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 x 10 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 NH(3) 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 CO(2) evolution from [2-C]UA. The magnitude of anaerobic uricolysis [0.67 pmol of UA catabolized/(gut x 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.     

Heterotrophic bacteria present in hindguts of wood-eating termites [Reticulitermes flavipes (Kollar)]

Strict anaerobic culture techniques were used to quantitate heterotrophic bacteria present in hindguts of Reticulitermes flavipes. The grand mean number of viable cells per hindgut was 0.4 X 10(5) (first-instar larvae), 1.3 X 10(5) (third-instar larvae), 3.5 X 10(5) (workers), and 1.5 X 10(5) (soldiers). Of a total of 344 isolates, 66.3% were streptococci that were always obtained regardless of the origin of termites, their developmental stage or caste, or their length of captivity. Most of the remaining isolates were strains of Bacteroides and Enterobacteriaceae. A small percentage were strains of Lactobacillus, Fusobacterium, and unidentified anaerobic gram-positive rods. Recovery of bacteria from worker hindguts was 13.0% of the direct microscopic count. Isolations performed aerobically failed to reveal strict aerobes. Attempts to isolate cellulolytic bacteria were uniformly unsuccessful. Of 145 streptococcal strains isolated from freshly collected termites, almost all were Streptococcus lactis and S. cremoris. Enterobacteriaceae isolates from the same termite specimens were indole-positive Citrobacter, citrate-negative Citrobacter, and Enterobacter cloacae. The possibility of in situ interspecies lactate transfer, between lactate producers (e.g., streptococci) and lactate fermenters (Bacteroides), is discussed.     

Beobachtungen an der eingeschleppten Gelbfüssigen Bodentermite (Reticulitermes flavipes Kollar)

Summary Since 1937 we have been aware of the occurrence of the subterranean termite Reticulitermes in Hamburg; this was probably introduced from the U.S.A. Control of this dangerous wood-destroying insect was carried out between 1954 and 1961. In 1966 the same termite species was found in Munich and control started immediatly. During this control work there was occasion to observe and to investigate some ecological factors such as correlation between temperature and termite infestation, the occurrence of winged termites (primary sexual stages), and the nutrition requirements (dead and living wood).     

Flight Speed of Tethered Reticulitermes flavipes (Kollar) (Isoptera: Rhinotermitidae) Alates

Alates of the Eastern subterranean termite, Reticulitermes flavipes (Kollar) were collected over two flight seasons (2002 and 2004) and flown on flight mills. Data were collected to test if alate mass, colony origin, or gender influenced flight speed. Flight speed ranged from 3.14 to 69.12 cm s⁻¹ and the maximum distance flown by an alate was 458.3 m. Alate mass (P = 0.9406), gender (P = 0.3976), colony origin (P = 0.1244), and the interaction of gender and colony (P = 0.7093) did not significantly influence flight speed. Additionally, an electronic counting device was used to provide instantaneous flight speeds and allowed flight speed to be modeled during acceleration, cruising, and deceleration periods of flight. Mean (±SEM) flight speeds in 2004 were 20.64 (±2.21) cm s⁻¹ (n = 13) for males and 17.76 cm s⁻¹ (n = 1) for the single female flown, falling within the range of the 2002 values.     

In situ morphology of the gut microbiota of wood-eating termites [Reticulitermes flavipes (Kollar) and Coptotermes formosanus Shiraki].

Light microscopy and scanning and transmission electron microscopy were used to examine the in situ morphology of the gut microbiota of Reticulitermes flavipes and Caoptotermes formosanus. Laboratory-maintained termites were used and, for R. flavipes, specimens were also prepared immediately after collection from a natural infestation. The latter endeavor enabled a study of different castes and developmental stages of R. flavipes and revealed differences in the microbiota of field versus laboratory specimens. The termite paunch microbiota consisted of an abundance of morphologically diverse bacteria and protozoa. Thirteen bacterial morphotypes in the paunch were described in detail: seven were observed only in R. flavipes, three were observed only in C. formosanus, and three were common to both termite species. The paunch epithelium was densely colonized by bacteria, many of which possessed holdfast elements that secured them tightly to this tissue and to other bacterial cells. Besides bacteria, the protozoan Pyrsonympha vertens adhered to the paunch epithelium of R. flavipes by means of an attachment organelle. Cuplike indentations were present on the paunch epithelial surface and were sites of bacterial aggregation. Ultrastructural features of cups suggested their involvement in ion absorption. In addition to the paunch, the midgut was also colonized by bacteria that were situated between epithelial microvilli. Results suggest that bacteria are an integral part of the gut ecosystem.     

