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. 2022 Apr 9;12(5):109. doi: 10.1007/s13205-022-03172-9

Myracrodruon urundeuva leaf lectin damages exochorionic cells and binds to the serosal cuticle of Aedes aegypti eggs

Robson Raion Vasconcelos Alves 1, Gabryella Borges Prazeres 1, Abdênego Rodrigues da Silva 1, Amanda Luiza Tomaz Soares da Silva 1, Jéssica Silva Nascimento 2, Roberto Araújo Sá 3, Gabriel Gazzoni Araújo Gonçalves 4,5, Fábio André Brayner 4,5, Luiz Carlos Alves 4,5, Daniela Maria do Amaral Ferraz Navarro 2, Paulo Euzébio Cabral Filho 6, Adriana Fontes 6, Thiago Henrique Napoleão 1,✉, Patrícia Maria Guedes Paiva 1
PMCID: PMC8994808  PMID: 35462951

Abstract

In recent years, lectins have been identified as alternative agents against Aedes aegypti during the aquatic phases of its life cycle. For example, chitin-binding lectin from Myracrodruon urundeuva leaf (MuLL) can function as a larvicide. In this study, we investigated whether MuLL can also act as an ovicide against this insect. Aedes aegypti eggs were incubated with MuLL for 72 h to determine the concentration at which the hatching rate reduces by 50% (EC50). The effects of MuLL on the egg surface structure were evaluated using scanning electron microscopy (SEM), and the possible interaction of MuLL with the internal structures of eggs and embryos was investigated using MuLL-fluorescein isothiocyanate (FITC) conjugate. MuLL acted as an ovicidal agent with an EC50 of 0.88 mg/mL. The SEM analysis revealed that eggs treated with MuLL for 24 and 48 h no longer had tubercles and did not show a well-defined exochorionic network. In addition, deformation and degeneration of the surface were observed after 72 h. Fluorescence microscopy showed that MuLL penetrated the eggs 48 h after incubation and was detected in the upper portion of the embryo’s gut. After 72 h, MuLL was observed in the serosal cuticle and digestive tract. In conclusion, MuLL can function as an ovicidal agent against A. aegypti through damage to the surface and internal structures of the eggs.

Keywords: Insecticidal activity, Dengue mosquito, Chitin-binding proteins, Egg structures

Introduction

Arboviruses comprise a group that includes the causative agents of yellow fever (YFV), dengue (DENV), chikungunya (CHIKV), and zika (ZIKV), which are diseases transmitted by the mosquito Aedes aegypti and have been a public health concern in Brazil and worldwide (Donalisio et al. 2017). In recent years, the incidence of arboviruses has increased remarkably, primarily due to disorganized urbanization and lack of basic sanitation, favoring the dispersion of their vectors (Gregianini et al. 2017; Gould et al. 2017).

Aedes aegypti is primarily controlled using insecticides with adulticidal and larvicidal effects and is controlled on a small scale using ovicidal agents (Zara et al. 2016; Alves et al. 2020). Aedes aegypti is dispersed mainly through its eggs, which are resistant to drought for several months, allowing rapid reconstitution of the population. Thus, the eggs are essential targets for mosquito control (Russell et al. 2001; Pontual et al. 2014). The resistance of this insect to various chemical insecticides has led to the usage of high concentration of insecticides over a brief period. In addition, the most commonly used insecticides cause several types of damage to the environment (Rodriguez et al. 2007; Perry et al. 2011). Thus, the search for alternatives to control A. aegypti has focused on plants as sources of natural compounds with insecticidal action. Among these are lectins, proteins that bind specifically to carbohydrates and glycoconjugates (Coelho et al. 2017). Studies have shown the insecticidal activity of plant lectins against A. aegypti, which are associated with morphological changes and modulation of enzymatic activities in the larval gut and delaying development (Coelho et al. 2009; Santos et al. 2012; Agra-Neto et al. 2014; Silva et al. 2019b).

