Role of bacteria in maintaining the redox potential in the hindgut of termites and preventing entry of foreign bacteria

The hindgut of the lower termites, Mastotermes darwiniensis and Coptotermes lacteus and the higher termite Nasutitermes exitiosus were made aerobic by exposure of the termites to pure oxygen, a procedure which killed their spirochaetes and their protozoa (lower termites only). The time taken for the hindgut to become anaerobic after the termites were restored to normal atmospheric conditions ranged from 2 to 4.5 hr. After oxygen treatment the number of gut bacteria increased some six- to ten-fold in all termite species, indicating that the bacteria are poised to use oxygen entering the gut. Removal of all the hindgut microbiota by feeding tetracycline caused the hindgut to become aerobic in M. darwiniensis and N. exitiosus. The transferring of M. darwiniensis to fresh wood, free of antibiotic, resulted in the return of the normal flora and the eventual establishment of anaerobic conditions in the hindgut. Thus the bacteria appear to be important in maintaining anaerobic conditions in the gut. Attempts to determine whether the protozoa (in the lower termites) played any part in maintaining the Eh of the hindgut were unsuccessful. Serratia marcescens failed to colonise the gut of normal C. lacteus and transiently colonized (for 5 days) the gut of normal N. exitiosus. Transient colonization by S. marcescens (from 6 to 10 days) occurred in N. exitiosus when its hindgut spirochaetes were killed and in C. lacteus when its spirochaetes and protozoa were killed, indicating a possible role for the spirochaetes and/or protozoa in influencing the bacteria allowed to reside in the hindgut. Exposure of normal termites to Serratia provoked an increase in the numbers of the normal gut bacteria.     

Aerobic state of gut of Nasutitermes exitiosus and Coptotermes lacteus, high and low caste termites

The redox potential and pH of the gut of the termites Nasutitermes exitiosus and Coptotermes lacteus was investigated by feeding the insects with redox dyes and pH indicators. For N. exitiosus the E′₀ (pH 7.0) of the foregut was above +200 mV; the midgut was about +100 to +150 mV and the hindgut was in the region of ?20 to +30 mV. For C. lacteus the fore- and midgut were about +30 to +50 mV and the hindgut about ?20 to +20 mV. The colours of the ingested dyes indicated that the gut was aerobic in both termites. The pH of the whole gut ranged from 6.5 to 7.5 for N. exitiosus and 6.0 to 7.0 for C. lacteus.     

Dependence of the higher termite, Nasutitermes exitiosus and the lower termite, Coptotermes lacteus on their gut flora

The importance of the gut microorganisms in the termites Nasutitermes exitiosus and Coptotermes lacteus was investigated by feeding them with antibiotics. With N. exitiosus, antibiotics which killed both the bacteria and the spirochaetes (ampicillin, kanamycin, chloramphenicol, erythromycin, cephaloridine, tetracycline) reduced the life span of the termite from 250 days to about 13 days, whereas antibiotics which had little effect on the flora (penicillin, methicillin) did not greatly reduce the life span of the termite. The essential role of the spirochaetes in N. exitiosus was shown by feeding metronidazole, or exposing the termites to pure oxygen. Both treatments killed the spirochaetes, but not the bacteria, resulting in a life span for the termite of 13–22 days. Acid fuchsin did not kill the spirochaetes. Fungi were not essential for N. exitiosus. In C. lacteus all treatments, except that with acid fuchsin, killed the protozoa, thereby reducing the life span of the termite from 69 days to 6–29 days.     

The Selection of an Isolate of the Hyphomycete Fungus,Metarhizium anisopliae,for Control of Termites in Australia

Ninety-three isolates ofMetarhizium anisopliae,mostly derived from a survey of termite material, were screened for activity againstNasutitermes exitiosusandCoptotermes frenchiorC. acinaciformisusing a grooming assay technique. Twenty-six of the most promising isolates were further evaluated by bioassay againstN. exitiosusandC. acinaciformis.All isolates were pathogenic withCoptotermesspp. being more susceptible thanN. exitiosus.A group of nine isolates, chosen for their level of pathogenicity for one or other genus of termites and to represent a genetically diverse group, was finally compared in a minicolony test using termite colonies in 1 liter jars. The isolate, code-named FI-610 (derived from nest-mound material ofC. lacteusin SE New South Wales), was one of the most effective isolates against termites from both of the two colonies tested. This isolate also grew relatively well on agar plates at 36°C. FI-610 was thus selected for field trials and was found to be effective in killing colonies ofC. acinaciformiswhen 10 g (=3 × 10¹¹conidia) or more of conidial powder was blown into the center of the large mound colonies.     

