Termite cellulases: potential target for screening of natural termiticidesAbstract
Among the methods tested for termite eradication, termiticides derived from plants are particularly noteworthy. These termiticides represent an effective strategy for insect management, causing minimal environmental harm and posing less risk to human health than widely used chemical controls. The present study aims to obtain crude protein extracts from termites and to characterize their cellulolytic activities for their use in the bioprospecting of natural termiticides. Adult individuals were collected in wood fragments, identified, selected, and sectioned to obtain two protein extracts: one sample contains the entire exoskeleton. The other contains only the abdominal portion of the termites. The extract obtained exclusively from the termite abdomen (ABE) presented the highest concentration of proteins (670 mg/mL) compared to the crude extract of whole termites (EWT) (495 mg/mL) and the extract of the fungus Trichoderma viride isolated from termites (TvE) (397 mg/mL). All extracts showed enzymatic activities of total cellulase, endoglucanase, and exoglucanase. However, TvE showed the highest activities for all kinds of enzymes. Although the cellulolytic activities of the crude termite extracts were relatively low, they were measurable and stable. This stability permits their use in screening potential natural termiticides.
Key words
Total cellulase; Endoglucanase; Exoglucanase; Termites; Natural termiticides
INTRODUCTION
Termites are insects widely found in nature, particularly in tropical regions. They are classified in the taxonomy in a single order, the Termitoidae (Isoptera). Approximately 3,500 species of termites have been described (Engel 2011), and more than 300 species are considered urban and agricultural pests (Guan et al. 2011). They play a crucial ecological role as primary consumers and decomposers in natural ecosystems. However, their way of feeding, composed of cellulosic materials, causes several economic losses throughout the national territory (Freyman et al. 2010). In São Paulo, an estimated $10 to $20 million is spent annually on repairing termite-damaged materials. The species Coptotermes gestroi, among the subterranean termites in urban areas, is noted for its exceptionally high destructive capacity (Fontes & Milano 2002, Bernardini 2005). In the rural environment, Cornitermes sp. is responsible for mechanical damage to machinery used in agriculture, as well as rendering part of the soil surface unusable due to its ability to injure parts of plants such as roots, stems, and leaves, and may or may not lead to a loss in production (Constantino 2002).
Chemical control through synthetic insecticides is commonly used to minimize the effects caused by these insects. However, due to their high toxicity and chemical stability, incorrect use can lead to tissue bioaccumulation in living organisms and their persistence in the environment. Additionally, the widespread use of these insecticides has led to increased resistance in pest populations, further complicating control efforts (Braga & Valle 2007). At the same time, the use of pesticides in insect management presents itself as a public health problem due to the intoxication rates caused by chemical control. Brazil has been pointed as a major consumer of these products since 2008 (INCA 2022). According to the National Cancer Institute (INCA) and the International Labor Organization (ILO), 70,000 cases of intoxication, which can potentially lead to death, are identified each year. In addition, the inappropriate use of these chemical agents extends to the ecosystems in which they are inserted, as it results in an imbalance in local biodiversity, requiring new management alternatives so that these problems are minimized (Chagnon et al. 2015, Lopes & Albuquerque 2018).
Natural termiticides derived from secondary metabolites with potential bioactive molecular patterns stand out among the methods tested for termite elimination. These plant-based termiticides offer an effective strategy for insect management, causing minimal harm to the environment and human health compared to the chemical controls currently in use. Cellulases are enzymes that play a crucial role in the digestion of cellulosic materials, releasing glucose, an important energy source for termites and other living beings. In this sense, cellulases have been used in industrial biotechnological processes and research as promising targets for substances with termiticide potential (McCann & Carpita 2008, Singh & Sharma 2020).
In this context, the pharmaceutical industry focuses on discovering new drugs and has made substances of natural origin the target of new research (O’Neill & Lewis 1993). Thus, screening new natural-origin pesticides has been the focus of several research efforts. Characterizing the enzymatic activity of cellulases present in termites is crucial for fully understanding the digestion mechanisms and their potential applications in biotechnology. This study aimed to obtain protein extracts from termites, determine the cellulase activities within these extracts, and compare them with the extract from Trichoderma viride, a symbiotic fungus of termites, to use them as a tool in the screening of natural termiticides.
MATERIALS AND METHODS
Obtaining and maintenance of termites
The colony of adult individuals was collected from wood fragments of an infested residential property in Pampulha (-19.8376422; -43.9919717), an urban area of Belo Horizonte, Minas Gerais, Brazil. After collection, the individuals were preserved in 80% alcohol (v/v) and stored in a freezer at -20°C.
