Cohnella is a highly cellulolytic bacterial genus, which can be found in a variety of habitats. The aim of this study was to assess its presence in the digestive tract of termite species collected in North-eastern Argentina: Nasutitermes aquilinus, N. corniger and Cortaritermes fulviceps. Gut homogenates were incubated with cellulosic substrate for bacterial growth. Bacterial 16S rDNA was partially amplified using new primers for Cohnella spp. and cloned. Sequences obtained showed highest similarity (97.2–99.9%) with those of Cohnella spp. previously reported from diverse environments. Phylogenetic analysis tended to group the clones according to their host species and sampling sites. These results indicate the association of Cohnella-related intestinal symbionts with three common Neotropical termites. Their potential industrial application encourages further research.
Cohnella es un género de bacterias celulolíticas que puede ser encontrado en una variedad de hábitats. El propósito de este estudio fue registrar su presencia en el tracto digestivo de termitas (Nasutitermes aquilinus, N. corniger y Cortaritermes fulviceps) colectadas en el noreste argentino (NEA). Se incubaron homogenados de intestinos en sustrato celulósico para multiplicar las bacterias. Utilizando nuevos cebadores para Cohnella spp., se amplificó una porción del ADN ribosomal 16S bacteriano, el cual fue posteriormente clonado. Las secuencias obtenidas mostraron su mayor porcentaje de similitud (97,2-99,9%) con Cohnella spp., previamente reportadas en diversos ambientes. El análisis filogenético tendió a agrupar a los clones de acuerdo a la especie hospedante y al sitio de muestreo. Estos resultados indican que especies de termitas frecuentes en el NEA albergan simbiontes intestinales relacionados con el género Cohnella. Las potenciales aplicaciones industriales de estos microorganismos animan a profundizar los estudios.
Termites depend on both endogenous (host) and exogenous (symbiont) enzymes to accomplish lignocellulose digestion. In higher termites, exogenous cellulolytic activity is mainly due to a variety of bacterial endosymbionts hosted in the guts3,14. Such diversity is thought to be influenced in part by the taxonomic position of the termite species, its diet and surrounding environment3. Throughout the last decades, there has been an increasing interest in this insect group as a valuable source of novel cellulolytic enzymes for the biofuel industry. South America produces large amounts of biomass from agricultural waste, and Neotropical termites could presumably provide useful glycosyl hydrolases for bioethanol production1,11. Much work is still to be done in this field. A previous research dealing with termite-symbiotic bacteria grown on cellulose-rich media indicated the presence of the highly cellulolytic genus Cohnella6 in the digestive tract of Nasutitermes aquilinus (Holmgren, 1910) specimens collected in North-eastern (NE) Argentina2. The ability of different Cohnella strains to degrade the plant cell wall2,7,10, makes these ubiquitous bacteria particularly interesting in view of their biotechnological potential. Thus, the aim of this study was to further investigate the occurrence of intestinal putative Cohnella spp. in different populations of N. aquilinus and two additional Nasutitermitinae species: N. corniger (Motschulsky, 1855) and Cortaritermes fulviceps (Silvestri, 1901). These species are often found in anthropized environments of NE Argentina and neighboring countries, and exploit dissimilar nutritional resources: wood for Nasutitermes spp. and various substrates (comprising gramineous plants and wood) for C. fulviceps.
Collection sites were located in urban landscapes (Corrientes city, Corrientes, Argentina) separated by 1.4 (min)–6.5 (max) km. Samples of arboreal- (N. aquilinus and N. corniger) and epigeal- (C. fulviceps) termite nests were taken to the laboratory in November 2015, and worker specimens were selected for processing. Three populations of N. aquilinus (Boca Unidos, Jardín and Laprida districts), two of C. fulviceps (Jardín and Campus) and one of N. corniger (Regional) were surveyed for the presence of Cohnella-related symbionts. Insects were surface-sterilized with 70% alcohol, entire guts were dissected under binocular microscope and grounded in distillate water. Homogenates corresponding to every species and collection site (10 guts/sample for the two Nasutitermes spp. and 15 guts/sample in the case of the smaller C. fulviceps individuals) were added to 40ml of minimal medium (MM) containing pretreated sugarcane bagasse (SCB)2 as cellulosic substrate. Cultures were incubated on a rotary shaker (37°C, 250rpm). Six days after, bacterial growth was clearly visible. No bacterial proliferation was observed in the negative controls included in the experiment (MM+SCB, no gut homogenate added). After substrate sedimentation at 200×g for 5min, bacterial cultures were transferred to polypropylene microtubes and stored at −20°C for posterior processing. Bacterial pellets were obtained by centrifugation at 10000×g for 10min and resuspended in CTAB extraction buffer. DNA was extracted as mentioned elsewhere2. Both the quantity and quality of the purified bacterial DNA were estimated by spectrophotometry and agarose-gel electrophoresis.