Passage and Survival of Chlamydospores of Phytophthora cinnamomi Rands, the Causal Agent of Forest Dieback Disease, Through the Gastrointestinal Tracts of Termites and Wild Birds

Chlamydospores of Phytophthora cinnamomi Rands have been shown to survive in the intestinal tracts of termites (Nasutitermes exitiosus) and two species of forest birds indigenous to West Australian jarrah forests. Viable chlamydospores were recovered from bird feces within the normal rate of passage time for food through the gut. The above factors would allow these creatures to function as vectors for the spores.     

Influence of laboratory maintenance,relative humidity and coprophagy on [14C]lignin degradation by Nasutitermes exitiosus

[¹⁴C](lignin)-Acer rubrum L. was produced by infusing stems of A. rubrum with [¹⁴C](3′-side chain)-cinnamic acid. Groups (1g) of Nasutitermes exitiosus (Hill) were placed in sealed flasks which were aspirated over a 2-week period. These released up to 8.3% of the [¹⁴C](lignin)-A. rubrum as ¹⁴CO₂. Termites maintained under standard conditions as 50 g non-breeding groups for 4 months or more showed diminished ability to degrade lignin. Optimal lignin degradation and survival of N. exitiosus was at 90 and 96% relative humidity (r.h.). At 75, 80 and 85% r.h., fungal growth in bioassay flasks was seen, but lignin degradation did not increase. At 100% r.h. where bacterial growth in faeces may have been encouraged due to the development of free water, termite survival was poor and lignin degradation decreased. Starved termites contained much more radioactivity (21.8 and 30.8%) than fed termites (1.6% radioactivity), probably due to greater coprophagy on deprivation of food. However, lignin degradation was only marginally higher in starved termites, suggesting lignin becomes progressively more resistant to termite degradation after passage through the gut.     

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).     

Termite gut symbiotic archaezoa are becoming living metabolic fossils

Over the course of several million years, the eukaryotic gut symbionts of lower termites have become adapted to a cellulolytic environment. Up to now it has been believed that they produce nutriments using their own cellulolytic enzymes for the benefit of their termite host. However, we have now isolated two endoglucanases with similar apparent molecular masses of approximately 36 kDa from the not yet culturable symbiotic Archaezoa living in the hindgut of the most primitive Australian termite, Mastotermes darwiniensis. The N-terminal sequences of these cellulases exhibited significant homology to cellulases of termite origin, which belong to glycosyl hydrolase family 9. The corresponding genes were detected not in the mRNA pool of the flagellates but in the salivary glands of M. darwiniensis. This showed that cellulases isolated from the flagellate cells originated from the termite host. By use of a PCR-based approach, DNAs encoding cellulases belonging to glycosyl hydrolase family 45 were obtained from micromanipulated nuclei of the flagellates Koruga bonita and Deltotrichonympha nana. These results indicated that the intestinal flagellates of M. darwiniensis take up the termite's cellulases from gut contents. K. bonita and D. nana possess at least their own endoglucanase genes, which are still expressed, but without significant enzyme activity in the nutritive vacuole. These findings give the impression that the gut Archaezoa are heading toward a secondary loss of their own endoglucanases and that they use exclusively termite cellulases.     

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.     