Taxonomic identification of termites
Taxonomic identification was performed through the morphological differentiation of soldiers and workers in a stereoscopic microscope (Medilux®) with an 80x magnification, using illustrative identification keys, according to Constantino (1999, 2002).
Preparation of termite protein extracts
Termites were decapitated and dissected using thin needles attached to syringes. Some specimens were separated into head/thorax and abdomen to create two distinct extracts: Complete Exoskeleton Extract (CEE) and Abdomen Extract (ABE). The CEE extract was prepared by crushing 45g of whole termites in liquid nitrogen and resuspending them in 450 ml of 0.1 M sodium acetate buffer, pH 5.5. The ABE extract was prepared by crushing and resuspending 5g of abdomens in 50 ml of the same 0.1 M sodium acetate buffer, pH 5.5. Both preparations were homogenized using a vortex, centrifuged at 14,000 RPM for 15 minutes at 4°C, and the supernatants were collected and filtered, producing the crude extracts named Complete Exoskeleton Enzyme Extract (CEE) and Abdomen Enzyme Extract (ABE). The extracts were aliquoted in fractions of 1.5 mL, stored in a freezer at -20°, and used as a source of cellulases in subsequent enzymatic assays.
A third protein extract was obtained from the fungus Trichoderma viride, isolated from termites, and subjected to chromatographic procedures to enrich enzymatic activity. The Laboratory of Biochemistry and Molecular Biology (Instituto de Ciências Exatas e Biológicas/Universidade Federal de Ouro Preto) kindly provided this extract, which was used as a control for comparing the results.
Determination of total protein concentration
The protein concentration in the enzymatic extracts from CEE, ABE, and TvE was assessed using the Lowry colorimetric method, employing Folin-Ciocalteau reagent and Bovine Serum Albumin (BSA) as a standard (Lowry et al. 1951).
Enzymatic assays
All enzymatic assays were conducted in triplicate using sodium acetate buffer (50 mM, pH 4.8) at 50°C, and the mean values were calculated. The relative standard deviations of the measurements were found to be below 5%.
Total reducing sugars
FPase and endoglucanase activities followed previously established standard conditions (Ghose & Bisaria 1987). For the FPase assay, 500 μL of appropriately diluted enzyme solution was added to 1000 μL of sodium citrate buffer and 50 mg of Whatman #1 filter paper (1x 6 cm). The reaction mixture was then incubated at 50°C for 60 minutes, then adding 10% (v/v) trichloroacetic acid (TCA) to halt hydrolysis.
For the endoglucanase assay, 500 μL of appropriately diluted enzyme solution was mixed with 500 μL of 2% (w/v) carboxymethyl cellulose (CMC) in sodium citrate buffer and incubated at 50°C for 30 minutes. The reaction was terminated using a boiling bath. Negative controls were prepared by substituting 500 μL of enzymatic extract with a buffer solution.
The total reducing sugars released during the enzymatic assays were quantified using the Glucose Oxidase Method from the Bioclin® Assay Kit, with glucose as the standard. For this, 30 μl of the final reaction mixture from the enzymatic assays, including controls, were combined with 300 µl of the kit’s enzymatic reagent and incubated at 37°C for 10 minutes in a water bath. Subsequently, the assays were subjected to reading at 490 nm using an ELISA Reader (Molecular Devices). One unit of enzymatic activity (U) was defined as the amount of enzyme that released 1 μmol of the corresponding product per minute under the assay conditions employed.
ρNp assays
Exoglucanase activity was measured using ρNPC as substrates. Under the assay conditions employed, one unit of enzymatic activity was defined as the amount of enzyme that released 1μmol of pNP per minute.
RESULTS AND DISCUSSION
Identification and selection of termites
Termites belonging to the worker caste were identified and isolated for sample preparation, as seen in Figure 1.
Selection of termites for the preparation of enzyme extracts. Left Panel - Green surrounded worker individuals; Red surrounded soldier individuals; Below: whole population with miscellaneous individuals. Right Panel - sub populations of workers and soldiers in larger augment.
Only the termites of the worker caste were selected since they are considered reservoirs of cellulases due to their primary consumer role of cellulose in the colony (Zhou et al. 2008). These wood-feeding termites are the main cellulose digesters in natural biomass ecosystems, able to degrade lignocellulose with their unique enzymatic complexes (Talia & Arneodo 2018).
Determination of protein concentration and enzymatic activities in extracts
Characterizing the crude extracts of termites is documented in the literature as a crucial method for assessing enzymatic activity. Cellulases play a direct role in catalyzing the hydrolysis of cellulose into glucose during the digestion process of this polymer within termite organisms.
