Evaluation of Endogenous and Symbiotic Cellulase Activities in Subterranean Termites: Their Role in Biomass Conversion and Termite Control Strategies

Abstract

Lower subterranean termites primarily feed on wood and wood products due to their dual cellulolytic systems comprising endogenous and symbiotic origin. The distribution of total cellulase, endoglucanase, exoglucanase, β-glucosidase, and xylanase activities were mapped out throughout the gut of termite species Heterotermes indicola and Reticulitermes flavipes using specific substrates. Native SDS-PAGE confirmed the cellulase activity distribution patterns by exhibiting the relative protein bands distribution of specific size throughout the termite gut. Seven partial genes encoding the two endogenous and five symbiotic cellulases belonging to different glucohydrolase families GHFs 1, 7, 9, 42, 45, 11 and 7 in H. indicola and R. flavipes. These genes encode proteins that significantly share similarity with previously reported cellulase genes including endoglucanase, β-glucosidase, and exoglucanases belonging to different GHFs. Quantitative (qRT-PCR) revealed that β-glucosidase and endoglucanase genes which encode GHF1 and GHF9 cellulases significantly expressed higher in salivary glands as compared to other gut parts respectively, whereas exoglucanases (GHF7) and other cellulases (GHF 42, 45, 11 and 7-5) expressed only in hindgut region where cellulolytic protozoa reside. Cellulase genes’ expression positively correlated with respective specific types of cellulase activities and t-test insignificant differences between H. indicola and R. flavipes. In second objective, protein-level in vitro and in vivo saccharification potential to biofuel producing feedstocks sugarcane bagasse, pinewood, cottonwood (Populus spp.), corn stover, rice husk by the guts of H. indicola and R. flavipes. The focus of this objective was (i) to measure the saccharification potential of feedstocks with respect to control diet (filter paper) by termites’ gut, and to determine impacts of feedstock on foraging activities and survival behavior. Sugarcane bagasse, pinewood, cottonwood, and corn stovers were saccharified at significantly high levels as compared to the control diet and rice husk. The gut protein extract from both termite species saccharified all lignocellulose material regardless of feedstock loading. However, R. flavipes showed significantly (p<0.05) high saccharification potential than H. indicola. Consumption of the sugarcane bagasse, pinewood, and corn stover was maximum by R. flavipes, whereas H. indicola exposed cottonwood and sugarcane feedstocks as the most palatable food source with 100% survivability. Results supported the “diet adaptation” hypothesis (Karl and Scharf, 2015) by indicating β-glucosidase, exoglucanase, and xylanase activities significantly highest for sugarcane 26 bagasse, pinewood, and cottonwood as compared to rice husk and control diets in both termite species. These results are important from the perspective of physiological changes in termites that lead to gut microbial environments changes and 100% survivability on exclusive sugarcane bagasse, pinewood, and cottonwood feedstocks. In the third objective, a cellulolytic bacterial strain was isolated from the gut of building infesting termite species H. indicola and R. flavipes for the production of ionic-solvent tolerant cellulase. After screening by congo red, strain HI-08 was identified as Bacillus licheniformis using 16S rDNA sequence analysis. It was accounted for maximum cellulase production on 2% sugarcane bagasse (1156 U/mL). The crude cellulase was purified by (NH4)2SO4 precipitation, affinity and size exclusion chromatography, with 12.02 purification fold and 32.33% overall recovery. Approximately 55 kDa molecular weight of gel-purified cellulase was revealed by SDS PAGE analysis. The optimum temperature and pH for maximum cellulase activity were determined as 45°C and 7.0, respectively. Kinetic analysis of purified cellulase showed the Km and Vmax be 2.24 mg/mL and 454.05 µg/mL/min, respectively. Statistical optimization using RSM indicated that sugarcane bagasse was the most useful agricultural waste for cellulase production, which proved its candidature in industrial bio ventures. In the fourth objective of this project, dry lab and wet lab approaches were conducted to cut off the lignocellulose activities of termites by targeting termite cellulases including endoglucanase, exoglucanase, and β-glucosidase using ecofriendly carbohydrate-based cellulase inhibitors. Before conducting wet laboratory bioassays, in dry lab studies 3D structure of three main cellulases endoglucanase, exoglucanase, and β-glucosidase from R. flavipes were modeled using Modeler 9.24v and validated via Structure Analysis and Verification Server (SEVES) comprising on the package of different programs such as PROCHECK, Verify3D, ERRAT, and Ramachandran plot. Interactive binding of carbohydrate-based cellulase inhibitors (cellobiomidazole, fluoromethyl cellobiose, fluoromethyl glucose, glucose, and cellobiose) with enzymes was assessed by GOLD scoring. The results of in silco studies revealed cellobiomidazole, cellobiose, and fluoromethyl cellobiose showed higher promising binding affinities with exoglucanase and β-glucosidase as compared to endoglucanase based on GOLD docking scores. These results were supported by wet-lab studies, in which the efficacy of these inhibitors was investigated by conducting biochemical and feeding bioassays. Cellobiomidazole, cellobiose, and 27 fluoromethyl cellobiose significantly (p < 0.05) inhibited the activities of exoglucanase and β glucosidase during in vitro assays. Feeding bioassays revealed significant effects of cellobiomidazole, cellobiose, and fluoromethyl cellobiose inhibitors reduced the gene expression levels, termite foraging activity, protozoal communities resulting in termite mortality. Results indicated the efficacy of Cellobiomidazole, Cellobiose, and Fluoromethyl cellobiose as termite control agents and can be applied in field conditions using baiting technology. In the last objective of the present study, silver oak (Grevillea robusta Cunn.) (Proteaceae) leaf extract was investigated for its termiticidal properties against the termite H. indicola in the laboratory tests. An ethanol-solvent system was used to remove the extract from dry leaf powder via the Soxhlet apparatus. A dose-dependent foraging response and mortality were observed after continuous exposure to a dose range of 1 to 20 mg/mL of extract based on dry extract weight. Results indicated that maximum (> 90%) termite mortality occurred at 20 mg/mL along with a significant reduction in termite tunneling activity. The LC50 was calculated at 5.47 mg/mL using the concentration series data. In filter paper antifeeding and repellency bioassays, a significant reduction in the total population of gut protozoa compared to untreated and negative controls (solvent) was observed. After 2 weeks of no-choice continuous feeding on filter paper treated at 20 mg/mL, the hindgut protozoan population was reduced by 62.90%. The extract induced resistance against termite feeding activities to the non-durable cottonwood and pinewood after vacuum pressure treatment. At the highest concentration tested, 100% mortality was recorded after exposing termites to cottonwood and pinewood treated with silver oak leaf extract. HPLC-DAD ESI-MS results of the extract showed a high level of phenolic compound, alkyl-resorcinol. Results indicated that silver oak leaf extract has termiticidal properties and could be of value in the development of botanical insecticides for termite control in wood products