Symbiotic Cellulolytic Bacteria from the Gut of the Subterranean Termite Psammotermes Hypostoma Desneux and Their Role in Cellulose Digestion Huda R
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Ali et al. AMB Expr (2019) 9:111 https://doi.org/10.1186/s13568-019-0830-5 ORIGINAL ARTICLE Open Access Symbiotic cellulolytic bacteria from the gut of the subterranean termite Psammotermes hypostoma Desneux and their role in cellulose digestion Huda R. K. Ali1*, Nada F. Hemeda2 and Yasser F. Abdelaliem3 Abstract The subterranean termite Psammotermes hypostoma Desneux is considered as an important pest that could cause severe damage to buildings, furniture, silos of grain and crops or any material containing cellulose. This species of termites is widespread in Egypt and Africa. The lower termite’s ability to digest cellulose depends on the association of symbiotic organisms gut that digest cellulose (fagellates and bacteria). In this study, 33 diferent bacterial isolates were obtained from the gut of the termite P. hypostoma which were collected using cellulose traps. Strains were grown on carboxymethylcellulose (CMC) as a sole source of carbon. Cellulolytic strains were isolated in two diferent cellulose medium (mineral salt medium containing carboxymethylcellulose as the sole carbon source and agar cel- lulose medium). Five isolates showed signifcant cellulolytic activity identifed by a Congo red assay which gives clear zone. Based on biochemical tests and sequencing of 16s rRNA genes these isolates were identifed as Paenibacillus lactis, Lysinibacillus macrolides, Stenotrophomonas maltophilia, Lysinibacillus fusiformis and Bacillus cereus, that depos- ited in GenBank with accession numbers MG991563, MG991564, MG991565, MG991566 and MG991567, respectively. Keywords: Termite, Psammotermes hypostoma, Symbiosis, Cellulose degrading bacteria, 16S rRNA gene, Phylogenetic analysis Introduction economic damage than food combined with fre (Eggle- Termites are social insects occurring in tropical, subtropical, ton 2011). Predominantly by feeding on structural tim- and temperate regions of the world. Presently, about 2800 bers they can use dead wood making them a major pest species are known. Termites are regarded as harmful insects for timber used for construction purposes, both inside because of their ability to destroy all materials contain- buildings and outdoors (Nobre and Nunes 2007). ing cellulose. Several species of wood-feeding termites are In the USA, the presence of the destructive subterra- known for causing serious economic damage. In the United nean termite Coptotermes formosanus species can cost States, the costs for damage repair and termite control may a multimillion-dollar termite control industry, therefore reach up to 5 billion dollars per year (NPMA 2006). the tolerance threshold for termites is approaching zero Te dangers of termites are known to most of peo- in countries with a high relative standard of living (Su ple. Troughout the tropical regions everyone knows and Schefrahn 1998). Eight species of subterranean ter- that termites are voracious eaters of houses, living trees mites were detected in Egypt. Te subterranean termite, and crops or any material contains cellulose, they can P. hypostoma Desneux is the most serious pest, causing cause severe damage. In the USA termites cause more damage to any materials containing cellulose, the annul control and repairing of damage costs millions of pounds, essentially in upper Egypt and the new valley (Ahmed *Correspondence: [email protected] 1 Plant Protection Research Institute, A. R. C, Giza, Egypt et al. 2014). Depending on the termiticide sales in 2010, Full list of author information is available at the end of the article the world wide annually controls and repair cost was © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Ali et al. AMB Expr (2019) 9:111 Page 2 of 9 evaluated and data displayed that the global economic species of termite. Tese bacterial isolates can be used in impact of termite pests has increased to $40 billion. Sub- the efective and novel control of subterranean termite in terranean termites accounted for ≈ 80% of the costs ($35 the future research to minimize pesticides usage. Since billion) (Rust and Su 2012). most previous studies are studying termite control using Termites can be divided into two categories: higher pesticides. termites (Termitidae) and lower (all families except Ter- Tis study aims to isolate and identify cellulolytic bac- mitidae) based on absence or presence of fagellate in teria from the gut of P. hypostoma and to confrm their their hindgut, respectively. Lower termites have many role in cellulose degradation, because the ability to dis- types of bacteria in addition to protozoa, while higher rupt this function has potential use for termite control. termites usually have only the bacteria and a more elab- orate anatomy while lacking the protozoa (Varma et al. Materials