Evaluation of novel and traditional measures for vigor of laboratory-cultured termites, Reticulitermes flavipes (Kollar)

The current study was undertaken to consider predictive methods for describing the vigor of Reticulitermes flavipes (Kollar) termites stored in a laboratory under conditions similar to control groups in bioassay. These novel methods were based on measurements for levels of biological molecules (uric acid, soluble proteins, lipid, and glycogen), percent water content, live weight, and running speed. Also considered were two established, non-predictive methods for determining vigor, survivorship and consumption rate. Of the novel measures tested, lipid and body water percentage show promise in distinguishing weak from vigorous groups of termites, with body water percentage a more practical means of measurement. Low body water percentage was concluded to be an indicator of weak groups of termites.     

Immunomarking reveals food flow and feeding relationships in the eastern subterranean termite, Reticulitermes flavipes (Kollar)

Trophallaxis and feeding relationships in the eastern subterranean termite, Reticulitermes flavipes (Kollar), were examined using a novel marking technique, rabbit IgG protein coupled with an enzyme linked immunosorbent assay (ELISA) to detect the marker. Transfer experiments in small dishes evaluated the trophallactic transfer of the marker from donor workers fed IgG-treated paper to recipient workers or larvae. Worker donors rapidly acquired the marker, and 100% of donors tested positive within 24 h. Trophallactic transfer from donors to recipients was relatively inefficient, and 51 +/- 2% of recipient workers and 31 +/- 2% of recipient larvae tested positive at 72 h. Based on the mean optical density counts, approximately 27% of marker ingested by the donors was passed on to the recipient workers in the first 24 h, 14% to recipient larvae, and 26% to recipient soldiers. The ability of soldiers to feed independently of workers was examined in dish assays. Soldiers showed no significant uptake of the marker when isolated from the workers, and uptake increased significantly when workers were present. The distribution of the marker was further studied in larger colony fragments composed of workers, soldiers, nymphs, and larvae. Marker acquisition by the different castes/developmental stages was highly variable, with workers and nymphs acquiring the marker at a faster rate than soldiers and larvae. The results of this study contribute to our understanding of the foraging ecology and social behavior in R. flavipes. In addition, they may help design improved control programs for subterranean termites based on baits.     

Infection of the Eastern Subterranean Termite,Reticulitermes flavipes (Kollar) with the fungusBeauveria bassiana (Balsamo) Vuill.

Observations were made on the histopathology of the eastern subterranean termite,Reticulitermes flavipes (Kollar) infected with the fungusBeauveria bassiana (Balsamo) Vuill. Initial symptoms of infected termites were sluggishness, anal discharge, passivity, and failure to respond to tactile stimuli. Infected fifth instar worker termites changed from a healthy creamy color to a light brown. Penetration of the integument by germ tubes ofB. bassiana occurred as early as 16 hours after inoculation. Infection through the digestive tract also resulted. Following penetration of the integument the fungus attacked the fat body, then the nervous tissue and finally the musculature.     

First report of the invasive Reticulitermes flavipes (Kollar, 1837) (Blattodea,Rhinotermitidae) in the Canary Islands

Reticulitermes flavipes, one of the most harmful subterranean termite pests, is reported for the first time from Tenerife (Canary Islands, Spain). Cytochrome oxidase II was sequenced from five specimens in order to confirm the identification. To date, this invasive species has been detected in a limited area in the northeast of the island, affecting buildings, crops and native plant species. Another colony with the identical haplotype found in the southwest, 60 km away from the main population, indicates that this invasive insect may be more widespread over the island.     

Anaerobic Degradation of Uric Acid by Gut Bacteria of Termites

A study was done of anaerobic degradation of uric acid (UA) by representative strains of uricolytic bacteria isolated from guts of Reticulitermes flavipes termites. Streptococcus strain UAD-1 degraded UA incompletely, secreting a fluorescent compound into the medium, unless formate (or a formicogenic compound) was present as a cosubstrate. Formate functioned as a reductant, and its oxidation to CO₂ by formate dehydrogenase provided 2H⁺ + 2e⁻ needed to drive uricolysis to completion. Uricolysis by Streptococcus UAD-1 thus corresponded to the following equation: 1UA + 1formate → 4CO₂ + 1acetate + 4NH₃. Urea did not appear to be an intermediate in CO₂ and NH₃ formation during uricolysis by strain UAD-1. Formate dehydrogenase and uricolytic activities of strain UAD-1 were inducible by growth of cells on UA. Bacteroides termitidis strain UAD-50 degraded UA as follows: 1UA → 3.5 CO₂ + 0.75acetate + 4NH₃. Exogenous formate was neither required for nor stimulatory to uricolysis by strain UAD-50. Studies of UA catabolism by Citrobacter strains were limited, because only small amounts of UA were metabolized by cells in liquid medium. Uricolytic activity of such bacteria in situ could be important to the carbon, nitrogen, and energy economy of R. flavipes.     

