Napavarn Noparatnaraporn

Intra- and Interspecific Comparisons of Bacterial Diversity and Community Structure Support Coevolution of Gut Microbiota and Termite Host

ABSTRACT We investigated the bacterial gut microbiota from 32 colonies of wood-feeding termites, comprising four Microcerotermes species (Termitidae) and four Reticulitermes species (Rhinotermitidae), using terminal restriction fragment length polymorphism analysis and clonal analysis of 16S rRNA. The obtained molecular community profiles were compared statistically between individuals, colonies, locations, and species of termites. Both analyses revealed that the bacterial community structure was remarkably similar within each termite genus, with small but significant differences between sampling sites and/or termite species. In contrast, considerable differences were found between the two termite genera. Only one bacterial phylotype (defined with 97% sequence identity) was shared between the two termite genera, while 18% and 50% of the phylotypes were shared between two congeneric species in the genera Microcerotermes and Reticulitermes, respectively. Nevertheless, a phylogenetic analysis of 228 phylotypes from Microcerotermes spp. and 367 phylotypes from Reticulitermes spp. with other termite gut clones available in public databases demonstrated the monophyly of many phylotypes from distantly related termites. The monophyletic “termite clusters” comprised of phylotypes from more than one termite species were distributed among 15 bacterial phyla, including the novel candidate phyla TG2 and TG3. These termite clusters accounted for 95% of the 960 clones analyzed in this study. Moreover, the clusters in 12 phyla comprised phylotypes from more than one termite (sub)family, accounting for 75% of the analyzed clones. Our results suggest that the majority of gut bacteria are not allochthonous but are specific symbionts that have coevolved with termites and that their community structure is basically consistent within a genus of termites.

Intracolony variation of bacterial gut microbiota among castes and ages in the fungus‐growing termite Macrotermes gilvus

The fungus‐growing termites Macrotermes cultivate the obligate ectosymbiontic fungi, Termitomyces. While their relationship has been extesively studied, little is known about the gut bacterial symbionts, which also presumably play a crucial role for the nutrition of the termite host. In this study, we investigated the bacterial gut microbiota in two colonies of Macrotermes gilvus, and compared the diversity and community structure of bacteria among nine termite morphotypes, differing in caste and/or age, using terminal restriction fragment length polymorphism (T‐RFLP) and clonal analysis of 16S rRNA. The obtained molecular community profiles clustered by termite morphotype rather than by colony, and the clustering pattern was clearly more related to a difference in age than to caste. Thus, we suggest that the bacterial gut microbiota change in relation to the food of the termite, which comprises fallen leaves and the fungus nodules of Termitomyces in young workers, and leaves degraded by the fungi, in old workers. Despite these intracolony variations in bacterial gut microbiota, their T‐RFLP profiles formed a distinct cluster against those of the fungus garden, adjacent soil and guts of sympatric wood‐feeding termites, implying a consistency and uniqueness of gut microbiota in M. gilvus. Since many bacterial phylotypes from M. gilvus formed monophyletic clusters with those from distantly related termite species, we suggest that gut bacteria have co‐evolved with the termite host and form a microbiota specific to a termite taxonomic and/or feeding group, and furthermore, to caste and age within a termite species.

Phylogenetic diversity, localization, and cell morphologies of members of the candidate phylum TG3 and a subphylum in the phylum Fibrobacteres, recently discovered bacterial groups dominant in termite guts.

ABSTRACT Recently we discovered two novel, deeply branching lineages in the domain Bacteria from termite guts by PCR-based analyses of 16S rRNA (Y. Hongoh, P. Deevong, T. Inoue, S. Moriya, S. Trakulnaleamsai, M. Ohkuma, C. Vongkaluang, N. Noparatnaraporn, and T. Kudo, Appl. Environ. Microbiol. 71:6590-6599, 2005). Here, we report on the specific detection of these bacteria, the candidate phylum TG3 (Termite Group 3) and a subphylum in the phylum Fibrobacteres, by fluorescence in situ hybridization in the guts of the wood-feeding termites Microcerotermes sp. and Nasutitermes takasagoensis. Both bacterial groups were detected almost exclusively from the luminal fluid of the dilated portion in the hindgut. Each accounted for approximately 10% of the total prokaryotic cells, constituting the second-most dominant groups in the whole-gut microbiota. The detected cells of both groups were in undulate or vibroid forms and apparently resembled small spirochetes. The cell sizes were 0.2 to 0.4 by 1.3 to 6.0 μm and 0.2 to 0.3 by 1.3 to 4.9 μm in the TG3 and Fibrobacteres, respectively. Using PCR screenings with specific primers, we found that both groups are distributed among various termites. The obtained clones formed monophyletic clusters that were delineated by the host genus rather than by the geographic distance, implying a robust association between these bacteria and host termites. TG3 clones were also obtained from a cockroach gut, lake sediment, rice paddy soil, and deep-sea sediments. Our results suggest that the TG3 and Fibrobacteres bacteria are autochthonous gut symbionts of various termites and that the TG3 members are also widely distributed among various other environments.

