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.     

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.     

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.     

Fipronil enhanced natural occurrence of Fusarium solani (Hypocreales: Nectriaceae) on building infesting termite Heterotermes indicola Wasmann (Blattodea: Rhinotermitidae)

The Southeast Asian subterranean termite, Heterotermes indicola Wasmann (Blattodea; Rhinotermitidae), is recognized as a building infesting lower termite species in urban environment. The extensive use of chemical termiticides against aerial mud tubes and underground nests of H. indicola beneath the buildings could not suppress its infestation; however, it enhanced the environmental contamination and insecticide resistance. In the present study, we tried to control termites using naturally occurring entomopathogenic fungi Fusarium solani (Mart.) Sacc., along with sublethal concentrations of termiticide fipronil in no-choice feeding pathogenecity bioassay for 20?days. Termite mortality after 20?days of continuous exposure to highest fungal treatment 1?×?10⁹?conidia/mL was 10% exclusively, whereas 100% mortality was calculated just after 16?days of concurrently exposure to 5?ppm of fipronil and highest rate of fungus 1?×?10⁹?conidia/mL. These results indicated that insecticidal stress declined the immune response of termites and reduced the repellency of termites against fungal conidia by breaching the primary defense mechanism (allogrooming). This co-application of F. solani at suitable sublethal concentrations of fipronil showed the promising potential against termites that may reduce the selection pressure of pesticides and resistance risk by targeted pests, but further investigations are necessary for developing field trials.     

Bacteria from the Gut of Australian Termites

The major gut bacteria of the worker caste of nine species of Australian termites, belonging to four families, were isolated and identified to generic level. All species were either facultative anaerobes or strict aerobes. A correlation appears to exist between the major gut bacterium and the family to which the termite belongs. The major bacterium from the two lowest termites, Mastotermes darwiniensis (family Mastotermitidae) and Cryptotermes primus (family Kalotermitidae), was Streptococcus; from four species belonging to the Rhinotermitidae (Heterotermes ferox, Coptotermes acinaciformis, C. lacteus, Schedorhinotermes intermedius intermedius) it was Enterobacter; and from three species of the Termitidae (Nasutitermes exitiosus, N. graveolus, N. walkeri) it was Staphylococcus. Enterobacter was a minor symbiont of M. darwiniensis, C. primus, and N. graveolus; Streptococcus was a minor symbiont of H. ferox, C. lacteus, S. intermedius intermedius, and N. exitiosus; and Bacillus was a minor symbiont of C. acinaciformis and S. intermedius intermedius. M. darwiniensis possessed another minor symbiont tentatively identified as Flavobacterium. C. acinaciformis from three widely separated locations possessed a similar microbiota, indicating some form of control on the composition of the gut bacteria. Bacteria, capable of growth on N-free medium in the presence of nitrogen gas, were isolated from all termites, except N. exitiosus and N. walkeri, and were identified as Enterobacter. No cellulose-degrading bacteria were isolated.     

Effect of laboratory containment on the nitrogen metabolism of termites

When Nasutitermes exitiosus, Nasutitermes walkeri and Coptotermes lacteus were brought into the laboratory they rapidly lost, within 24–48 hr, their ability to fix dinitrogen. With N. exitiosus and N. walkeri the loss was linear over the first 26–32 hr at a rate of about 3–4% per hour. N. walkeri completely lost its ability to fix dinitrogen and did not recover it during a further 11 days in the laboratory, whereas N. exitiosus and C. lacteus partially recovered their dinitrogen fixing ability to about 25–50% of the original rate. During laboratory storage of up to 60 days both C. lacteus and N. exitiosus gradually lost total nitrogen, while at the same time their uric acid content increased. The uric acid content of N. walkeri increased during 17 days in the laboratory while total nitrogen remained essentially constant. Xanthine dehydrogenase was not detected in freshly-collected N. walkeri but was detectable after two days of laboratory storage and reached a maximum activity in 8–10 days. The rate of dinitrogen fixation, total nitrogen and uric acid of field populations of N. exitiosus and N. walkeri (tested within 2 hr of collection) remained within close limits over a 6–8 week period, indicating that the changes in these parameters observed in populations kept in the laboratory did not occur in field populations. In field populations of N. walkeri the total nitrogen was about 1.4% of the fresh weight (6.7% of the dry weight) and the uric acid content was about 1.3% of the fresh weight (6.6% of the dry weight), with the amount of total nitrogen present as uric acid being about 31%. In N. exitiosus these values were: total nitrogen about 1.6% of the fresh weight (7.4% of the dry weight), uric acid about 0.6% of the fresh weight (2.9% of the dry weight), with uric acid accounting for about 13% of total nitrogen. When workers of N. walkeri were stored in a container near their nest they lost dinitrogen fixing ability to the same extent as workers brought into the laboratory, indicating that disruption of the nest was sufficient to affect dinitrogen fixation.     