Effect of Temperature and Termite Starvation on Phagocytosis by Protozoan Symbionts of the Eastern Subterranean Termite Reticulitermes flavipes Kollar

Abstract Many termite species rely on intestinal protozoan symbionts to digest their cellulosic foods. We examined cellulose acquisition by the symbionts of the Eastern subterranean termite Reticulitermes flavipes Kollar (Isoptera; Rhinotermitidae) by following their phagocytosis of red paper fed to the termite host. The effects of termite host starvation and environmental temperature on feeding activity were studied in the zooflagellates Trichonympha agilis Leidy (Trichonymphidae), Pyrsonympha vertens Leidy, Dinenympha fimbriata Kirby, and D. gracilis Leidy (Pyrsonymphidae), which are among the largest residents in R. flavipes' hindguts. Protozoans in termites starved for 24 h ingested red paper significantly sooner than protozoans in termites with continuous access to food. Trichonympha, Pyrsonympha, and Dinenympha all ingested red paper particles at approximately the same rate. Red paper appeared significantly sooner in protozoans in termites maintained at 32°C than in those maintained at 22°C or 26°C. At 32°C, numbers of Trichonympha per gut remained constant over 96 h. Pyrsonympha and Dinenympha cells were absent or significantly reduced in number by 72 h at that temperature. These results provide insight into the environmental factors that shape the termite–protozoan symbiosis. They may aid in the development of protozoicides used to control pest termites. Received: 1 August 1997; Accepted: 26 November 1997     

Purification and characterization of trans-permethrin metabolizing microsomal esterases from workers of the eastern subterranean termite, Reticulitermes flavipes (Kollar)

Three alpha-naphthyl acetate hydrolyzing esterase isozymes were purified from microsomes prepared from Reticulitermes flavipes workers. The two step process involved sequential preparative IEF followed by continuous elution preparative electrophoresis on a 5% non-denaturing polyacrylamide gel. The first IEF run resulted in 5.4-fold purification with a yield of 46.1%. Subsequent IEF further purified the esterases 14.3-fold and 12% yield. Preparative electrophoresis of the pooled IEF fractions produced three major peaks of alpha-naphthyl acetate hydrolyzing activity. The esterases were correspondingly designated microsomal esterase (ME) 1, ME 2, and ME 3 based on increasing molecular retention on a native PAGE gel. ME 1, ME 2, and ME 3 were acidic proteins with pI values of 4.61, 4.70, and 4.77, respectively. Molecular mass as determined by gel filtration chromatography of ME 1, ME 2, and ME 3 was 69, 64, and 62 kDa, respectively. SDS-PAGE gels produced a single band for each of the isozymes with a molecular mass of 63 kDa indicating that the esterases were monomers. Specific activities of ME 1, ME 2, and ME 3 increased with increasing pH and the enzymes were active over a broad temperature range (25-55 degrees C). The three purified isozymes were inhibited at low concentration by paraoxon (10(-10) M), chlorpyrifos (10(-6) M), DEF (10(-6) M), and PMSF (10(-6) M) indicating that they were "B" type serine esterases. Conversely, inhibition was not observed at 10(-4) M eserine, PHMB, or CaCl(2), further supporting the conclusion that the microsomal esterases were of the "B" type. None of the isozymes was inhibited by 10(-4) M imidacloprid, fipronil, or PBO. Quantitatively, ME 1, ME 2 and ME 3 metabolized t-permethrin at 21.8, 21.0, and 38.8 nmol/h/mg protein, representing a purification factor of 333-, 318-, and 591-fold over microsomes, respectively. The three isozymes produced the same type and number of t-permethrin metabolites.     