The cellulases in the digestive system of termites include endo-β-1,4-glucanases (EC 3.2.1.4), which hydrolyze cellulose chains randomly in a non-processive manner. Exoglucanases, such as cellobiohydrolases (EC 3.2.1.74) or cellodextrinases (EC 3.2.1.91), depolymerize cellulose chains from either the reducing or non-reducing ends processively or sequentially. Finally, β-glucosidases (EC 3.2.1.21) break down cellulose oligosaccharides, mainly cellulose, to release glucose (Ni & Tokuda 2013).
The results obtained from the quantification assays of total proteins and the enzymatic activities of total cellulases, endoglucanase, and exoglucanase are summarized in Table I below and indicate the presence of important cellulases in extracts from termites and the symbiotic fungus T. viride.
Protein concentration and activities of cellulases (U/mL) of the enzymatic extracts analyzed. FPase: total cellulase activity (Filter paper unit per milliliter).
Upon observation, it is evident that all cellulolytic activities measured in the enzymatic extracts of termites (Coptotermes sp.) obtained in this study—Complete Exoskeleton Enzyme Extract (CEE) and Abdomen Enzyme Extract (ABE)—were notably lower compared to the T. viride enzyme extract (TVE). The total cellulolytic activity was approximately five times and eight times lower. In comparison, the endo-β-1,4-glucanase activity was lower by about 23 times and 14 times, and the exoglucanase activity was lower by approximately 30 times and 22 times for CEE and ABE, respectively.
The concentration of total proteins was also determined and used to calculate the specific activity of each enzyme in each of the extracts. This parameter considers the degree of enrichment of activities as a function of the total protein content in the extract. Based on this parameter, it is evident that the TVE extract exhibits higher specific activity for all cellulases. Among the extracts from termite, CEE demonstrated higher specific activity for total cellulase activity and similar specific activity for exoglucanase and endoglucanase compared to ABE. These facts indicate the importance of subsequent processes of protein purification as a way to improve the catalytic efficiency of the enzymes present in each extract. Hence, it is imperative to underscore the significance of employing protein purification techniques on crude termite extracts and thoroughly characterizing their biochemical properties. This approach can significantly enhance the potential of utilizing these enzymes as a biotechnological tool.
It is known that termite biotechnology falls into two categories: a) biotechnology directed to termites for pest management purposes and b) biotechnology modeled on termites for use in various industrial applications. The initial category encompasses various mechanisms of action of potential termiticide candidates, including interference RNA, augmentation of antimicrobial pathogens, and inhibition of digestive enzymes such as cellulases. The second category encompasses termite digestomes (Scharf & Tartar 2008), which offer promising resources for host lignocelluloses, symbionts, and other enzymes with diverse industrial applications, primarily concentrated on biomass processing.
However, in the future, one of the most critical approaches to accelerate advances in both categories of termite biotechnology will be to consider the host and symbiote together as a single functional unit (Scharf 2015). Specific methods, such as the one elucidated by Zhou et al. (2008), are highlighted as pivotal tools for identifying compounds that inhibit cellulase enzymes within the termite digestive system. These enzymes are essential molecules for cellulose digestion in these organisms. Characterizing the cellulolytic activity of enzymes from termites allows the elaboration of enzymatic inhibition studies with substances that target cellulases. Molecular modeling, for example, allows the synthesis of new molecules in a rational way and can also be applied to molecules of natural origin to optimize their relationship structure activity and improve the interaction of the substance with its target receptor (Barreiro & Rodrigues 1997).
CONCLUSIONS
Pursuing natural termiticides has emerged as a pivotal strategy in identifying prototype substances to substitute the heavily utilized chemical management methods. This pursuit is driven by the myriad repercussions of untrained agricultural workers conducting management practices that compromise their health and cause environmental damage.
While the cellulolytic activities observed in the crude extracts analyzed are diminished compared to the purified extract from termite commensal fungus, they are nevertheless quantifiable and enable their utilization in studies and screening for potential natural termiticides. This tool facilitates the exploration of active metabolites to achieve more targeted control measures that inflict minimal environmental harm.
ACKNOWLEDGMENTS
Authors want to thank the Conselho Nacional de Desenvolvimento Tecnológico e Científico (CNPq) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the financial support.
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Publication Dates
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Publication in this collection
17 Mar 2025 -
Date of issue
2025
History
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Received
17 July 2024 -
Accepted
26 Nov 2024