Acromyrmex Leaf-Cutting Ants Have Simple Gut Microbiota with Nitrogen-Fixing Potential

Ants and termites have independently evolved obligate fungus-farming mutualisms, but their gardening procedures are fundamentally different, as the termites predigest their plant substrate whereas the ants deposit it directly on the fungus garden. Fungus-growing termites retained diverse gut microbiota, but bacterial gut communities in fungus-growing leaf-cutting ants have not been investigated, so it is unknown whether and how they are specialized on an exclusively fungal diet. Here we characterized the gut bacterial community of Panamanian Acromyrmex species, which are dominated by only four bacterial taxa: Wolbachia, Rhizobiales, and two Entomoplasmatales taxa. We show that the Entomoplasmatales can be both intracellular and extracellular across different gut tissues, Wolbachia is mainly but not exclusively intracellular, and the Rhizobiales species is strictly extracellular and confined to the gut lumen, where it forms biofilms along the hindgut cuticle supported by an adhesive matrix of polysaccharides. Tetracycline diets eliminated the Entomoplasmatales symbionts but hardly affected Wolbachia and only moderately reduced the Rhizobiales, suggesting that the latter are protected by the biofilm matrix. We show that the Rhizobiales symbiont produces bacterial NifH proteins that have been associated with the fixation of nitrogen, suggesting that these compartmentalized hindgut symbionts alleviate nutritional constraints emanating from an exclusive fungus garden diet reared on a substrate of leaves.     

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.     

A comparison of two methods for sampling assemblages of subterranean,wood-eating termites (Isoptera)

Abstract Within a 50 × 50 m area of wandoo Eucalyptus capillosa woodland in the Western Australian wheatbelt, the diversity and frequency of occurrence of wood-eating termite species was assessed at two food types. Over a 12 month period, monthly termite activity was determined: (i) at sound/undecayed artificial baits (seasoned wooden stakes of Jarrah, Karri, Pine, Batu, Oregon; Jarrah sawdust; paper rolls); and (li) at naturally occurring timber, fallen logs and branches of wandoo, in varying stages of decay. Termite diversity was 11 species at baits, 18 species at wandoo out of an overall site richness of 21 species. Karri attracted the most species (9); sawdust attracted none. At wandoo, Nasutitermes exitiosus, Coptotermes acinaciformis and Occasitermes occasus accounted for 59% of samples where termites were recorded. At baits, Heterotermes occiduus accounted for a mean of 80% of samples across bait types, but was rarely sampled at wandoo (5% of samples). Only H. occiduus, C. acinaciformis and Amitermes neogermanus ate bait. Pine, Oregon and paper rolls were most effective in attracting foraging termites in terms of highest per cent of replicates showing bait consumption and highest consumption rates. Jarrah and Batu were least attractive to foraging termites. Samples from wandoo underestimated the relative frequency of occurrence of H. occiduus within the study site. Coptotermes acinaciformis, which attack large food items, and certain species of Amitermes, which forage on subterranean food, may have been underestimated by both sampling methods. These findings indicate that a proper understanding of the structure of wood-eating termite assemblages within a given area requires a composite sampling strategy which addresses termites that eat sound or decayed wood, as well as surface and subsurface foragers.     

Vertical transmission as the key to the colonization of Madagascar by fungus-growing termites?

The mutualism between fungus-growing termites (Macrotermitinae) and their mutualistic fungi (Termitomyces) began in Africa. The fungus-growing termites have secondarily colonized Madagascar and only a subset of the genera found in Africa is found on this isolated island. Successful long-distance colonization may have been severely constrained by the obligate interaction of the termites with fungal symbionts and the need to acquire these symbionts secondarily from the environment for most species (horizontal symbiont transmission). Consistent with this hypothesis, we show that all extant species of fungus-growing termites of Madagascar are the result of a single colonization event of termites belonging to one of the only two groups with vertical symbiont transmission, and we date this event at approximately 13 Mya (Middle/Upper Miocene). Vertical symbiont transmission may therefore have facilitated long-distance dispersal since both partners disperse together. In contrast to their termite hosts, the fungal symbionts have colonized Madagascar multiple times, suggesting that the presence of fungus-growing termites may have facilitated secondary colonizations of the symbiont. Our findings indicate that the absence of the right symbionts in a new environment can prevent long-distance dispersal of symbioses relying on horizontal symbiont acquisition.     

