidins and hydrolysable tannins) evaluated. Phytochemical evaluation of lichen methanolic extract was performed aiming to analyze the potential presence of terpenoids. TLC revealed bands corresponding to b-amirin and b-sitosterol standards and additional bands of smaller polarity that characterize triterpenes and steroids, which reacted with Liebermann–Burchard’s reagent. No other secondary metabolite was detected in the methanolic extract. 3.3. Termiticidal activity of lichen extract and fraction LE, F1 and ClaveLL in PBS, samples free of secondary metabolites, were submitted to insecticidal assay against N. corniger aiming to evaluate termiticidal activity. LE, F1 and ClaveLL in all tested concentrations induced 100% mortality of workers and soldiers. Also, in all assays survival rate of 50% was detected in a smaller time than that obtained for the control. LE and F1 containing 20, 2 or 1 mg ml1 of protein (corresponding to 320, 32 or 16 mg of protein cm2, respectively) induced 100% mortality of workers after 11, 13 and 14 days (LE) and 7, 10 and 12 days (F1), in respective concentrations. Survival rate of 50% for LE treatment was observed after 2 days (20 mg ml1) and at fifth and sixth days (2 and 1 mg ml1, respectively). F1 treatments’ survival rate of 50% was reached after 2 days (20 mg ml1) and between fourth and fifth days (with 2 and 1 mg ml1, respectively). Mortality induction on workers by LE and F1 was related to protein concentration (Fig. 1A and C). In negative control, 100% of mortality was observed after 17 days and survival rate was lower than 50% around 7 days. There was a significant difference among LE treatment levels 20, 2 and 1 mg ml1 in the induction of worker’s death. LE and F1 samples also induced 100% mortality of soldiers after 7, 12 and 14 days (LE) and 7, 9 and 13 days (F1) at 20, 2 or 1 mg ml1 of protein, respectively. Survival rate of 50% was observed at 2, 6 and 7 days for LE and after 2, 5 and 6 days for F1 (concentrations above cited, respectively). Mortality induction on soldiers by LE and F1 was also related to protein concentration (Fig. 1B and D). In negative control, 100% mortality of soldiers was observed after 16 days and 50% survival rate after 7 days. LE and F1 LC50 values after 4 days for workers and soldiers were shown in Table 1. Although LC50 values indicate that LE was more efficient than F1, comparison between LE and F1 effects on workers or soldiers showed statistically significant differences only at a concentration of 20 mg ml1 (320 mg cm2), as illustrated in mortality data for workers and soldiers compared to negative control. 3.4. Termiticidal activity of ClaveLL Analysis of worker’s survival revealed that treatment with ClaveLL at 0.6 mg ml1 (9.6 mg cm2) induced death of all workers after 15 days. Treatments with 0.4 (6.4 mg cm2), 0.2 (3.2 mg cm2) and 0.05 (0.8 mg cm2) mg ml1 of lectin promoted 100% mortality after 19, 23 and 26 days, respectively. Survival rate of 50% for these treatments was observed around 9 and 10 days for treatment with 0.6 and 0.4 mg ml1 ClaveLL, and around 12 and 14 days for treatments with 0.2 and 0.05 mg ml1 ClaveLL. Mortality induction on N. corniger workers by ClaveLL was also proportional to protein concentration (Fig. 2A). LC50 value for workers after 12 days was 0.196 mg ml1 (or 3.13 mg cm2). In negative control 100% of

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Fig. 1. Survival percentile of Nasutitermes corniger workers (A and C) and soldiers (B and D) castes in presence of lichen extract (LE) or fraction 0–30% (F1). Treatments were at 20 (C), 2 (B) and 1 (-) mg ml1 concentration of protein. PBS was used as control (x). Each point represents the mean of five repetitions. Statistically significant difference was observed between 20 mg ml1 and control (A, B, C and D), and between 20 and 1 mg ml1 (B).