Carbon dioxide release in Coptotermes formosanus Shiraki and Reticulitermes flavipes (Kollar): effects of caste, mass, and movement

Movement and carbon dioxide (CO(2)) release of individual Formosan, Coptotermes formosanus Shiraki and Eastern, Reticulitermes flavipes (Kollar) subterranean termites were recorded simultaneously in real time. Worker, soldier, and pre-alate (nymph) caste termites were recorded over 1-h periods at ambient temperature and normoxia in dry, CO(2)-free air. No evidence of discontinuous gas exchange cycles (DGCs) was observed in 344 recordings. Intensity of movement was constant in video tape recordings of termites under respirometry conditions. Duration of movement did not have a significant effect on residuals of &Vdot;(CO(2)) regressed on mass. Thus, movement did not effect &Vdot;(CO(2)) for these two species. Overall CO(2) release values were calculated for all recordings resulting in mean &Vdot;(CO(2)) (ml CO(2) g(-1) h(-1)), and compared among caste, colony, and species with a nested ANOVA. There was significant interaction (P=0.0161) only for species. Mean CO(2) release was significantly greater for R. flavipes (0.507 ml CO(2) g(-1) h(-1)) than C. formosanus (0.310 ml CO(2) g(-1) h(-1)). Mass scaling of termite &Vdot;(CO(2)) was investigated by regressing log(10)&Vdot;(CO(2)) on log(10)mass. The overall model combining species gave a mass scaling coefficient of 0.861(+/-0.0791), which approximates a previously published value for the arthropods as a whole (0.825).     

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.     

Influence of Guidelines and Passageways on Tunneling Behavior of Reticulitermes flavipes (Kollar) and R. virginicus (Banks) (Isoptera: Rhinotermitidae)

Tunneling behavior of laboratory-maintained cultures of Reticulitermes flavipes (Kollar) and R. virginicus (Banks) was examined to determine (1) if the termites build tunnels along preexisting wires or tunnels, and (2) whether tunnels are arranged to optimize search efficiency. Tunnel patterns were considered optimal if, for the number of tunnels present, the maximum area was explored. Termites entered either control arenas or arenas in which they encountered a wire or a pre-formed tunnel. Analyses revealed that R. flavipes and R. virginicus almost always follow pre-formed tunnels, but do not follow wires as readily. Within each species, the distributions of tunnels in treatment arenas were different from distributions in control arenas, most often when pre-formed tunnels were the treatment. Optimal tunnel arrangements in control arenas were found in 62% of R. flavipes patterns with 2 tunnels and in 43% of R. virginicus patterns with 2 tunnels. None of the 3-tunnel patterns from control arenas of R. flavipes and 29% of those of R. virginicus had optimal arrangements. Overall, the spatial arrangement of tunnels in control arenas was significantly different between R. flavipes and R. virginicus.     

Hydrogen Concentration Profiles at the Oxic-Anoxic Interface: a Microsensor Study of the Hindgut of the Wood-Feeding Lower Termite Reticulitermes flavipes (Kollar)

Molecular hydrogen is a key intermediate in lignocellulose degradation by the microbial community of termite hindguts. With polarographic, Clark-type H(inf2) microelectrodes, we determined H(inf2) concentrations at microscale resolution in the gut of the wood-feeding lower termite Reticulitermes flavipes (Kollar). Axial H(inf2) concentration profiles obtained from isolated intestinal tracts embedded in agarose Ringer solution clearly identified the voluminous hindgut paunch as the site of H(inf2) production. The latter was strictly coupled with both a low redox potential (E(infh) = -200 mV) and the absence of oxygen, in agreement with the growth requirements of the cellulolytic, H(inf2)-producing flagellates located in the hindgut paunch. Luminal H(inf2) partial pressures were much higher than expected (ca. 5 kPa) and increased more than threefold when the guts were incubated under a N(inf2) headspace. Radial H(inf2) concentration gradients showed a steep decrease from the gut center towards the periphery, indicating the presence of H(inf2)-consuming activities both within the lumen and at the gut epithelium. Measurements under controlled gas headspace showed that the gut wall was also a sink for externally supplied H(inf2), both under oxic and anoxic conditions. With O(inf2) microelectrodes, we confirmed that the H(inf2) sink below the gut epithelium is located within the microoxic gut periphery, but the H(inf2)-consuming activity itself, at least a substantial part of it, was clearly due to an anaerobic process. These results are in accordance with the recently reported presence of methanogens attached in large numbers to the luminal side of the hindgut epithelium of R. flavipes. If the oxygen partial pressure was increased, O(inf2) penetrated deeper and H(inf2) production was suppressed; it ceased completely as soon as the gut was fully oxic. In experiments with living termites, externally supplied H(inf2) (20 kPa) stimulated methane formation five- to sixfold to 0.93 (mu)mol (g of termite)(sup-1) h(sup-1), indicating that the methanogenic activity in R. flavipes hindguts is not saturated for hydrogen under in situ conditions. This rate was in good agreement with the H(inf2) uptake rates exhibited by isolated hindguts, which would account for more than half of the CH(inf4) formed by living termites under comparable conditions.