Growth and flocculation of a marine photosynthetic bacterium Rhodovulum sp.

Abstract A marine photosynthetic bacterium (PS88), identified as Rhodovulum sp., with flocculating ability was isolated from the sea sediment mud of a shrimp cultivation farm in Thailand. This bacterium flocculated in glutamate/malate medium during aerobic dark or anaerobic light cultivation. The flocculating ability was enhanced with the increase of NaCl concentration to 6% (w/v). When PS88 was grown in glutamate/malate medium containing 3.5% NaCl, protein, RNA and DNA were produced exocellularly and there was flocculation. The yields of DNA, RNA and protein were 8.3, 62.5 and 48.5 mg/g dry cell, respectively. The flocculated cells were deflocculated by treatment with a nucleolytic enzyme such as RNase or DNase, while amylase, protease, trypsin, cellulase and pectinase had no deflocculating effect. These results suggest that the exocellular nucleic acids are active in flocculation.

Comparison of bacterial communities in the alkaline gut segment among various species of higher termites

The first proctodeal (P1) segment in the hindgut of certain higher termites shows high alkalinity. We examined the bacterial diversity of the alkaline P1 gut segments of four species of higher termites by T-RFLP and phylogenetic analyses based on PCR-amplified 16S rRNA genes. The bacterial community of the P1 segment was apparently different from that of the whole gut in each termite. Sequence analysis revealed that Firmicutes (Clostridia and Bacilli) were dominant in the P1 segments of all four termites; however, the phylogenetic compositions varied among the termites. Although some of the P1 segment-derived sequences were related to the sequences previously reported from the alkaline digestive tracts of other insects, most of them formed phylogenetic clusters unique to termites. Such “termite P1 clusters” were distantly related to known bacterial species as well as to sequences reported from alkaline environments in nature. We successfully obtained enrichment cultures of Clostridia- and Bacilli-related bacteria, including putative novel species under anaerobic alkaline conditions from the termite guts. Our results suggest that the alkaline gut region of termites harbors unique bacterial lineages and are expected to be a rich reservoir of novel alkaliphiles yet to be cultivated.

Symbiotic Fungi Produce Laccases Potentially Involved in Phenol Degradation in Fungus Combs of Fungus-Growing Termites in Thailand

ABSTRACT Fungus-growing termites efficiently decompose plant litter through their symbiotic relationship with basidiomycete fungi of the genus Termitomyces. Here, we investigated phenol-oxidizing enzymes in symbiotic fungi and fungus combs (a substrate used to cultivate symbiotic fungi) from termites belonging to the genera Macrotermes, Odontotermes, and Microtermes in Thailand, because these enzymes are potentially involved in the degradation of phenolic compounds during fungus comb aging. Laccase activity was detected in all the fungus combs examined as well as in the culture supernatants of isolated symbiotic fungi. Conversely, no peroxidase activity was detected in any of the fungus combs or the symbiotic fungal cultures. The laccase cDNA fragments were amplified directly from RNA extracted from fungus combs of five termite species and a fungal isolate using degenerate primers targeting conserved copper binding domains of basidiomycete laccases, resulting in a total of 13 putative laccase cDNA sequences being identified. The full-length sequences of the laccase cDNA and the corresponding gene, lcc1-2, were identified from the fungus comb of Macrotermes gilvus and a Termitomyces strain isolated from the same fungus comb, respectively. Partial purification of laccase from the fungus comb showed that the lcc1-2 gene product was a dominant laccase in the fungus comb. These findings indicate that the symbiotic fungus secretes laccase to the fungus comb. In addition to laccase, we report novel genes that showed a significant similarity with fungal laccases, but the gene product lacked laccase activity. Interestingly, these genes were highly expressed in symbiotic fungi of all the termite hosts examined.

Biosorption of cadmium ions using a photosynthetic bacterium, Rhodobacter sphaeroides S and a marine photosynthetic bacterium, Rhodovulum sp. and their biosorption kinetics.