Lectins have been isolated from bark, heartwood, and leaves of Myracrodruon urundeuva. The study with this plant started aiming to investigate the presence of a lectin in the heartwood of M. urundeuva, which is strongly resistant to attack by fungi and insects. A lectin (MuHL) was found in this tissue and, when isolated, showed antifungal and termiticidal activities (Sá et al. 2008, 2009a). These data stimulated the investigation of the lectin presence in other tissues/organs of the plant. Sá et al. (2009a) and Napoleão et al. (2011) detected and purified lectins from bark (MuBL) and leaves (MuLL), respectively, which also showed insecticidal activity against termites. The detection of termiticidal activity prompted the evaluation of the effects of these proteins on other insects.

Sá et al. (2009b) reported that MuBL and MuLL are larvicidal agents against A. aegypti. Later, MuLL was also reported as larvicidal molecule on this mosquito and the authors also described that this lectin is resistant to proteolysis by larval digestive enzymes, as well as modulated protease and amylase activities (Napoleão et al. 2012). In line with the facts that M. urundeuva lectins are chitin-binding proteins as well as chitin is a major component of the A. aegypti eggs, Alves et al. (2020) evaluated the ovicidal activity of MuHL and MuBL, demonstrating that both lectins were able to alter the external chorionic structure and penetrate the eggs, reaching the digestive tract of the embryos. However, to our knowledge, there are no reports on whether and how MuLL interacts with A. aegypti eggs and embryos.

In the present study, we investigated whether MuLL could also act as an ovicidal agent against A. aegypti. In addition, we evaluated the effect of MuLL on the ultrastructure of the egg surface and its potential ability to penetrate the eggs and reach the internal structures of the embryo.

Materials and methods

Isolation of MuLL

Myracrodruon urundeuva leaves were collected in the city of Caxias, Maranhão, Brazil, as authorized (no. 38690) by the Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio). The voucher specimen was deposited under number 054 at the Aluísio Bittencourt herbarium, located at the Universidade Estadual do Maranhão. The project was recorded (A7777F8) in the Sistema Nacional de Gestão do Patrimônio Genético e do Conhecimento Tradicional Associado (SisGen).

MuLL was isolated following the protocol established by Napoleão et al. (2011). Briefly, a protein extract was obtained by homogenizing M. urundeuva leaf powder in 0.15 M NaCl to a proportion of 10% (w/v) for 16 h at 25 °C, followed by centrifugation at 9000×g for 15 min. Thereafter, the extract was treated with ammonium sulfate and the precipitated fraction obtained during the 60–80% saturation step was applied to a chitin (Sigma-Aldrich, MO, USA) column (7.5 × 1.5 cm) equilibrated with 0.15 M NaCl at a flow rate of 0.3 mL/min. After washing with equilibration solution, the lectin was eluted with 1.0 M acetic acid and dialyzed against distilled water. The protein concentration was determined according to Lowry et al. (1951) by employing a standard curve of bovine serum albumin (0.031–0.5 mg/mL). The ability of MuLL to bind carbohydrates was evaluated using a hemagglutinating activity assay according to Paiva and Coelho (1992). This required a suspension (2.5%, v/v) of rabbit erythrocytes (fixed with glutaraldehyde) in 0.15 M NaCl, which was obtained as approved by the Ethics Committee on Animal Use of the Universidade Federal de Pernambuco (23076.033782/2015-70). The hemagglutinating activity was quantified as the reciprocal of the highest sample dilution that promoted agglutination. The specific hemagglutinating activity was defined as the ratio between hemagglutinating activity and protein concentration (mg/mL).

Evaluation of MuLL binding to carbohydrates

A hemagglutinating activity inhibition assay was used to evaluate the possible interaction between MuLL carbohydrate-binding sites and mono- and disaccharides. In this assay, MuLL (50 µL) was incubated with a solution (0.4 M) containing d-arabinose, d-cellobiose, d-fructose, l-fucose, lactose, d-mannose, d-methyl-glycopyranoside, d-methyl-mannopyranoside, N-acetyl-d-glucosamine, d-ribose, sucrose, or d-xylose (Sigma Aldrich, St. Louis, MO, USA) for 15 min, and then an erythrocyte suspension was added.

