Molecular phylogeny of three oxymonad genera: Pyrsonympha,Dinenympha and Oxymonas

Oxymonads are a morphologically well-characterized and highly diverse lineage of protists. They are, however, under sampled at a molecular level. It has recently been demonstrated that a genus of oxymonads, Pyrsonympha, is phylogenetically related to the excavate taxon Trimastix. Here, we addressed issues of internal oxymonad evolution. Pyrsonympha and Dinenympha are shown, by fluorescent in situ hybridization and phylogenetic evidence, to be separate genera and not morphotypes of the same organism. We demonstrated that three genera of oxymonads, Dinenympha, Pyrsonympha, and Oxymonas are each monophyletic and together form a clade which excludes other known eukaryotes. We have presented a taxonomic scheme of oxymonads taking into account their sisterhood with Trimastix and speculated on morphological evolution of oxymonads, particularly of their attachment apparatuses. Our biogeographical analysis with Japanese and Canadian Pyrsonympha and Dinenympha suggests that these genera diverged before the separation of termites that inhabit Eastern Asia and Western North America.     

Symbiotic spirochetes in the termite hindgut: phylogenetic identification of ectosymbiotic spirochetes of oxymonad protists

Some species of protists inhabiting the hindgut of lower-termites have a large number of ectosymbiotic spirochetes on the cell surface. The phylogenetic positions of the ectosymbiotic spirochetes of three oxymonad protists, Dinenympha porteri in the gut of Reticulitermes speratus, and Pyrsonympha sp. and Dinenympha sp. in Hodotermopsis sjoestedti, were investigated without cultivation of these organisms. Protist fractions carefully collected with a micromanipulator were used as templates for the amplification of small subunit ribosomal RNA genes (SSU rDNA). The phylogenetic tree inferred from the nucleotide sequences of the SSU rDNA showed that they were affiliated with the Treponema cluster of spirochetes and they were divided into two clusters. One was grouped together with the spirochetal sequences reported previously from the gut of termites and the other was related to the Treponema bryantii subgroup of treponemes (denoted as termite Treponema clusters I and II, respectively). Whole-cell in situ hybridization using a fluorescent-labeled oligonucleotide probe specific for the group of sequences in cluster II identified most of the ectosymbiotic spirochetes of the oxymonad protists in the gut of R. speratus and H. sjoestedti. However, not all of the ectosymbiotic spirochetes could be detected by means of this cluster II group-specific probe and the population of ectosymbiotic spirochetes of cluster II was different among the oxymonad species. In the case of D. porteri, an oligonucleotide probe specific for one member of cluster II recognized a portion of the ectosymbiotic spirochetes of cluster II, and their population was also different depending on the cell-type of D. porteri in terms of the attachment of ectosymbiotic spirochetes. The results indicate that the spirochetes of cluster II and probably those of a part of cluster I can be assigned to ectosymbiotic species of oxymonad protists and that the population of ectosymbiotic spirochetes associated with a single protist consists of at least three species of phylogenetically distinct spirochetes.     

Repellent and Insecticidal Activity of Naphthoquinones from the Heartwood of <i>Tectona grandis</i> on <i>Incisitermes marginipennis</i> (Latreille)

Wood is a lignocellulosic material that, because of its organic nature, is a source of nutrients for bacteria, fungi, and insects. Incisitermes marginipennis (Latreille) is an interesting dry wood-boring termite. Because it is an extremely destructive pest, difficult to control due to its cryptic lifestyle, and for its role in the biogeochemical cycle of carbon and other elements; it is a fundamental organism. The use of less toxic and eco-friendly substances is important for protecting domestic, cultural, and historical heritage made of wood or lignocellulosic materials. The aim of this study was to evaluate the biological activity of various naphthoquinones extracted from the heartwood of T. grandis on I. marginipennis. First, heartwood flour was used for Soxhlet-type extractions to isolate and characterize the heartwood compounds by thin-layer chromatography. To identify the heartwood compounds, nuclear magnetic resonance and gas chromatography coupled to a mass spectrometer were used. The information obtained was supported using the Interactive Spectroscopy software. The identified heartwood compounds were lapachol, tectoquinone, deoxylapachol, dehydro-α-lapachone, tectol and tecomaquinone-I. Using nymphal termites of I. marginipennis as a biological model, the isolated and identified molecules were under three different tests (repellent, insecticide and protective) and their biological activity was individually evaluated. The results showed that the molecules obtained have a repellent-protective effect against I. marginipennis, although not in all cases, these molecules have high percentages of mortality and decreased the weight loss of the treated material exposed to I. marginipennis.     