Identifying the core microbial community in the gut of fungus‐growing termites

Gut microbes play a crucial role in decomposing lignocellulose to fuel termite societies, with protists in the lower termites and prokaryotes in the higher termites providing these services. However, a single basal subfamily of the higher termites, the Macrotermitinae, also domesticated a plant biomass‐degrading fungus (Termitomyces), and how this symbiont acquisition has affected the fungus‐growing termite gut microbiota has remained unclear. The objective of our study was to compare the intestinal bacterial communities of five genera (nine species) of fungus‐growing termites to establish whether or not an ancestral core microbiota has been maintained and characterizes extant lineages. Using 454‐pyrosequencing of the 16S rRNA gene, we show that gut communities have representatives of 26 bacterial phyla and are dominated by Firmicutes, Bacteroidetes, Spirochaetes, Proteobacteria and Synergistetes. A set of 42 genus‐level taxa was present in all termite species and accounted for 56–68% of the species‐specific reads. Gut communities of termites from the same genus were more similar than distantly related species, suggesting that phylogenetic ancestry matters, possibly in connection with specific termite genus‐level ecological niches. Finally, we show that gut communities of fungus‐growing termites are similar to cockroaches, both at the bacterial phylum level and in a comparison of the core Macrotermitinae taxa abundances with representative cockroach, lower termite and higher nonfungus‐growing termites. These results suggest that the obligate association with Termitomyces has forced the bacterial gut communities of the fungus‐growing termites towards a relatively uniform composition with higher similarity to their omnivorous relatives than to more closely related termites.     

IDENTIFICATION OF THE FGL‐AMIDE ALLATOSTATIN GENE OF THE PRIMITIVE TERMITE Mastotermes darwiniensis AND THE WOODROACH Cryptocercus darwini

Allatostatins with the C‐terminal ending Tyr/Phe‐Xaa‐Phe‐Gly‐Leu/Ile‐amide (FGLa/ASTs) are widespread neuropeptides with multiple functions. The gene encoding the FGLa/AST polypeptide precursor was first isolated from cockroaches and since then could be identified in many insects and crustaceans. With its strictly conserved regions in combination with variable regions the gene seems to be a good candidate for phylogenetic analyses between closely and distantly related species. Here, the structure of the FGLa/AST gene of the most primitive termite, the giant northern termite Mastotermes darwiniensis Froggatt, was identified. The FGLa/AST gene of the woodroach Cryptocercus darwini was also determined. Precursor sequences of both species possess the general organization of dictyopteran FGLa/AST precursors containing 14 putative FGLa/AST peptides. In M. darwiniensis, only 11 out of the 14 FGLa/AST‐like peptides possess the C‐terminal conserved region Y/FXFGL/I/V/M and four of the putative peptide structures are not followed by a Gly residue that would lead to nonamidated peptides. Phylogenetic analyses show the high degree of similarity of dictyopteran FGLa/AST sequences. The position of termites, nested within the Blattaria, confirms that termites have evolved from primitive cockroaches.     

Nitrogen mineralization, denitrification, and nitrate ammonification by soil-feeding termites: a 15N-based approach

Soil-feeding termites are abundant and play important roles in the biogeochemical processes in tropical soils. Previous studies indicated that they preferentially utilize the peptidic components of soil organic matter as a nutrient resource. Here, we determined the corresponding mineralization fluxes and elucidated other N transformation processes that occur during soil gut passage using ¹⁵N tracer techniques. Termite-based rates of N mineralization by Cubitermes umbratus and Cubitermes ugandensis in soil microcosms amended with ¹⁵NH₄ ⁺ were 6.6 and 9.2 nmol N day^?1 (g fresh wt)^?1, which means that the soil peptides fuel about 20 and 40% of the respiratory activity of these insects. Considering the areal biomass of soil-feeding termites in humid savannahs, soil-feeding termites should mineralize about 3% of the total N in their food soil per year. In addition to producing ammonia from ingested ¹⁵NO₃ ^? at approximately 10% of the mineralization rate, C. umbratus also formed N₂ at similar rates. The formation of labelled N₂ in microcosms amended with ¹⁵NH₄ ⁺ seems to be at least partially due to nitrification activity in the soil; evidence for the formation of nitrate in the posterior hindgut remains inconclusive. However, the so far unexplained increase of ¹⁵N abundance in the ammonia pools of the posterior hindgut compartments manifests additional hitherto unknown metabolic processes in this gut region. Collectively, our results not only reinforce the concept of nitrogenous soil components as an important dietary resource for soil-feeding termites, but also allow us to predict that N mineralization and nitrate ammonification activities in the termite gut should positively affect the dynamics of N in tropical soil.     