and methods 1994; Eggleton 2011). It is also known that the higher ter- Termite collection and identifcation mites degrade cellulose by using their own enzyme which Te Subterranean termite was collected using cellulose were secreted by their gut and salivary glands (Breznak traps according to the method of El-Sebay (1991) which and Brune 1994; Ohkuma 2003; Ramin et al. 2008). Most were located in Al-Haraga village, Fayoum Governorate, termite gut fagellates are associated with bacterial sym- Egypt on February 2017. Te termite samples were then bionts. Te bacteria can be found connected with the transported immediately to the microbiology lab and plasma membrane of the fagellates (ectosymbionts) washed with tap water, to get rid of any adherent dirt. and/or located in the cytoplasm or the nucleus of their Te termite was identifed based on its morphologi- hosts (endosymbionts). Te symbiosis between the fagel- cal characteristic at Department of Termite and Wood lates and bacteria seems to be highly specifc (Stingl et al. Borers, Plant Protection Research Institute Agricultural 2005; Ohkuma 2008). Microbial communities in hindgut Research Center, Giza, Egypt. are associated with wood digestion in termite and play an essential role in supplementing this nutrient-poor food Isolation of cellulase producing bacteria source. Symbionts stimulate reactions involved in the One hundred termite workers were surface sterilized breakdown of all three major ingredients of wood (cel- with 70% ethanol then washed with sterile distilled water. lulose, hemicellulose, and lignin phenolics) and supple- After cutting their heads using a syringe needle, the ment this diet by synthesizing other essential nutrients entire guts were removed and macerated on sterilized (Husseneder 2010; Peterson and Scharf 2016). sand. Cellulase producing bacteria were isolated accord- Several strains of cellulolytic bacteria were isolated from ing to the method of Gupta et al. (2012), the guts samples the termite. Ramin et al. (2008) isolated three intestinal were inoculated in broth mineral salt medium (NaNO3 bacteria from the hind gut of the subterranean termite 2.5 g; KH2PO4 2 g; MgSO4 0.2 g; NaCl 0.2 g; CaCl2·6H2O Coptotermes curvignathus, identifed them as (Enterobac- 0.1 g in a liter) containing 0.5% carboxymethylcellulose ter aerogenes, E. cloacae and Clavibacter agropyri) and (CMC) as a sole carbon source, the pH of the medium is demonstrated their roles in cellulose degradation. Eight adjusted to 7.0. Te inoculated fasks were incubated for bacterial and fve fungal cellulose degrading isolates were 2 weeks at 37 °C. Subsequently, subculture was repeated isolated from the termite gut, the isolated organisms were using same broth mineral salt medium (MSM) and using identifed as Bacillus sp., Cellulomonas sp., Enterobacter the same conditions. sp., and Aspergillus sp. Te Congo red screening assay for To select the cellulolytic bacteria and estimate the abil- cellulase production showed the widest zone of hydrolysis ity of the bacteria to digest cellulose, 1 mL of the culture (38 mm) for Aspergillus sp. (Sharma et al. 2015). was spread on cellulose agar plates medium composed of Sreena et al. (2015) isolated fve efective cellulose K2HPO4 (0.2 g/L), KH2PO4 (0.2 g/L), MgSO4 (0.2 g/L), degrading bacteria from the hind gut of the termites NaCl (0.2 g/L), NaNO 3 (1 g/L), CaCO 3 (0.01 g/L), yeast genus Heterotermes and Odontotermes, three of them extract (0.5 g/L), CMC (10 g/L), Agar (15 g/L), pH 7.0 belonged to Bacillus and one each to Staphylococcus and and incubated for 48 h at 37 °C. All bacterial isolates were Enterobacter sp. purifed by re-streaking on CMC agar plates. Te most Tere are 2800 termite species, but few have been promising cellulase producing isolates were selected for examined to determine their gut fora. Few cellulolytic- further investigation and characterization. Isolates were bacteria were isolated and identifed from certain termite stored at − 80 °C in sterilized glycerol (20%). species because of the difculty in isolating and cultivat- ing a large number of gut microorganisms. Screening for cellulolytic bacteria To the best of our knowledge this is the frst study on Te pure colonies from each isolates were grown in bacteria that have mutualistic relationships with this 50 mL medium containing (g/L) 0.2 K2HPO4, 0.01 Ali et al. AMB Expr (2019) 9:111 Page 3 of 9 MgSO4, 1 NaCl, 0.5 NaNO3, 0.05% yeast extract and and forward primer: 5′-AAG GAG GTG ATC CAG CCG 10 g CMC according to Sharma et al. (2015). Cultures CA-3′) according to Cheng et al. (2010). Te PCR was were incubated at 37 °C for 72 h. At the end of incuba- performed in the thermal cycle 2720 (Applied Biosys- tion 1 mL of each culture suspension was centrifuged at tem, USA) in a total volume of 25 μL containing 1 μL of 5000 rpm for 10 min and the pellets were re-suspended each primer, 3 μL of template DNA (50 ng/μL), 12.5 μL in fresh medium.