Evaluation of mold,decay and termite resistance of pine wood treated with zinc- and copper-based nanocompounds

In this work, the resistance of black pine wood (Pinus nigra L.) vacuum-treated with zinc oxide, zinc borate and copper oxide nanoparticles against mold and decay fungi and the subterranean termites was evaluated. Some of the nanocompounds tested were forced with acrylic emulsions to avoid leaching. Results showed that mold fungi were slightly inhibited by nanozinc borate, while the other nanometal preparations did not inhibit mold fungi. Mass loss from fungal attack by Trametes versicolor was significantly inhibited by the zinc-based preparations, while the brown-rot fungus, Tyromyces palustris was not inhibited by the nanometal treatments. Notably, nanozinc borate plus acrylic emulsion imparted very high resistance in pine wood to the white-rot fungus, T. versicolor with a mass loss of 1.8%. Following leaching, all pine specimens treated with nanozinc borate, with or without acrylic emulsion, strongly inhibited termite feeding, i.e. mass losses varying at 5.2–5.4%. In contrast, the copper-based treatments were much less effective against the subterranean termites, Coptotermes formosanus. In general, nanozinc borate possessed favorable properties, that is, inhibition of termite feeding and decay by T. versicolor.     

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).     

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.     

Field Evaluations of Subterranean Termite Preference for Sap-Stain Inoculated Wood

Few studies have focused on interactions between subterranean termites and the ophiostomatoid fungal associates of pine bark beetles or root feeding weevils. Field stake tests were employed at four locations throughout Mississippi to determine the feeding preference of subterranean termites for blue-stained, unstained, and partially decayed southern pine sapwood stakes. This study also utilized wood decayed by Gloeophyllum trabeum, a fungus previously shown to elicit a positive subterranean termite feeding response, as a positive control. Stakes inoculated with G. trabeum received significantly more attacks than all other treatments after 16 weeks. Of the stakes attacked by subterranean termites, stakes inoculated with Ophiostoma minus were degraded faster than any other treatment. Subterranean termite preference for stakes treated with either of two Leptographium spp. and the untreated negative controls did not differ; however, each was fed upon less than all other treatments. The feeding rate on stakes inoculated with O. ips and G. trabeum being fed upon by subterranean termites was not significantly different. These results represent the first evidence of wood containing non-structurally degrading fungi (O. ips and O. minus) eliciting a feeding preference from subterranean termites greater than that of decayed wood. The implications of these results are particularly relevant to pine forest ecology, nutrient cycling, subterranean termite control, and the utilization of blue-stained southern pine building products in the southeastern U.S.     

Utilizing rabbit immunoglobulin G protein for mark-capture studies on the desert subterranean termite, Heterotermes aureus (Snyder)

Mark-capture dispersal studies were conducted to investigate the feasibility of marking the southwestern desert subterranean termite, Heterotermes aureus (Snyder) with rabbit immunoglobulin G (IgG). In turn, short-range dispersal patterns of H. aureus were measured across a 20-m diameter desert landscape at three distinct field locations. Each location consisted of 51 termite feeding stations containing corrugated cardboard. The central feeding station (CFS) at each location was impregnated with rabbit IgG. A circular grid was then constructed around each CFS that consisted of 50 additional unmarked cardboard feeding stations strategically placed around the CFS at distances of 1.5, 2.0, 4.0, 7.0 or 10.0 m. Termites self-marked with rabbit IgG by feeding on the marked bait. The CFS and the 50 peripheral feeding stations were sampled for marked termites twice at each location 17–65 days after the marked bait was placed at the CFS to determine the spatial dispersal patterns of H. aureus within each research grid. Termites that self marked by feeding on rabbit IgG marked bait were detected by an anti-rabbit IgG enzyme-linked immunosorbent assay (ELISA). Generally, the CFSs contained the highest frequency of marked termites with 28.0% of the individuals assayed from the CFSs containing rabbit IgG. Over the course of the study, 39 of the unmarked peripheral feeding stations contained at least one marked termite. Of the termites assayed from the peripheral stations (n = 2,955), 124 or 4.2% of the individuals contained the mark. The average distance traveled by the marked termites collected at the peripheral feeding stations was 5.7 ± 3.3 m from the CFSs. We also examined single nucleotide polymorphisms (SNPs) from termites collected at each field site. Data indicated that each field site were genetically distinct and therefore non-related termites. We discuss the advantages and limitations of marking termites with rabbit IgG for dispersal studies.     