Phylogenetic Position and In Situ Identification of Ectosymbiotic Spirochetes on Protists in the Termite Gut

Phylogenetic relationships, diversity, and in situ identification of spirochetes in the gut of the termite Neotermes koshunensis were examined without cultivation, with an emphasis on ectosymbionts attached to flagellated protists. Spirochetes in the gut microbial community investigated so far are related to the genus Treponema and divided into two phylogenetic clusters. In situ hybridizations with a 16S rRNA-targeting consensus oligonucleotide probe for one cluster (known as termite Treponema cluster I) detected both the ectosymbiotic spirochetes on gut protists and the free-swimming spirochetes in the gut fluid of N. koshunensis. The probe for the other cluster (cluster II), which has been identified as ectosymbionts on gut protists of two other termite species, Reticulitermes speratus and Hodotermopsis sjoestedti, failed to detect any spirochete population. The absence of cluster II spirochetes in N. koshunensis was confirmed by intensive 16S ribosomal DNA (rDNA) clone analysis, in which remarkably diverse spirochetes of 45 phylotypes were identified, almost all belonging to cluster I. Ectosymbiotic spirochetes of the three gut protist species Devescovina sp., Stephanonympha sp., and Oxymonas sp. in N. koshunensis were identified by their 16S rDNA and by in situ hybridizations using specific probes. The probes specific for these ectosymbionts did not receive a signal from the free-swimming spirochetes. The ectosymbionts were dispersed in cluster I of the phylogeny, and they formed distinct phylogenetic lineages, suggesting multiple origins of the spirochete attachment. Each single protist cell harbored multiple spirochete species, and some of the spirochetes were common among protist species. The results indicate complex relationships of the ectosymbiotic spirochetes with the gut protists.     

The redox state of the gut of termites

The redox potential of the gut of nine species of termites was investigated by feeding the insects with redox dyes. The fore- and mid-gut of all species was aerobic with an E′_o probably in excess of + 100 mV. whereas the paunch and colon were anaerobic with an E′_o of about ?230 to ?270 mV, except in Coptotermes lacteus and Nasutitermes exitiosus whose colons were at a E′_o of ?50 to ?125 mV. In four species (Incisitermes barretti, Glyptotermes brevicornis, Stolotermes victoriensis, Coptotermes lacteus) the rectum was aerobic (E′_o about +60 mV), whereas the rectum of the other species was anaerobic (E′_o from about ?125 to ?270 mV).     

Enzymes of acetate and glucose metabolism in termites

Extracts of tissues of the lower termites, Reticulitermes flavipes and Coptotermes lacteus, and the higher termite, Nasutitermes exitiosus, possess acetyl-CoA synthetase and all the enzymes of the tricarboxylic acid cycle and are thus able to oxidize acetate to CO₂. The specific activities of these enzymes in R. flavipes are sufficient to cope with the rate of acetogenesis by the gut microbiota. The presence of the malic enzyme and malate dehydrogenase, but not pyruvate carboxylase or phosphoenolpyruvate carboxykinase, indicates that they may be important as anaplerotic enzymes for the conversion of pyruvate to oxalacetate. An apparent absence of pyruvate dehydrogenase in all termites suggests that they do not convert pyruvate to acetyl-CoA, but rather convert acetate (transported from the hindgut) to this compound. All the enzymes of glycolysis were present in termite extracts. Thus any glucose absorbed from the midgut, and originating from hydrolysis of cellulose by salivary or midgut enzymes, can be metabolized by termites as an energy source.     