Identification of the isoelectric point (pI) of MuLL

Two-dimensional electrophoresis was performed to identify the isoelectric point (pI) of MuLL. The lectin (250 μg) was solubilized in a rehydration buffer (GE Healthcare Life Sciences) containing IPG buffer (1%, v/v, pH 3–10). This solution was used to rehydrate the strip (7 cm, linear pH gradient 3–10) for 24 h at 28 °C. Isoelectric focusing was performed in a Ettan IPGPhor III system (GE Healthcare Life Sciences) at 25 °C. Next, the strip was submitted to SDS-PAGE (Laemmli, 1970). Staining was performed with Coomassie Brilliant Blue R-250 at 0.02% (w/v) in 10% (v/v) acetic acid. After discoloration, the analysis was performed using ImageMaster software (GE Healthcare Life Sciences).

Ovicidal assay

The ovicidal assay was performed according to Santos et al. (2012) using A. aegypti eggs (Rockefeller strain) stored at 27 ± 2 °C for a maximum of 3 months at the Laboratório de Ecologia Química at the Universidade Federal de Pernambuco. Intact eggs were selected by examining filter papers pieces that females used as oviposition support using a Leica KL300 stereomicroscope (Leica Microsystems, Wetzlar, Germany). MuLL was diluted in filtered tap water (pH 6.8) to produce 20 mL test solutions at 0.5, 0.6, 0.8, 1.0, and 1.4 mg/mL. The filter papers were cut in pieces containing 50 eggs and, in each assay, 1 piece was immersed in the solution. Controls contained distilled water plus filtered tap water. Two independent experiments were conducted in triplicate, and the number of hatched larvae was recorded after 72 h of incubation. The concentration of MuLL required to reduce the hatching rate by 50% (EC50), and the 95% confidence intervals were calculated using probit analysis in the program MedCalc (MedCalc Software bvba, Ostend, Belgium).

Scanning electron microscopy (SEM)

Changes in the surface of A. aegypti eggs were investigated using SEM. The eggs (50 per assay) were exposed to 0.88 mg/mL of MuLL for 24, 48, and 72 h. At the end of each time period, the eggs were washed in 0.1 M cacodylate buffer pH 7.2 and fixed for 30 min at 28 °C in 2.5% glutaraldehyde, 4% paraformaldehyde, and 5 mM CaCl2 in cacodylate buffer. After washing again with the same buffer, the eggs were allowed to adhere to poly-l-lysine coated slides and were fixed for 1 h with 1% osmium tetroxide/0.8% potassium ferricyanide/5 mM CaCl2 in 0.1 M cacodylate buffer pH 7.2. Next, they were dehydrated in graduated ethanol, submitted to CO2 critical point drying, covered with a 20-nm-thick gold layer, and observed using a JEOL JSM-5600 LV scanning electron microscope (JEOL, Peabody, MA, USA). Eggs from the control assay were also observed.

Conjugation of MuLL with fluorescein isothiocyanate (FITC)

A 5.0 mg/mL stock solution of FITC (Sigma Aldrich) in dimethyl sulfoxide (DMSO) was prepared according to the manufacturer’s instructions. MuLL (5.0 mg/mL) was dialyzed overnight against an inhibition buffer (0.1 M bicarbonate/carbonate pH 9.0, containing 0.4 M N-acetyl-D-glucosamine). Next, the dialyzed lectin (20 mg) was mixed with the FITC solution (1.0 mL) and 0.1 M sodium bicarbonate/carbonate buffer pH 9.0 (5.0 mL). After mixing the solutions, the reaction vessel was covered with aluminum foil and constantly stirred in a dark environment for 2 h at 28 °C. The reaction mixture was then loaded onto a Sephadex G-25 (GE Healthcare Life Sciences, Sweden) column (20 × 1.9 cm) previously equilibrated (0.5 mL/min) with phosphate-buffered saline (PBS) to separate MuLL-FITC conjugates from unconjugated molecules. The molar ratio between fluorescence and protein (F/P) was calculated as the ratio between the absorbances at 495 and 280 nm. These values are expected to range from 5 to 10 (Lyerla and Hierholzer, 1975). The conjugate MuLL-FITC was dialyzed against distilled water to remove the carbohydrates and then evaluated for hemagglutinating activity as described above.

