Biodiversity of Entozoic Fauna of Odontotermes obesus and Heterotermes indicola with the Screening of Phytochemicals as a Potential Source of Termite Control

Abstract

The subterranean termites, Odontotermes obesus and Heterotermes indicola are highly infesting wood pests causing an economic loss of billions of dollars throughout the world, especially in the tropical and subtropical regions. The O. obesus is a higher termite and harbors bacteria as symbionts in the hindgut which aids in wood digestion along with its cellulases secreted by gut wall and provides a mechanism for recycling of nitrates, acetates, etc. whereas H. indicola is a lower termite which harbors entozoic flagellates along with bacteria. Both of these termite genera are commonly found throughout Pakistan especially in the temperature moderate areas and cause serious damage to woods, wooden materials, and forest. There are certain woods and trees which are not attacked by termites as they have certain chemicals which are either termite repellent or toxic. The present study is focused to isolate and identify such chemical/s which could be a potential source for preparing termiticides either targeting directly to insect host or via its gut symbionts. For this purpose two termite species „O. obesus‟ and „H. indicola‟ were collected from the vicinity of Quaid-i-Azam University Islamabad, Pakistan and identified based on 36 morphological characters i.e. length and width of the body, thorax, abdomen, head, mandibles, pronotum, postmentum, antenna (scape, pedicle, flagellum) and legs. Protozoan fauna residing in the hindgut of “H. indicola” was also identified morphologically based on length and width of the body, the diameter of the nucleus, length of flagella, length and width of the bulge, length of axostyle and centroblepharoplast. Resultant values of both (termites and protozoa) were analyzed using "Student t-test” to investigate significant differences among various parameters using Minitab version 18. The largest and most abundant protozoan species, Pseudotrichonympha grassii was further confirmed by molecular analysis. Bacterial fauna isolated from the hindgut of O. obesus and H. indicola was characterized based on morphology, Gram staining, biochemical identification, amplification and sequencing of 16S rRNA gene. For the evaluation of plants having significant mortality effects on termites and their gut symbionts, eight plants were selected including Carica papaya, Eucalyptus camaldulensis, Osmium basilicum, Grevillea robusta, Eucalyptus globulus, Pongamia pinnata, Mentha longifolia, and Melia azedarach for screening as anti-termite chemicals. Crude leaves extracts of plants were prepared in ethanol by Soxhlet extraction apparatus and evaluated for their toxicity against termites and their gut endosymbionts (protozoa and bacteria) at subsequent concentrations i.e. 100, 500, 1000, 1500 and 2000ppm to find LC50 in no-choice bioassay for termiticidal and protozocidal activity and agar well diffusion method for antibacterial activity. Leaves extract of the most effective plant was fractionated through column and thin layer chromatography in different solvents (n-hexane, ethyl acetate, and methanol). Twenty-one fractions were collected and applied for termiticidal, protozocidal and antibacterial activities at the concentration range of 100-2000ppm. The effective plant and their active fractions were further characterized by preliminary phytochemical tests, Ultraviolet-visible spectroscopy (UV-Vis), Fourier transforms infrared spectroscopy (FTIR) and Gas chromatography coupled mass spectroscopy (GC-MS). Morphometric measurements of termites showed that majority of external characters of soldier cast were not significantly different from each other. However, slight differences were observed in the measurements of different parts of flagellum (pedicle, scape) and legs (trochanter, tarsus, tibia, and claw) which represent the adaptive traits that help an insect to adapt themselves to the changing environmental conditions. Nine species of flagellates belonging to three genera i.e. Holomastigotes, Holomastigotoides, and Pseudotrichonympha were identified. These include Holomastigotes metchnikowi, Holomastigotes campanula and Holomastigotes annandalei, Holomastigotoides