Lignocellulose degradation by microorganisms from termite hills and termite guts: A survey on the present state of art

Abstract: In several aspects termites are a fascinating group of insects having attracted the interest of many researchers. They exhibit a complex social behavior and caste differentiation occurring elsewhere only among the hymenoptera. In an enlarged part of the hindgut, the paunch, termites have established a unique symbiotic association with prokaryotic and eukaryotic microorganisms. A similar flora is also found in wood-eating roaches of the genus Cryptocercus . The study of symbiosis between termites and their intestinal microbes is of general interest, because due to this symbiotic interaction termites can feed on complex biopolymers such as wood. Flagellates and bacteria occur in the gut of lower termites, while higher termites possess only bacteria. In particular spirochetes are abundant in the termite gut. Apart from spirochetes and other more common bacteria, actinomycetes, yeasts and fungi have also been isolated from different species of termites. This review summarizes the distinct role of the intestinal flora in degradation of wood components such as cellulose, hemicellulose and lignin.     

Uric Acid-Degrading Bacteria in Guts of Termites [Reticulitermes flavipes (Kollar)]

Uricolytic bacteria were present in guts of Reticulitermes flavipes in populations up to 6 × 10⁴ cells per gut. Of 82 strains isolated under strict anaerobic conditions, most were group N Streptococcus sp., Bacteroides termitidis, and Citrobacter sp. All isolates used uric acid (UA) as an energy source anaerobically, but not aerobically, and NH₃ was the major nitrogenous product of uricolysis. However, none of the isolates had an absolute requirement for UA. Utilization of heterocyclic compounds other than UA was limited. Fresh termite gut contents also degraded UA anaerobically, as measured by ¹⁴CO₂ evolution from [2-¹⁴C]UA. The magnitude of anaerobic uricolysis [0.67 pmol of UA catabolized/(gut × h)] was entirely consistent with the population density of uricolytic bacteria in situ. Uricolytic gut bacteria may convert UA in situ to products usable by termites for carbon, nitrogen, energy, or all three. This possibility is consistent with the fact that R. flavipes termites from UA, but they do not void the purine in excreta despite the lack of uricase in their tissues.     

Phylogenetic position and in situ identification of ectosymbiotic spirochetes on protists in the termite gut

Phylogenetic relationships, diversity, and in situ identification of spirochetes in the gut of the termite Neotermes koshunensis were examined without cultivation, with an emphasis on ectosymbionts attached to flagellated protists. Spirochetes in the gut microbial community investigated so far are related to the genus Treponema and divided into two phylogenetic clusters. In situ hybridizations with a 16S rRNA-targeting consensus oligonucleotide probe for one cluster (known as termite Treponema cluster I) detected both the ectosymbiotic spirochetes on gut protists and the free-swimming spirochetes in the gut fluid of N. koshunensis. The probe for the other cluster (cluster II), which has been identified as ectosymbionts on gut protists of two other termite species, Reticulitermes speratus and Hodotermopsis sjoestedti, failed to detect any spirochete population. The absence of cluster II spirochetes in N. koshunensis was confirmed by intensive 16S ribosomal DNA (rDNA) clone analysis, in which remarkably diverse spirochetes of 45 phylotypes were identified, almost all belonging to cluster I. Ectosymbiotic spirochetes of the three gut protist species Devescovina sp., Stephanonympha sp., and Oxymonas sp. in N. koshunensis were identified by their 16S rDNA and by in situ hybridizations using specific probes. The probes specific for these ectosymbionts did not receive a signal from the free-swimming spirochetes. The ectosymbionts were dispersed in cluster I of the phylogeny, and they formed distinct phylogenetic lineages, suggesting multiple origins of the spirochete attachment. Each single protist cell harbored multiple spirochete species, and some of the spirochetes were common among protist species. The results indicate complex relationships of the ectosymbiotic spirochetes with the gut protists.     