Fluorescence microscopy

The eggs were incubated with MuLL-FITC at 0.88 mg/mL for 24, 48, and 72 h. After the incubation period, the eggs were washed with distilled water to remove unbound conjugates and placed in 5% sodium hypochlorite solution for chorion dissolution, allowing the embryo to be visualized inside the egg (Mortenson 1950). The eggs were monitored under a stereomicroscope and rinsed with distilled water to avoid complete dissolution by sodium hypochlorite. Next, slides containing the eggs were observed using a fluorescence microscope DMI4000B (Leica, Wetzlar, Germany). Samples were excited using a 480/40 nm bandpass filter (BP), and fluorescence was detected using a 527/30 nm BP filter. Control samples (eggs exposed to water) were also evaluated. Several images for each treatment were acquired using the same acquisition parameters.

Results

MuLL was isolated using chitin column chromatography, as established by Napoleão et al. (2011), showing a specific hemagglutinating activity (HA) level of 256. A single 14-kDa polypeptide band was observed on polyacrylamide gel electrophoresis under denaturing conditions (data not shown), similar to reported by Napoleão et al. (2011), confirming the homogeneity of MuLL. Table 1 shows that the monosaccharides N-acetyl glucosamine and methyl-mannopyranoside were the most efficient in inhibiting the hemagglutinating activity of MuLL. At the same time, fructose, fucose, lactose, and mannose were poorly recognized by lectin. Isoelectric focusing revealed that the pI of MuLL is ca. 9.7 (Fig. 1).

Table 1.

Evaluation of the inhibitory effect of carbohydrates on the hemagglutinating activity (HA) of MuLL. HA of MuLL in the absence of carbohydrates is 256

Sample HA
Arabinose 64
Cellobiose 32
Fructose 128
Fucose 128
Lactose 128
Mannose 128
Methyl-mannopyranoside 8
Methyl-glucopyranoside 16
N-acetyl-d-glucosamine 4
Ribose 16
Sucrose 64
Xylose 16
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Fig. 1.

Fig. 1

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Isoelectric focusing of Myracrodruon urundeuva leaf lectin. An isoelectric point of ca. 9.7 was identified

MuLL was able to reduce the hatching rate of the eggs in a dose-dependent way, thus indicating ovicidal activity (Table 2). The EC50 value was 0.880 [0.779–0.995] mg/mL. The images obtained by SEM showed that in the control samples, the eggs had a standard shape and characteristic surface, a well-defined exochorionic network, and preserved morphology of the external exochorionic cells, with central and peripheral tubercles at all time periods evaluated (24, 48, and 72 h) (Fig. 2A–C). The exochorionic network and tubercles were absent in some regions of eggs treated with MuLL (EC50) for 24 and 48 h (Fig. 2D, E), making it possible to observe the surface of the endochorium. Eggs exposed to lectin for 72 h showed severe deformation and degeneration of the eggshell constituents (Fig. 2F). These data indicate that MuLL was able to destroy the external egg structures (exochorionic network, central and peripheral tubercles).

Table 2.

Number of unhatched eggs of A. aegypti after incubation with Myracrodruon urundeuva leaf lectin (MuLL)

Assay Unhatched eggs
Number Percentage

Control

MuLL

 0.0 0.0
 0.5 mg/mL 2.4 ± 1.0 4.8 ± 2.0
 0.6 mg/mL 5.0 ± 1.5 10.0 ± 3.1
 0.8 mg/mL 22.8 ± 2.9 45.7 ± 5.8
 1.0 mg/mL 35.7 ± 3.2 71.5 ± 6.4
 1.4 mg/mL 100.0 100.0
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The values correspond to the mean ± standard deviation of two independent experiments carried out in triplicate

Fig. 2.

Fig. 2

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Representative scanning electron microscopy images of Aedes aegypti eggs from control samples (A–C) or samples incubated with MuLL at 0.880 mg/mL (D–F) for 24 (A, D), 48 (B, E), and 72 (C, F) hours. Control eggs showed standard shape and characteristic surface, with a preserved exochorionic network (EN) and exochorionic cells with central (CT) and peripheral (PT) tubercles. EN, CT and PT were not observed in some eggs treated with MuLL for 24 and 48 h. Exposure of eggs to this lectin for 72 h led to severe deformation and degeneration of eggshell constituents

MuLL-FITC showed the same level of hemagglutinating activity (256) as unconjugated lectin, indicating that the presence of the fluorescent compound did not block or affect the carbohydrate-binding site. MuLL-FITC was chromatographed on a Sephadex G-25, and the chromatographic profile showed a single overlapping peak (Fig. 3). This confirmed that MuLL was efficiently conjugated to FITC (molar ratio 9.1), since the protein and fluorescent compound were detected in the same fractions.