Specific features of nitrogen fixation in the termite Reticulitermes lucifugus

Nitrogenase activity, abundance of diazotrophic bacteria, and structural and functional parameters have been determined in microbial complexes of three populations of the termite Reticulitermes lucifugus and their nest materials. These data have been used for comparative analysis of nitrogen-fixing microorganism communities in termite guts and in nest materials from different termite populations. Similarities in the structure and other parameters of these communities have been revealed. The taxonomic composition of microbial communities differs among the populations, but the functional properties of these communities are almost identical. It is concluded that no symbiotic (mutualistic) relationship exists between nitrogen-fixing intestinal bacteria and their host termites.     

Draft genome sequence of the termite,Coptotermes formosanus: Genetic insights into the pyruvate dehydrogenase complex of the termite

Termites are generally deficient in pyruvate dehydrogenase (PDH), which links glycolysis to the Krebs cycle; however, one termite species, Coptotermes formosanus, has some PDH activity, though it is not enough to maintain the respiration of the termite by itself. We obtained a high quality annotated draft genome of C. formosanus. We found that all genes constituting the PDH complex and controlling PDH activity are present in the genome of C. formosanus, except for the PDH protein X component that is essential for a functional PDH complex. Additionally, we found that C. formosanus has three endo-ß-1,4-glucanases (EGs), for which the amino acid sequences differ from those of previously reported EGs. Despite the ability of termites to convert cellulose to glucose and the resulting glucose to pyruvate, PDH is likely to function poorly due to a missing X component of the PDH complex.     

Fungal nutrition allocation enhances mutualism with fungus-growing termite

Fungal nodules and aged fungus gardens are products of termite fungiculture systems, and are the diets of termites. To understand the nutrition flow in fungiculture, we quantified the number and mass of fungal nodules produced along with fungus garden maturation and analysed the α-amino acid and fatty acid compositions of fungal nodules, fungus gardens, and termite tissues of a fungus-growing termite, Odontotermes formosanus. 1 g of fungus garden produced 5,148 fungal nodules (∼68.0 mg). Approximately 7.0% of α-amino acids were allocated to the fungal nodules and the rest (∼93.0%) remained in the fungus gardens. The compositions of α-amino acids or fatty acids in aged fungus gardens and fungal nodules were more similar to that of termite tissues than fresh fungus gardens, which supports the idea that termites nutritionally depend on the fungal products. Among the 18 α-amino acids, tryptophan was an essential amino acid and was the only one missing from fresh and aged fungus gardens, but found in fungal nodules at significantly higher concentrations. Hence, termites must consume fungal nodules to obtain tryptophan for survival. Furthermore, the fungus spores incorporated in nodules, were transferred when nodules were ingested by termites. We propose that allocating tryptophan in fungal nodules is crucial to enhance the mutualism between the fungus and termite.     

Diazotrophs in the digestive tract of termite Neotermes castaneus

Normal vital activity of termites Neotermes castaneus requires the presence and continuous replenishment of transient nitrogen-fixing bacteria in their digestive tract, which is realized by coprophagy and repeated utilization of substrate enriched in termite feces. This is the first demonstration of significant changes in the complex of aerobic and facultatively anaerobic nitrogen fixers mediated by an extension of microbial group composition and a shift in dominant taxa in termites that cannot reutilize their feces.     

Estimating termite population size using spatial statistics for termite tunnel patterns

Subterranean termites build underground tunnels for foraging. The obtained food is transported to the nest through these tunnels, and consumed to maintain the termite colony. In this process, termites can cause damage to wooden structures. To develop effective control strategies to reduce termite damage, it is important to know the sizes of the termite populations in the tunnels. In this study, we proposed a method for estimating the termite population size using the spatial statistic indices including fractal dimension (FD), local density (LD), and join count statistic (JCS) for the tunnel patterns. However, the method needs further improvement to be applied in field conditions. For the method, we generated 8,000 tunnel pattern images (1,000 images for each N) using an agent-based model based on experimental data. Here, N (= 3, 4, ..., 10) represents the number of termites participating in tunnel construction in the simulation. Subsequently, we calculated the FD, LD and JCS values of the tunnel pattern and trained and verified the k-nearest neighbors (KNN) algorithm, using 5,600 and 2,400 images, respectively. The population size (N) was estimated based on the FD, LD and JCS using the KNN algorithm. The estimated accuracy for all N was 60% to 97% in the range of k = 1 to 300. If the model for tunnel pattern generation includes heterogeneous environmental conditions, the proposed method could be used to effectively estimate the actual number of termite populations. Finally, we briefly discuss the challenges affecting our model, and how these could be overcome.     