Effects of antibiotics on hydrogen production and gut symbionts in the Formosan subterranean termite Coptotermes formosanus (Isoptera: Rhinotermitidae)

Abstract Symbiotic microorganisms that inhabit the gut of Coptotermes formosanus enable this termite to degrade lignocelluloses and further produce hydrogen as an important intermediate to be recycled in its hindgut or as a byproduct to be emitted to the atmosphere. Both symbiotic protists and prokaryotes in the guts of termites demonstrated some different roles with respect to hydrogen production. In this study, the effects of two antibiotics, ampicillin and tetracycline, on hydrogen emission and the gut symbionts of C. formosanus were investigated. Hydrogen emission from termite guts was significantly enhanced when termites fed on wood diets treated with either ampicillin or tetracycline. The greatest H₂ emission rates, 2 519 ± 74 and 2 080 ± 377 nmol/h/g body weight, were recorded with the treatments of ampicillin and tetracycline, respectively, which showed 6–7 times more H₂ production than that of controls. Antibiotic‐treated diets negatively affected the prokaryotic communities and reduced their abundances, particularly on those ectosymbionts inhabiting the gut walls or in the gut fluid of C. formosanus, such as spirochetes. However, no significant reductions in the counts of gut cellulolytic protists, Pseudotrichonympha grassii and Holomastigotoids hartmanni, were recorded; and with a further observation by confocal laser scanning microscopy, the endosymbionts inhabiting P. grassii generally survived the antibiotic treatments. These results suggest that some prokaryotes may serve as the main hydrogen consumers, while P. grassii, together with its endosymbionts, may function as the main contributors for hydrogen production in the hindgut of C. formosanus.     

The Enterobacterium Trabulsiella odontotermitis Presents Novel Adaptations Related to Its Association with Fungus-Growing Termites

Fungus-growing termites rely on symbiotic microorganisms to help break down plant material and to obtain nutrients. Their fungal cultivar, Termitomyces, is the main plant degrader and food source for the termites, while gut bacteria complement Termitomyces in the degradation of foodstuffs, fixation of nitrogen, and metabolism of amino acids and sugars. Due to the community complexity and because these typically anaerobic bacteria can rarely be cultured, little is known about the physiological capabilities of individual bacterial members of the gut communities and their associations with the termite host. The bacterium Trabulsiella odontotermitis is associated with fungus-growing termites, but this genus is generally understudied, with only two described species. Taking diverse approaches, we obtained a solid phylogenetic placement of T. odontotermitis among the Enterobacteriaceae, investigated the physiology and enzymatic profiles of T. odontotermitis isolates, determined the localization of the bacterium in the termite gut, compared draft genomes of two T. odontotermitis isolates to those of their close relatives, and examined the expression of genes relevant to host colonization and putative symbiont functions. Our findings support the hypothesis that T. odontotermitis is a facultative symbiont mainly located in the paunch compartment of the gut, with possible roles in carbohydrate metabolism and aflatoxin degradation, while displaying adaptations to association with the termite host, such as expressing genes for a type VI secretion system which has been demonstrated to assist bacterial competition, colonization, and survival within hosts.     

低等白蚁肠道共生微生物的多样性及其功能

低等白蚁肠道里存在着复杂的微生物区系,包括真核微生物鞭毛虫和原核生物,细菌及古细菌。低等白蚁的后肠以特别膨大的囊形胃及其氢氧浓度的明显梯度分布和丰富的微生物区系为特征,是白蚁进行木质纤维素消化的主要器官。后肠内的鞭毛虫能将纤维素水解并发酵为乙酸,二氧化碳和氢,为白蚁提供营养和能源。系统发育研究表明,低等白蚁肠道共生细菌的主要类群为白蚁菌群1、螺旋体、拟杆菌,低G C mol%含量的革兰氏阳性菌和紫细菌等。而古细菌主要为甲烷短杆菌属的产甲烷菌。共生原核生物与二氧化碳的还原和氮的循环等代谢有关。但肠道共生微生物的具体功能和作用机制还有待进一步的揭示。     

Surface morphology of Saccinobaculus (Oxymonadida): implications for character evolution and function in oxymonads

Examination of surface morphology of the oxymonad genus Saccinobaculus from the gut of the wood-feeding cockroach Cryptocercus punctulatus with scanning and transmission electron microscopy reveals several new characters not observable with light microscopy. These include small concavities covering the external surface, a glycocalyx, coated pinocytotic vesicles, and, in one species, unidentified, membrane-bounded organelles with a granular matrix that may represent peroxisomal or mitochondrial derivatives. Unlike representatives of some other oxymonad families, Saccinobaculus lacks extracellular surface structures, a holdfast, and, generally, ectobiotic bacteria. We examined the evolution of these and other characters in light of previously published phylogenies of oxymonads based on molecular data. The presence of concavities in Saccinobaculus and families Pyrsonymphidae and Oxymonadidae strengthens support for a clade comprising these three families. A glycocalyx appears to be a synapomorphy of all oxymonads, and the presence of ectobiotic bacteria also appears to be ancestral to oxymonads, but lost in Saccinobaculus. A holdfast appears to have arisen multiple times. We hypothesize that concavities may play a role in a two-step mechanism for the accumulation and internalization of specific solutes, and that the highly motile and morphologically plastic nature of Saccinobaculus cells limits the possibility of retaining a covering of ectobiotic bacteria.     