mortality was observed only after 42 days and survival rate lower than 50% around 26 days. ClaveLL treatment at 0.6 mg ml1 (9.6 mg cm2) induced death of all soldiers after 16 days. Mortality of 100% was promoted by treatments with ClaveLL at 0.4, (6.4 mg cm2), 0.2 (3.2 mg cm2) and 0.05 (0.8 mg cm2) after 19, 24 and 28 days, respectively. Survival rate of 50% was observed around 11, 17, 20 and 23 days for treatments with 0.6, 0.4, 0.2 and 0.05 mg ml1, respectively. Lectin mortality induction on N. corniger soldiers was also proportional to protein concentration (Fig. 2B). LC50 value for soldiers was 0.5 mg ml1 (8.01 mg cm2) after 10 days. In negative control, 100% mortality was observed after 41 days and survival rate of 50% after 35 days. Results with all lectin treatments for workers and soldiers showed statistically significant differences in relation to control. Treatment with 0.6 mg ml1 ClaveLL showed statistical difference in relation to 0.2 and 0.05 mg ml1, as well as 0.4 mg ml1 ClaveLL was statistically different in relation to 0.05 mg ml1. Statistical analyses of ClaveLL effect on insects considering workers and soldiers together showed that induction of termite death by ClaveLL was proportional to protein concentration (Fig. 2C). Effect of 0.6 mg ml1 concentration was significantly different in relation to 0.2 and 0.05 mg ml1 regarding promotion of death of all termites. 3.5. Repellence assay LE, F1 and ClaveLL samples showed no repellent property against workers or soldiers since the insects continuously and randomly visited peripheral wells containing disks of filter paper soaked with lectin samples as well as negative control (PBS). Insects built galleries randomly, with no differences observed between samples and negative controls. Termites maintained galleries open and active throughout the experiment duration.

4. Discussion Lichens have been source of compounds with several biological activities, including insecticidal activity on larvae of Aedes aegypti (Kathirgamanathar et al., 2006) and Culex pipiens mosquitoes (Cetin et al., 2008), and growth retarding activity on polyphagous herbivorous insect Spodoptera littoralis (Giez et al., 1994). C. verticillaris is a lichen of cosmopolitan distribution on the world and represents a rich lectin source. PBS was able to extract high protein concentration; the simple and efficient protocol used to isolate ClaveLL in this work yielded milligram quantities of lectin (18 mg from 20 g of lichen flour). One gram of lichen flour contains 0.93 mg of ClaveLL, i.e. 2 and 5 times greater concentration than the LC50 determined for soldiers and workers, respectively. The easiness to obtain the lichen, the feasibility of ClaveLL isolation, and the previous reports of insecticidal activity of lectins and compounds extracted from lichens stimulated the evaluation of ClaveLL termiticidal activity. It is known that chemical plant defense against microorganisms, insects or herbivore predators can be due to secondary metabolites of Table 1 Termiticidal activity lectin preparations from C. verticillaris lichen on N. corniger. Termite caste

LE

F1

ClaveLL

Soldiers LC50 (mg ml1) LC50 (mg cm2) SE LC50a

6.5 103.59 18.17

7.7 121.46 34.87

0.5 8.01 0.24

Workers LC50 (mg ml1) LC50 (mg cm2) SE LC50a

3.0 47.42 15.59

3.26 51.97 25.41

0.196 3.13 0.34

a Standard error for LC50 values expressed as mg cm2. LC50 determined for LE and F1 after 5 days; after 12 days for ClaveLL.