Fig. 3.

Fig. 3

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Sephadex G-25 chromatography of the MuLL-FITC conjugates. Fractions were monitored for absorbance at 280 nm (black circles) and 495 nm (gray squares)

Fluorescence microscopy of untreated eggs revealed low autofluorescence in the embryo’s serosal cuticle and head (Fig. 4A–C). After incubation for 24, 48, and 72 h with MuLL-FITC, the fluorescence intensity in these regions increased, indicating the accumulation of lectin. After 24 h, the increase in fluorescence intensity was detected only in the embryo's head (Fig. 4D). After 48 h, the increase in fluorescence intensity was detected in both the head and the upper portion of the digestive tract (Fig. 4E). After 72 h, the increase in fluorescence intensity was detected in the head, serosal cuticle, and throughout the digestive tract (Fig. 4F).

Fig. 4.

Fig. 4

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Fluorescence microscopy of Aedes aegypti eggs after removal of the chorion with sodium hypochlorite. Control eggs (A–C) and eggs incubated with MuLL-FITC (D–F) were observed after 24 (A, D), 48 (B, E), and 72 h (C, F). The head (H) and gut (G) regions are indicated in the images. The white arrows indicate regions with higher intensity of fluorescence in eggs incubated with MuLL-FITC and include the embryo head, gut region, and serosal cuticle (SC)

Discussion

Careless use of synthetic insecticides to control A. aegypti has resulted in the emergence of insects resistant to these insecticides (Zara et al. 2016). Reports on larvicidal and ovicidal activities of plant compounds have demonstrated their potential as alternative insecticides (Sá et al. 2009a, b; Santos et al. 2012; Napoleão et al. 2012; Alves et al. 2020; Sarma et al. 2020; Souza et al. 2020). Myracrodruon urundeuva lectins were reported to be effective against immature forms of A. aegypti. MuBL, MuHL, and MuLL are effective larvicidal agents against this mosquito (LC50 of 0.125, 0.04, and 0.202 mg/mL, respectively) and MuBL and MuHL showed ovicidal activity (EC50 of 0.26 mg/mL and 0.80 mg/mL, respectively) (Sá et al. 2009a, b; Napoleão et al. 2012; Alves et al. 2020). In the present study, leaf lectin (MuLL) also reduces the hatching rate of A. aegypti eggs and highlights its potential as an ovicidal agent in ovitraps. The ovicidal activity of MuLL was similar to that of MuHL and lower than that of MuBL.

The pI found for MuLL in this study is consistent with the results reported by PAGE for native proteins (Napoleão et al. 2011). This means that this protein contains more positively charged side chain amino acids. Identifying the isoelectric point of proteins is essential in defining their biotechnological use since their solubility is higher at pH values ​​above or below the pI (Hristova and Zhivkov 2019). The isoelectric point of MuLL gives this protein adequate solubility in water, where the biological cycle of A. aegypti occurs.

The hemagglutinating activity of MuLL confirms that the carbohydrate-binding sites of the lectin molecules in the preparation used in this study were suitable to bind glycans at cell surfaces (Silva et al. 2019a). MuLL is known to bind chitin (N-acetyl-d-glucosamine polymer) and the glycoproteins azocasein, casein, fetuin, ovalbumin, and thyroglobulin; however, it does not have the hemagglutinating activity inhibited by N-acetyl-d-glucosamine at 0.2 M (Napoleão et al. 2011). Here, we evaluated the carbohydrate-binding ability of MuLL using another set of monosaccharides and disaccharides as well as N-acetyl-d-glucosamine at a higher concentration (0.4 M). At this concentration, the ability of MuLL to interact with this monosaccharide was revealed. MuLL can bind both N-acetyl-d-glucosamine and its polymer. Chitin-binding lectins have been reported as insecticidal agents due to their ability to interfere with the digestion and absorption of nutrients, penetrating the hemolymph, and causing systemic effects after crossing the epithelial barrier, as well as being able to interact with glycosylated structures, including the peritrophic matrix (composed by chiton, glycoproteins, and proteoglycans) in the midgut (Napoleão et al. 2011, 2013, 2019).