Colonization of termite hindgut walls by oxymonad flagellates and prokaryotes in Incisitermes tabogae,I. marginipennis and Reticulitermes flavipes

We studied the colonization of the paunch wall of three lower termites, Reticulitermes flavipes, Incisitermes tabogae, and Incisitermes marginipennis, by light and electron microscopy. In addition to various prokaryotes, oxymonad flagellates were attached to the wall of the paunch in all three species. The prokaryotic layer found in R. flavipes is relatively thin, since most organisms are attached laterally. Large members of the flagellate genus Pyrsonympha protrude into the gut lumen. The prokaryotes are very abundant on the gut wall in I. tabogae and I. marginipennis, forming a thick carpet of mostly vertically attached rods and wavy spirochetes. The adhering oxymonads are relatively small and almost hidden in the thick bacterial biofilm. Three small morphotypes were seen in I. tabogae; two possessing a short rostellum and one amoeboid. The only oxymonad found in I. tabogae so far, Oxymonas clevelandi, is not identical to any of the present oxymonads. I. marginipennis contains a mid-sized oxymonad with ectobiotic spirochetes, probably identical to Oxymonas hubbardi, and a tiny unknown morphotype. The spatial organization of the pro- and eukaryotic microorganisms on the gut wall of the three termites is described and discussed concerning oxygen stress.     

Cloning and Heterologous Expression of Insecticidal-Protein-Encoding Genes from Photorhabdus luminescens TT01 in Enterobacter cloacae for Termite Control

Enterobacter cloacae, one of the indigenous gut bacteria of the Formosan subterranean termite (Coptotermes formosanus), was genetically modified with a transposon Tn5 vector containing genes (tcdA1 and tcdB1) encoding orally insecticidal proteins from the entomopathogenic bacterium Photorhabdus luminescens subsp. laumondii TT01, a symbiont of the entomopathogenic nematode Heterorhabditis bacteriophora, for termite control. In the laboratory, termites were fed filter paper inoculated with the recombinant bacteria. The chromosomal expression of the introduced genes showed that there were insecticidal activities against termite workers and soldiers challenged with the transformed bacteria. After termites were fed recombinant bacteria, the termite mortality was 3.3% at day 5, and it increased from 8.7% at day 9 to 93.3% at day 29. All the dead termites contained the recombinant bacteria in their guts. Transfer of the recombinant bacteria occurred between donor workers (initially fed recombinant bacteria) and recipient workers (not fed). More than 20% of the recipient termites ingested recombinant bacteria within 2 h, and 73.3% of them had ingested recombinant bacteria after 12 h. The method described here provides a useful alternative for sustainable control of the Formosan subterranean termite (C. formosanus) and other social insects, such as the imported red fire ant (Solenopsis invicta).     

Asymmetric interaction specificity between two sympatric termites and their fungal symbionts

Abstract.  1. Fungus-growing termites live in an obligate mutualistic symbiosis with Termitomyces fungi. The functions of the fungal symbiont have been hypothesised to differ between species and to range from highly specific roles of providing plant-degrading enzymes complementary to termite gut enzymes, to non-specific roles of providing protein-rich food to the termites.
2. Termite species with unspecialised fungal symbionts are predicted to be associated with a wider range of symbionts than species with specialised symbionts. Recent DNA data have confirmed this prediction, but evidence for differences in functional specificity has been sparse and indirect.
3. Here the consequences of symbiont interaction specificity are experimentally tested by reciprocally exchanging the fungal symbionts of sympatric colonies of Macrotermes natalensis and Odontotermes badius , which were inferred to have specialised and non-specialised symbionts respectively.
4. As expected, survival of O. badius termites on M. natalensis fungus was not significantly worse than on their own fungus, but survival of M. natalensis termites on O. badius fungus was significantly reduced.
5. This asymmetric result confirms that symbiont roles differ significantly between macrotermitine genera and indicates that symbiont transplantation experiments are a powerful tool for testing the functional details of mutualistic symbioses.