Based on sequences available in GenBank, the following primers were designed to amplify an 846-bp-long fragment of 16S rDNA from Cohnella spp.: Cohnella Fw (5′-TAAGTCTGGTGTHTAAGTGCGG-3′) and Cohnella Rv (5′-CGRCTTCGGGTGTTGTAAACTC-3′). PCR conditions were set at 95°C for 5min; 35 cycles of 95°C for 1min, 57°C for 30s, 72°C for 1min; and 72°C for 2min. A highly purified Taq polymerase free of bacterial DNA (INBIO, Tandil, Argentina) was used. Under this restrictive annealing temperature, PCR amplifications yielded a single band of the expected size in every sample tested. Amplicons from each bacterial culture were cloned using the pGEM-T vector (Promega Corp., Madison, USA). Thirty-nine clones corresponding to N. aquilinus (13 from Boca Unidos, 14 from Jardín and 12 from Laprida), 34 to C. fulviceps (16 from Jardín and 18 from Campus) and 15 to N. corniger (from Regional) were sequenced in an ABI PRISM 3500 XL genetic analyzer (Applied Biosystems, USA) at the Instituto de Biotecnología – INTA (Hurlingham, Argentina). Obtained sequences were screened for chimeras using DECIPHER15 and compared to those in public databases. All sequences were deposited at GenBank with accession numbers KY230519 to KY230606. As presumed, BLAST search revealed highest sequence similarity with previously reported Cohnella spp., with identity values ranging from 97.2 to 99.9%. These data proved the efficacy of the primer set and evidenced the occurrence of Cohnella-related bacteria in all three termite species studied. A phylogenetic tree was constructed with the sequenced clones by MEGA512 using the maximum likelihood method with 1000 bootstrap replicates. Bacterial sequences from GenBank that showed maximum identity percentages by BLAST were also incorporated in the analysis. The Firmicute Clostridium frigoris served as outgroup. In general, the investigated 16S rDNA sequences tended to group together according to the sample, i.e. host species and collection site (Fig. 1). A few exceptions were observed within Nasutitermes spp.: some clusters were formed by bacterial sequences obtained from distinct samples (different districts and/or host species). Concerning the 16S rDNA clones from C. fulviceps, sequences were grouped by district origin in more homogeneous clusters.
Phylogenetic tree inferred by the maximum likelihood method based on the partial bacterial 16S rDNA sequences obtained in this work. Host species, collection site and clone identification are given at the branch tips. Reference and previously reported sequences sharing highest nucleotide identity are highlighted by a black circle. All sequences are identified by their corresponding accession numbers. Branches with <50% Bootstrap support are collapsed (values indicated at the nodes).
Since its separation from the genus Paenibacillus6, several species have been characterized within the genus Cohnella, most of them with cellulolytic or xylanolytic activities2,7,10. Members of Cohnella were isolated from a variety of environments, including industrial samples, plant root nodules, soil, water and animal feces6,7,10. With respect to Nasutitermitinae, metagenomic analyses regarding the microbial diversity of N. corniger revealed the regular presence of Firmicutes, though at comparatively low abundance compared to other bacterial phyla8,9,14. None of the latter studies referred to Paenibacillus or Cohnella spp. More recently, however, a Paenibacillus sp. has been reported in Nasutitermes arborum by 16S rRNA gene pyrosequencing5, and Paenibacillus nasutitermitis was described as an intestinal symbiont of Nasutitermes sp13. In a preceding study restricted to a single N. aquilinus nest, Cohnella was shown to form part of the cellulolytic microbial community associated to this wood-eating termite2. The present paper supports this finding and demonstrates that such situation is a common feature in different N. aquilinus colonies. Moreover, 16S rDNA sequences affiliated with Cohnella spp. were consistently determined in the taxonomically close N. corniger and C. fulviceps. In all cases, DNA amplification and sequencing was made after bacterial multiplication in cellulose medium. When PCR was directly performed on total gut DNA extractions, only a few samples gave positive reactions (not shown). This could be due to their relatively low abundance in the termite guts, counteracted by elevated growth rates in enrichment media. In this sense, a recent survey carried out on a wood-feeding lower termite showed that the largest amount of culturable cellulolytic bacteria belonged to the genera Bacillus and Paenibacillus4. In digestive symbioses, different symbiont transmission routes have been postulated to play a concomitant role in shaping the intestinal microbiota3. Social exchanges (including stomodeal trophallaxis) have been proposed as a chief way of gut-specific bacterial colonization in higher termites. In addition, the possibility of direct acquisition of facultative symbionts from the environment cannot be excluded3,5. Whether culturable Cohnella-related bacteria associated to Nasutitermitinae also exist as free-living organisms in nature is to be elucidated.
Conflict of interestThe authors declare that they have no conflicts of interest.
This work was funded by Agencia Nacional de Promoción Científica y Tecnológica through Grant PICT-2013-1454, Instituto Nacional de Tecnología Agropecuaria, Secretaría General de Ciencia y Técnica – Universidad Nacional del Nordeste (Argentina), Bilateral Project MinCyT-DST SA/13/10 (Argentina/South Africa), the National Research Foundation UID 91990 and North-West University (South Africa). Authors are indebted to Facundo Felice (IB-INTA) for Technical Assistance.
