Termite Reticulitermes Speratus Research Articles

Environmental cDNA analysis of the genes involved in lignocellulose digestion in the symbiotic protist community of Reticulitermes speratus

To clarify the lignocellulolytic process of the lower termite symbiotic protistan system, we constructed a cDNA library from an as yet uncultivated symbiotic protist community of the lower termite Reticulitermes speratus. The library was constructed by the biotinylated CAP trapper method and analyzed by one-pass sequencing. Phylogenetic analysis of actin orthologs confirmed that the resulting library reflected the intact organismal and mRNA composition of the symbiotic system. The contents of the library included abundant numbers of lignocellulolytic genes of the glycosyl hydrolase family orthologs (families 3, 5, 7, 8, 10, 11, 26, 43, 45 and 62). Our results clearly indicated that a multiple family of glycosyl hydrolase enzymes was involved in the protistan cellulose degradation system. The data also suggested that the most extensively expressed enzyme was glycosyl hydrolase family 7, a cellobiohydrolase ortholog. This family of enzymes enables the degradation of crystalline cellulose, the principal component of wood biomass.

Size, hatching rate, and hatching period of sexually and asexually produced eggs in the facultatively parthenogenetic termite Reticulitermes speratus (Isoptera: Rhinotermitidae)

Facultative parthenogenesis, or condition-dependent alternation of sexual and asexual reproduction, is widespread in animals. Parthenogenesis enables unmated females to reproduce and thus has a great adaptive significance, especially under low pairing efficiency. In the termite Reticulitermes speratus Kolbe, females that fail to pair with males found colonies cooperatively with partner females and reproduce parthenogenetically. We compared the quality of parthenogenetic and sexual eggs in terms of size, hatching rate, and hatching period. We developed a method to culture isolated eggs until hatching under sterile conditions and in appropriate humidity. We successfully isolated, sterilized, and maintained the eggs on agar plates containing 200 ppm tetracycline. Females of female-female (FF) pairs began to lay eggs at the same time as those of female-male (FM) pairs. Nevertheless, the parthenogenetic eggs were significantly larger than sexual eggs. While the two types of eggs had similar hatching rates, parthenogenetic eggs had longer hatching periods (36.36±0.16 [SE] days) than sexual eggs (34.95±0.12 SE). We conclude that primary queens invest more resources into each parthenogenetic egg than each sexual egg, and that parthenogenetic eggs are as viable as sexual eggs.

Seasonal patterns of egg production in field colonies of the termite Reticulitermes speratus (Isoptera: Rhinotermitidae)

This is the first report on the annual egg production patterns in mature termite colonies in the field. Data on the seasonal patterns of egg production in field colonies are very important for understanding the annual colony growth schedule, resource allocation, and population dynamics of the termites. However, collecting the eggs from a sufficient number of colonies is extremely difficult in Reticulitermes termites because their multiple-site nesting makes it difficult to find the reproductive center of the colonies. Here, we first show the seasonal pattern of egg production in the subterranean termite Reticulitermes speratus by collecting the reproductive center of ten colonies each month from April through October. We had to destructively examine dozens of nests to find eggs from enough field colonies each month. Mature field colonies began to produce eggs in late May, soon after the swarming season, and the egg production rate (EPR) reached its maximum in early July. The eggs hatched until late October. The EPR was significantly correlated with the average monthly temperature. Additional investigation of the egg distributions in the nests showed that most eggs were kept around the royal cell, which contained the reproductives. The largest colony had 109 supplemental queens and 94,023 eggs, suggesting that each queen produced an average of 24.7 eggs per day, based on the known mean hatching period of an inseminated egg of 34.95±0.12 (SE) days.

Spatial distribution of bacterial phylotypes in the gut of the termite Reticulitermes speratus and the bacterial community colonizing the gut epithelium

The bacterial community colonizing the gut wall of the termite Reticulitermes speratus was characterized without cultivation. Analysis of 16S rRNA genes after fractionation of the gut revealed that the bacterial composition on the gut wall was diverse and significantly different from that able to move unconfined in the gut fluid or physically associated with the gut protists. Actinobacteria, Firmicutes and Bacteroidetes were dominant on the gut wall, but Spirochaetes and the Termite group 1 phylum, abundant in the gut lumen, were relatively rare. A sequence-specific probe enabled the in situ detection of a rod-shaped Actinobacteria member, abundantly colonizing the gut paunch epithelium.

Distribution of termite egg-mimicking fungi ("termite balls") in Reticulitermes spp. (Isoptera: Rhinotermitidae) nests in Japan and the United States

A termite egg-mimicking fungus was discovered in nests of the Japanese subterranean termite Reticulitermes speratus in 1997 and was reported for the first time in 2000. This fungus has a mutualistic relationship with termites. When termite workers recognize eggs laid by queens, they gather the eggs to tend them. The corticioid fungus Fibularhizoctonia sp. forms sclerotia that morphologically and chemically mimic termite eggs. By mimicking eggs, the fungus is protected and may be transported by termites to a competitor-free habitat. In turn, the sclerotia enhance egg survival, probably because the antifungal and antibacterial compounds produced by the fungus protect the eggs from putative pathogens. This novel termite-fungus interaction has previously been reported only in R. speratus in Japan. In this study, I conducted wide-range sampling in Japan and the United States to investigate the distribution of egg-mimicking fungi in five Reticulitermes spp. Here I show that R. flavipes and R. virginicus in the United States, as well as R. speratus, harbor the egg-mimicking fungus. No egg-mimicking fungi were found in R. okinawanus in Okinawa and R. hesperus in California. A BLAST homology search for rDNA sequences categorized the egg-mimicking fungi isolated from R. flavipes and R. virginicus as a species of Fibularhizoctonia.