The ovicidal activity of MuLL could be attributed to its ability to bind to chitin in the exochorionic cells and serosal cuticle of A. aegypti eggs (Noh et al. 2020), resulting in the damage observed using SEM. Fluorescence microscopy revealed that MuLL penetrated the eggs, reaching the serosal cuticle and the entire digestive tract of the embryos; thus, the ovicidal activity of MuLL may also be the result of damage to the embryo. The integrity of the digestive tract is essential for the embryo’s nutrition process, while the serosal cuticle protects it from damage caused by mechanical impacts, temperature changes, and water flow (Farnesi et al. 2015; Noh et al. 2020). If the serosal cuticle falls apart, the egg may become permeable and even break, although this was not observed within 72 h of incubation with MuLL. Chitin is the main component of the insect cuticle and is found in the exo- and endocuticle. It is always associated with proteins that determine the mechanical properties of the cuticle. The cuticle contains repeats of hydrophobic residues that seem to be associated with cuticle stiffness and waterproofing (Andersen 1979).

Alves et al. (2020) showed that the other chitin-binding lectins from M. urundeuva (MuBL and MuHL) also penetrated A. aegypti eggs. However, unlike MuLL, they were not detected in the serosal cuticle. These authors also reported that both MuBL and MuHL formed lumps corresponding to aggregates of lectin molecules that adhered to the egg surface, but MuHL formed a denser network of aggregates than MuBL. In addition, no disruption of the egg surface was visible in eggs treated with MuBL and MuHL; therefore, the disruption described here as a result of treatment with MuLL may be attributed to its ability to reach the serosal cuticle. Thus, MuBL, MuHL, and MuLL may differ in their mechanisms of action on A. aegypti eggs and embryos.

The results obtained here encourage the assessment of the possibility that MuLL may present ovicidal activity against other species of mosquitoes, such as from Culex and Anopheles genera which, unlike A. aegypti, even lay their eggs on the surface of water surface, where the lectin will be dissolved. On the other hand, MuLL may not have the same effect on eggs of these mosquitoes, as they have a lower amount of chitin on the egg surface (Farnesi et al. 2015). Thus, future studies in this sense are promising for the construction of a panel of the effects of lectins on mosquito eggs.

The determination of the larvicidal and ovicidal activities of MuLL against A. aegypti stimulates new evaluations related to the application of this lectin in the field for the control of this mosquito populations. A future step will be to verify the toxicity of this lectin to non-target insects and other animal species, to ensure whether or not MuLL poses an environmental risk. This is crucial because there are some reports on the toxicity of some lectins to non-target species. For example, Konrad et al. (2008) reported that transgenic plants expressing the Galanthus nivalis lectin can cause harmful effects on Osmia bicornis (lone bee) larvae if they are exposed to elevated levels of this protein. In another study, the M. oleifera seed lectin (WSMoL) was shown to be toxic to Danio rerio larvae (zebra fish), which makes its use in natural water bodies not recommendable (Silva et al. 2017). On the other hand, a larvicidal lectin from Annona muricata seeds against A. aegypti exhibited low toxicity in the non-target mosquito Chironomus costatus (Parthiban et al. 2020).

Conclusion

The study identified MuLL as a novel ovicidal agent against A. aegypti, capable of promoting damage to the egg surface, interacting with the embryo’s gut structures, and binding to the serosal cuticle. The results of this study suggest future evaluation of the use of ovicidal and larvicidal MuLL in ovitraps aiming to block the mosquito life cycle by reducing egg viability and killing any larvae that may hatch.

Acknowledgements

We would like to express their gratitude to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the research grants and fellowships (PECF, AF, THN, and PMGP) and financial support (407192/2018-2). We are also grateful to the Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE; APQ-0108-2.08/14) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES; Finance Code 001) for financial support. R.R.V. Alves would like to thank FACEPE (IBPG-1081-2.08/15) for graduate scholarship. We thank Carlos Eduardo Sales da Silva for technical assistance. The National Institute of Photonics (INFo) is also recognized.

Declarations

Conflict of interest

The authors declare no conflicts of interest.

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