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Fig. 2. Survival percentile of Nasutitermes corniger workers (A), soldiers (B) and both castes (C) in presence of ClaveLL. Treatments were at 0.6 (C), 0.4 (B), 0.2 (-) and 0.05 (,) mg ml1. PBS was used as control (x). Each point represents the mean of five repetitions. Statistically significant difference was observed between all treatments and control, between 0.6 and 0.2 mg ml1 (B), between 0.6 and 0.05 mg ml1 (B and C), and between 0.4 and 0.05 mg ml1 (B).

low molecular weight and many entomotoxic proteins such as lectins. Evaluated samples showed to be free of alkaloids, terpenoids, saponins, iridoids, polyphenols, condensed anthocyanidins, and hydrolysable tannins, compounds that can be responsible for insecticidal

activity in preparations obtained from plants. The absence of secondary metabolites in LE, F1 and isolated ClaveLL samples suggests that the termiticidal action evaluated in this work had no interference of these compounds and was due to lectin activity. Presence of terpenes in the methanolic extract proved the existence of secondary metabolites in C. verticillaris constitution, which were not extracted using PBS in the first step to obtain the lectin. ClaveLL was toxic on termites in all tested concentrations since the time of life for insects in contact with lectin was reduced regarding negative control. ClaveLL effect was related to lectin concentration since the minor and larger time to the total mortality did correspond to the largest and smaller offered concentrations, respectively. The insects reacted differently in the assays using purified ClaveLL or LE and F1 since 100% mortality was observed after 42 days in negative control (in the assays with ClaveLL); insects stayed alive during 17 days with negative control for LE and F1 assays. This difference was probably due to the fact that the bioassays were conducted at different times of the year. Sa´ et al. (2008a) reported that the lectin from M. urundeuva heartwood (MuHL) showed LC50 of 0.248 mg ml1 and 0.199 mg ml1 on N. corniger workers and soldiers, respectively, after 4 days. LC50 for LE, F1 and ClaveLL was calculated after 5 days (in bioassay with LE and F1) and 12 days (in bioassay with ClaveLL). The time used to calculate LC50 corresponded to 23% of total duration of experiments. LC50 values showed that ClaveLL was less effective on soldiers than MuHL but a little more efficient on workers. Similarly to MuHL, termiticidal effect of ClaveLL yielded different LC50 values for soldier and worker castes but, on the other hand, ClaveLL was more effective on workers while MuHL was more toxic for soldiers (Sa´ et al., 2008a). Treatment with 0.6 mg ml1 ClaveLL was the main concentration with toxic effect on termites, promoting death of all workers and soldiers at 37 and 39% of the experimental time, respectively. Lectin effects on the insects are expressed using different parameters to protein values according to experimental methods used that are generally based on bioassays with lectin incorporation into artificial diets offered to target insects. Lectin toxicity seems to depend upon both the necessary resistance of the molecule against assimilatory proteins and proteolytic degradation by the insect digestive enzymes as well as on the binding to insect gut structures (Coelho et al., 2007; Macedo et al., 2007; Sa´ et al., 2008b). ClaveLL, as well as MuHL, showed high affinity to N-acetylglucosamine as other lectins with insecticidal properties. Sa´ et al. (2008a) suggested that the termiticidal effect of MuHL may be due to lectin action on digestive tract of termite and showed that this lectin is resistant to proteolytic degradation by trypsin. In insects, the peritrophic matrix constitutes a membrane found in the midgut containing chitin, which separates the contents of the gut lumen from the digestive epithelial cells. It has been suggested that chitin and glycosylated proteins of peritrophic matrix can be targets for lectin binding (Tellam et al., 1999). Lectins able to bind N-acetylglucosamine or chitin (N-acetylglucosamine polymer) promoted larval mortality of dengue mosquito A. aegypti (Sa´ et al., 2008b), flour moth Anagasta kuehniella (Coelho et al., 2007) as well as bruchid beetle larvae from Zabrotes subfasciatus and Callosobruchus maculatus (Macedo et al., 2007). These authors suggested that insecticidal activity may be related to lectin binding to chitin components in the insect gut, interaction with glycoconjugates on the surface of epithelial cells along the digestive tract, binding to the sugar moiety of glycosylated digestive enzymes or assimilatory proteins and resistance to insect digestive proteases. ClaveLL may promote termite mortality through the mechanisms already proposed to these other lectins; according to subcellular targets of lectin, it may decrease food intake through its binding to the midgut epithelium or the peritrophic matrix (Fitches et al., 1997). Lectin may also cross the midgut epithelial barrier and