Volatile Fatty Acid Production by the Hindgut Microbiota of Xylophagous Termites

Acetate dominated the extracellular pool of volatile fatty acids (VFAs) in the hindgut fluid of Reticulitermes flavipes, Zootermopsis angusticollis, and Incisitermes schwarzi, where it occurred at concentrations of 57.9 to 80.6 mM and accounted for 94 to 98 mol% of all VFAs. Small amounts of C₃ to C₅ VFAs were also observed. Acetate was also the major VFA in hindgut homogenates of Schedorhinotermes lamanianus, Prorhinotermes simplex, Coptotermes formosanus, and Nasutitermes corniger. Estimates of in situ acetogenesis by the hindgut microbiota of R. flavipes (20.2 to 43.3 nmol · termite⁻¹ · h⁻¹) revealed that this activity could support 77 to 100% of the respiratory requirements of the termite (51.6 to 63.6 nmol of O₂ · termite⁻¹ · h⁻¹). This conclusion was buttressed by the demonstration of acetate in R. flavipes hemolymph (at 9.0 to 11.6 mM), but not in feces, and by the ability of termite tissues to readily oxidize acetate to CO₂. About 85% of the acetate produced by the hindgut microbiota was derived from cellulose C; the remainder was derived from hemicellulose C. Selective removal of major groups of microbes from the hindgut of R. flavipes indicated that protozoa were primarily responsible for acetogenesis but that bacteria also functioned in this capacity. H₂ and CH₄ were evolved by R. flavipes (usually about 0.4 nmol · termite⁻¹ · h⁻¹), but these compounds represented a minor fate of electrons derived from wood dissimilation within R. flavipes. A working model is proposed for symbiotic wood polysaccharide degradation in R. flavipes, and the possible roles of individual gut microbes, including CO₂-reducing acetogenic bacteria, are discussed.     

Variations in Diversity and Richness of Gut Bacterial Communities of Termites (Reticulitermes flavipes) Fed with Grassy and Woody Plant Substrates

Diets shape the animal gut microbiota, although the relationships between diets and the structure of the gut microbial community are not yet well understood. The gut bacterial communities of Reticulitermes flavipes termites fed on four individual plant biomasses with different degrees of recalcitrance to biodegradation were investigated by 16S rRNA pyrosequencing analysis. The termite gut bacterial communities could be differentiated between grassy and woody diets, and among grassy diets (corn stover vs. sorghum). The majority of bacterial taxa were shared across all diets, but each diet significantly enriched some taxa. Interestingly, the diet of corn stover reduced gut bacterial richness and diversity compared to other diets, and this may be related to the lower recalcitrance of this biomass to degradation.     

Phylogenetic diversity and physiology of termite gut spirochetes

The hindgut microbiota of termites includes an abundant andmorphologically diverse population of spirochetes. However,our understanding of these symbionts has remained meager sincetheir first observation in termite guts by Leidy over a centuryago, in part because none had ever been isolated in culture.Recently, this situation has changed dramatically with the applicationof cultivation-independent molecular methods to determine theirphylogeny, and with the isolation of the first pure cultures.The emerging picture is that earth's termites constitute anenormous reservoir of novel spirochetes, which possess metabolicproperties (H₂/CO₂-acetogenesis and N₂ fixation) hitherto unrecognizedin spirochetes and which contribute to the carbon, nitrogenand energy requirements of their termite host. These discoverieshelp to explain the enigmatic dominance of CO₂-reductive acetogenesisover methanogenesis in the hindgut of many termites, as wellas the old observation that elimination of spirochetes fromthe gut results in decreased termite survival.