We examined the biosorption characteristics of cadmium ions onto a photosynthetic bacterium, Rhodobacter sphaeroides S and a marine photosynthetic bacterium Rhodovulum sp. PS88 in a batch culture system. Both photosynthetic bacteria are capable of cadmium removal with 30 g/l sodium chloride and divalent cations (Mg2+ and Ca2+) in the culture medium. In particular, the strain PS88 shows a high removal ratio and high specific removal rate of cadmium ions from the culture medium under aerobic-dark (heterotrophic) and anaerobic-light (photoheterotrophic) conditions. The adsorption of cadmium onto strains PS88 and S is dependent on the cadmium concentration, and follows the Freundlich adsorption isotherm. In addition, biosorption isotherms for cadmium show that the strain PS88 exhibits higher values of the empirical constant for the cadmium adsorption capacity, Kr, than that of the strain S under both aerobic-dark (K(f)=17.44) and anaerobic-light (K(f)=1.270) conditions.

Culture of glochidia of the freshwater pearl mussel Hyriopsis myersiana (Lea, 1856) in artificial media

Abstract The freshwater pearl mussel, Hyriopsis myersiana (Limnoscapha) (Lea, 1856) was cultured in two artificial media at 23±2°C. Each artificial medium contained a mixture of M199, (Life Technologies, No. 71N0262) horse serum or fish ( Oreochromis niloticus ) artificial medium plasma as a protein source, and antibiotics/antimycotics at a ratio of 2:1:0.5. Glochidia were reared until they became juveniles, i.e. until the mantle and foot could be observed under a light microscope. The duration of glochidia development until the juvenile stage was 9–10 days in both media. After 1 month of controlled feeding with phytoplankton, the juveniles showed an elongate of shell with several growth lines. The more suitable artificial culture formula for the transformation from glochidia to juvenile stage was the medium containing protein from fish plasma. Survival from glochidia to juvenile stage was up to 85.3±3.9% in fish plasma, while it was equal to 46.2±12.7% in horse serum. The transformation from glochidia to juvenile stage was up to 84.3±2.3% in fish plasma, while it was equal to 44.3±8.9% in horse serum. Percentage survival and transformation from glochidia to juvenile stage were significantly higher in fish plasma than in horse serum ( P

Study of a suitable fish plasma for in vitro culture of glochidia Hyriopsis myersiana

Several organic and inorganic sources from the plasma of different fish species and horse serum were utilized as additives to the artificial culture M199 medium to improve glochidial survival and transformation of Hyriopsis myersiana. After 2–3 days of culturing in the medium containing plasma of Nile tilapia or hybrid catfish, striped catfish or horse serum, the glochidia presented significantly (P<0.05) lower percentage survival compared to medium containing common carp plasma. The highest (93.77±3.0) and lowest (32.42±5.85) percentage survival rates of glochidia were found with common carp and striped catfish plasma, respectively. After 10 days, relevant signs of glochidia transformation, such as the foot and mantle edge, were observed. In all assays, the glochidia transformation reached 100% most probably due to the exchange of the medium at the fifth day and the addition of 1 ml of distilled water at the ninth day of culturing. The intense mobility of juveniles in the medium containing the common carp plasma indicated excellent culture conditions. The ideal density for this plasma corresponded to 150–200 glochidia per culture dish. The present results suggest that M119 medium complemented with the common carp plasma and the medium exchange during culturing period may constitute a functional process to prepare an in vitro culture for freshwater mussels, particularly H. myersiana. The most relevant amino acids for a successful development are CIT, GLX, LEU, PRO, THR and ALA particularly with the contents in the common carp plasma.

Large-scale identification of transcripts expressed in a symbiotic fungus (Termitomyces) during plant biomass degradation

Fungus-growing termites have a symbiotic relationship with the basidiomycetes of the genus Termitomyces. This symbiotic system is able to degrade dead plant material efficiently. We conducted expressed sequence tag (EST) analysis of a symbiotic Termitomyces fungus degrading plant material in a field nest of the termite Macrotermes gilvus. A subtractive cDNA library was also investigated to facilitate the discovery of genes expressed specifically under the symbiotic conditions. A total of 2,613 ESTs were collected and resulted in 1,582 nonredundant tentative consensus sequences, of which approximately 59% showed significant similarity to known protein sequences. A number of homologous sequences to genes involved in plant cell wall degradation were identified and a majority of them encoded putative pectinolytic enzymes. Real-time quantitative reverse transcriptase polymerase chain reaction analyses confirmed significant upregulation of putative stress response genes under symbiotic conditions. The present ESTs database provides a valuable resource for molecular biological study of plant material degradation in the symbiosis between termites and fungi.