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pass into the insect circulatory system resulting in a toxic action interfering with endogenous lectins involved in self-defense present in the haemolymph (Fitches et al., 2001); it may be internalized by endocytotic vesicles into the epithelial cells and blocks nuclear localization and nuclear sequence-dependent protein import inhibiting cell proliferation (Yu et al., 1999). N-acetylglucosamine specific lectins with chitin-binding ability, in general, appear to own insecticidal property; however, plant lectins with different sugar specificities also acted as insecticidal molecules suggesting that this property is not exclusive to N-acetylglucosamine specific lectins. For example, XCL, a Xerocomus chrysenteron (mushroom) lectin with affinity for D-galactose and lactose and to a lesser extent, N-acetyl-D-galactosamine, had effect on Drosophila melanogaster and pea aphid Acyrthosiphon pisum showing LC50 values of 0.4 mg ml1 and 0.023 mg ml1, respectively (Trigueros et al., 2003). Gracilaria ornata (a red alga) lectin, GOL, only inhibited by some glycoproteins, caused a significant reduction (65.1%) in Callosobruchus maculates larval survival at 1% (w/w) in relation to control artificial seeds. Also, GOL significantly affected the percentage of adult emergence in relation to control (Leite et al., 2005). In other work, Sadeghi et al. (2006), using 14 plant lectins (of four lectin families) with several specificities at a concentration of 0.05% (w/v) solution, showed that all lectins had deterrent effect against oviposition of C. maculatus adults. Talisia esculenta seed lectin with chitin-binding properties and inhibited by mannose and glucose produced ca. 90% mortality to C. maculatus and Zabrotes subfasciatus larvae with LD50 and ED50 (decrease of 50% weight) of ca. 1% (w/w) for both insects (Macedo et al., 2002). The precise mechanism of insecticidal action of lectins is still unknown and it has been suggested that various modes of action exist to different lectins at the cellular levels not necessarily implying in the disruption of cellular function and lysis. Action mechanism of lectins as toxic molecules can also be related to interference with the functions of digestive enzymes and assimilatory proteins inhibiting digestion and absorption causing nutritional deprivation (Coelho et al., 2007; Sa´ et al., 2008b). N. corniger are insects that have the ability to build tunnels and galleries and on stress condition at the presence of possible toxic substances they can react closing these spaces to avoid physical contact (Su et al., 1982). The investigation of repellent activity revealed that ClaveLL and other samples (LE and F1) did not have repellent property on termites; the results suggested that the insects have eaten the paper disk containing lectin and that the insecticidal effect was due to action of lectin. Similarly to ClaveLL, MuHL did not show repellent properties (Sa´ et al., 2008a). C. verticillaris lichen lectin (ClaveLL) is a novel lectin easily purified in milligram quantities that may be developed as a biotechnical tool in termite management to facilitate plant species preservation and the use of wood in high risk environments. The effect of ClaveLL on N. corniger highlights the potential use of this bioactive protein to enhance termite resistance. Acknowledgements The authors express their gratitude to the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) and the Coordenaça˜o de Aperfeiçoamento de Pessoal de Nı´vel Superior (CAPES) for research grants and fellowship (CNPq, LCBBC). They are also deeply grateful to Maria Barbosa Reis da Silva and Joa˜o Antoˆnio Virgı´nio (for technical assistance). References Bandyopadhyay, S., Roy, A., Das, S., 2001. Binding of garlic (Allium sativum) leaf lectin to the gut receptors of homopteran pests is correlated to its insecticidal activity. Plant Science 161, 1025–1033.

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