Origin of termite eusociality: trophallaxis integrates the social,nutritional, and microbial environments

1. Numerous cladistic analyses have converged: termites are a monophyletic clade embedded within the paraphyletic cockroaches, and sister group to the biparental, wood‐feeding cockroach Cryptocercus. The latter is, therefore, an appropriate model for testing assumptions regarding early termite evolution. 2. The ground plan of the termite ancestor is reviewed based on shared characters of ecology, life history, and behaviour in Cryptocercus and incipient termite colonies, and includes two levels of dependence: a reliance of all individuals on gut microbiota, and dependence of early instars on parental care. Both these conditions co‐evolved with parent‐to‐offspring proctodeal trophallaxis. 3. The termite ancestor lived in a single log serving as food and nest. This ‘one‐piece’ nesting ecology prioritises nitrogen conservation and strongly influences interacting social, nutritional, and microbial environments. Each of these environments individually and in combination profoundly affect cockroach development. 4. Proctodeal trophallaxis integrates the social, nutritional, and microbial environments. A change in trophallactic behaviour, from parental to alloparental, can, therefore, shift developmental trajectories, ultimately adding a third level of dependence. The death of gut protists during the host molting period and consequent interdependence of family members shifted the hierarchical level at which selection acted; fixation of eusociality quickly followed. 5. The basic nesting ecology did not change when termites evolved eusociality, the change occurred in the allocation and use of existing resources within the social group, driven by nitrogen scarcity, mediated by trophallaxis, and made possible by a strongly lineage‐specific set of life history characteristics.     

Genetically engineered termite gut bacteria (Enterobacter cloacae) deliver and spread foreign genes in termite colonies

Indigenous gut bacteria of the Formosan subterranean termite (Coptotermes formosanus Shiraki, Isoptera: Rhinotermitidae) were used as shuttle systems to deliver, express and spread foreign genes in termite colonies. The gut bacterium Enterobacter cloacae was transformed with a recombinant plasmid (pEGFP) containing genes encoding ampicillin resistance and green fluorescent protein (GFP). In laboratory experiments, termite workers and soldiers from three colonies were fed with filter paper inoculated with transformed bacteria. Transformed bacteria were detected in termite guts by growing the entire gut flora under selective conditions and checking the cultures visually for fluorescence. We demonstrated that (1) transformed bacteria were ingested within a few hours and the GFP gene was expressed in the termite gut; (2) transformed bacteria established a persistent population in the termite gut for up to 11 weeks; (3) transformed bacteria were efficiently transferred throughout a laboratory colony, even when the donor (termites initially fed with transformed bacteria) to recipient (not fed) ratio was low; (4) transformed E. cloacae were transferred into soil; however, they did not accumulate over time and the GFP plasmid was not transferred to other soil bacteria. In the future, transgenic bacteria may be used to shuttle detrimental genes into termite colonies for improved pest control.     

Assessment of extracts from some tropical plants in the management of termite (Termitidae: Isoptera) in Ogun State,Nigeria

This study investigated the efficacy of extracts from four plants, namely: Azadirachta indica, Ocimum gratissimum, Vernonia amygdalina and Nicotiana tabaccum at managing the population of termites in the laboratory and in the field. The synthetic insecticide (Chlorpyrifos 0.1%) and water were included in the study as controls. In the laboratory, contact toxicity was conducted by topical application of the treatments to the thorax of soldier termite Macrotermes bellicossus and mortality noted. The repellent action of the treatments was tested by releasing 10 of the termites into the center of Petri dishes lined with treated filter paper. The number of insects present on the control and treatment halves was recorded and the percentage repellence values were computed. The residual effects of the treatments was studied by placing five termites in Petri dishes lined with filter paper that have been treated with the treatments 24 hours prior to its placement and insect mortality was noted. The studies were conducted using complete randomised design with four replicates. Field trial of the treatments was conducted on termitaria that were demolished and disinfested with five liters of the treatments. The results showed that all the treatments caused 95–100% mean insect mortality 10 hours after insect exposure and have repellence values of between 40 and 65%. The residual effects of the treatments caused termite mean mortality of 100% in Chlorpyrifos and N. tabacum-treated surfaces and between 50 and 65% in others. In the field, only A. indica and Chlopyrifos were effective at curtailing termite upsurge and rebuild of termitaria.