kempi, Holomastigotoides hemigynum, Holomastigotoides Koidzumi, Holomastigotoides hartmanni, Holomastigotoides metchnikowi, and Pseudotrichonympha grassii. The largest and the most abundant species P. grassii was further identified by amplifying SSU rRNA which indicated that Pakistani isolate was phylogenetically most closely related to the Japanese isolate. Bacterial fauna isolated from termite hindgut was also characterized by morphological, biochemical and molecular analysis that confirmed the presence of five bacterial genera from each termite species. The species Bacillus cereus, Escherichia coli, Lysinibacillus fusiformis were common to both termites where as Lysinibacillus xylanilyticus and Lysinibacillus macrolides were present only in O. obesus while Bacillus subtilis and Shigella sonnei in H. indicola only. Ethanolic leaves extract of all plant species exhibited termiticidal activities but G. robusta showed maximum mortality of 94% (LC50=1391.01ppm) and 90% (LC50=1607.95ppm) against O. obesus and H. indicola respectively. This extract also caused 95% mortality (LC50=502.64ppm) of hindgut flagellates of H. indicola. Similarly, G. robusta extracts also showed antibacterial activity against all the isolated bacteria from both termite species however maximum zone of inhibition appears against L. fusiformis was shown isolate O. obesus (27.50±1.02mm) and H. indicola (24.54±1.32mm). Column and thin layer chromatography were used for sepration of G. robusta leaves extract and separated 21 fractions (named as 2-22). Among all the 21 isolated fractions; 4,5,7,10 and 14 were found to be lethal for termites either directly or via symbionts (protozoa and bacteria) however fraction „5‟ was common having termiticidal, protozocidal and antibacterial potentials. LC50 of fraction 5 against O. obesus was 1342.81ppm, for H. indicola 1420.05ppm and for protozoa was 578.34ppm. Similarly fractions 5 was most active against bacteria of both termite species. The minimum inhibitory concentration of fraction 5 against the bacterial isolate of O. obesus was 100, 500 and 100ppm against isolates 1, 3 and 5 while 100ppm against the first 4 isolates and 500ppm against isolate 5 of H. indicola. Being the most effective, ethanolic extract of G. robusta was characterized by UV-Vis spectroscopy, FTIR and GC-MS analysis. The UV-Vis spectroscopic analysis was performed at a wavelength of 200 to 800nm and two absorption peaks were recorded at 208.94 and 282.97nm having absorption of 2.33 and 0.811 respectively. The FTIR analysis was performed for the identification of different functional groups based on characteristic peak values and the spectrum was recorded from 400 to 4000cm-1. Results confirmed the presence of amines, alcohols, alkanes, nitriles, aldehydes, ether and halo compounds. The GC-MS analysis revealed the presence of 15 phytochemicals with a high percentage of coumarin (30.64%), 7-hydroxy, methyl hexofuranoside (27.63%) and 9,12,15-octadecatrienoic acid (Z, Z, Z) (8.97%). Phytochemical screening of lethal fractions also showed presence of flavonoids, tannins, sterols, quinones, alkaloids, terpenoids and carbohydrates. The UV-Vis spectroscopic analysis revealed different absorption peaks with a common peak of all fractions at 665.50nm having different absorbance. The FTIR analysis of bioactive fractions (4,5,7,10 and 14) revealed the presence of alkanes, alkynes, aldehydes, amines, ether, amino acids, thiols, cyanides, nitriles, alcohols, carboxylic acids, thiocyanates, thiols, and azides. The GC-MS analysis confirmed the presence of some common chemical compounds including 3,7,11,15-tetramethyl-2-hexadecen-1-ol, 1,4-benzenedicarboxylic acid, bis(2- methylpropyl) ester, beta-sitosterol, 1,2-benzenedicarboxylic acid, diisodectyl ester, phytol, 1,2-benzenedicarboxylic acid, diisooctyl, hexadecanoic acid methyl ester, n hexadecanoic acid, octadecanoic acid, and p-arbutin. However, the compound 3,7,11,15- tetramethyl-2-hexadecane-1-ol were commonly found in all the active fractions along with other identified compounds. These natural phytochemicals having termiticidal, protozocidal and antibacterial potential being eco-friendly, can be used in termiticides preparation to control termite infestation and save a huge economic loss.