Sexual and asexual colony foundation and the mechanism of facultative parthenogenesis in the termite Reticulitermes speratus (Isoptera, Rhinotermitidae)

Facultative parthenogenesis has great adaptive significance, especially with regard to low pairing efficiency. In the termite Reticulitermes speratus, females that fail to mate with males reproduce parthenogenetically and found colonies cooperatively with partner females or even alone. Comparison of colony foundation success at 400 days between colonies founded by single females (F), female-female pairs (FF), and male-female pairs (FM) showed that female-female cooperation promoted colony survivorship over monogamous foundation. We report here for the first time the mode of parthenogenesis in Isoptera. Combining chromosome observations and genetic analysis using microsatellites, we show that the mode of parthenogenesis is diploid thelytoky and that the restoration of ploidy is most likely accomplished by terminal fusion. Parthenogens show a higher mortality and a longer egg-development time than sexually produced offspring, probably due to reduced heterozygosity. In addition Wolbachia bacteria were detected in R. speratus. However, since Wolbachia was also detected in non-parthenogenetic R. flavipes, it is unlikely that Wolbachia is the cause of parthenogenesis in R. speratus.

Effects of stilbenes from bark of Picea glehnii (Sieb. et Zucc) and their related compounds against feeding behaviour of Reticulitermes speratus (Kolbe)

The effects of stilbene glucosides and related compounds on termite feeding behavior were investigated using paper disc methods against the subterranean termite Reticulitermes speratus. The stilbene-rich fraction and isorhapontin (3′-methoxy-3,4′, 5-trihydroxystilbene-3-β-d-glucoside) from bark extracts of Picea glehnii showed avoidance by termites in choice tests. In the no-choice tests using compounds purified from the stilbene-rich fraction, the largest feeding deterrent effect was observed for piceid (3,4′,5-trihydroxystilbene-3-β-d-glucoside), followed by isorhapontin, and astringin (3,3′,4′,5-tetrahydroxystilbene-3-β-d-glucoside), at the concentrations from 0.63 to 2.5 µmol/disc. No change in activity was observed at retentions of more than 5.0 µmol/disc. When the activities of isorhapontin and its aglycone derivative (isorhapontigenin: 3′-methoxy-3,4′,5-trihydroxystilbene) were compared with that of taxifolin (3,3′,4′,5,7-pentahydroxyflavanone) in the no-choice test, the stilbenes exhibited a larger antifeedant potential. Methylation of isorhapontigenin increased its termiticidal activity.

CDNA cloning, expression, and enzymatic activity of a cellulase from the mulberry longicorn beetle, Apriona germari

A novel cellulase [β-1,4-endoglucanase (EGase), EC 3.2.1.4] cDNA belonging to glycoside hydrolase family (GHF) 45 was cloned from the mulberry longicorn beetle, Apriona germari. The cDNA encoding EGase of A. germari (Ag-EGase) is 711 bp long with an open reading frame of 237 amino acid residues. The Ag-EGase was closely related to another beetle, Phaedon cochleariae, cellulase and one symbiotic protist cellulase in the hindgut of the termite Reticulitermes speratus, those belonging to GHF 45. The catalytic sites of GHF 45 are conserved in Ag-EGase. Southern blot analysis of genomic DNA suggested the presence of Ag-EGase gene as a single copy and Northern blot analysis confirmed midgut-specific expression at transcriptional level. Similarly, the Ag-EGase enzyme assay exhibited high activity only in midgut tissue, suggesting that the midgut is the prime site where large quantities of EGase are synthesized for degrading the absorbed cellulose from the diet. The cDNA encoding Ag-EGase was expressed as a 29-kDa polypeptide in baculovirus-infected insect Sf9 cells and the culture supernatants of the recombinant baculovirus-infected cells showed EGase enzyme activity of 15.25 U/ml of medium containing 0.5×10 6 cells at 5 days post-infection (p.i.). The enzyme activity of the purified recombinant Ag-EGase expressed in baculovirus-infected insect cells was approximately 992 U per mg of recombinant Ag-EGase. The purified recombinant Ag-EGase showed the highest enzymatic activity at 50 °C and pH 6.0, and was stable at 55 °C at least for 10 min.

Endosymbiotic Methanobrevibacter species Living in Symbiotic Protists of the Termite Reticulitermes speratus Detected by Fluorescent In Situ Hybridization

Two species of cellulolytic protist, Dinenympha parva and Microjoenia, living in the guts of the lower termite Reticulitermes speratus are known to harbor endosymbiotic methanogens detectable with an epifluorescent microscope. DNA isolated from the guts of worker termites in a colony of R. speratus was amplified using archaea-specific primers and cloned, and partial 16S rRNA gene sequences were obtained. Archaeal PCR clones obtained from the guts of xylophagous insects in this and previous works formed four subgroups within the Methanobrevibacter branch of a phylogenetic tree; the sequences of the clones obtained in this report belonged to subgroups, designated XSAT1A and XSAT1D. Using a probe specific to each of the subgroups, 50 and 10 endosymbiotic Methanobrevibacter cells per protist respectively were detected with a probe specific to the subgroup XSAT1A by fluorescent in situ hybridization analysis in D. parva and Microjoenia. There was no observed hybridization to the endosymbiont with other subtype-specific probes including XSAT1D. Based on these results, endosymbionts in D. parva and Microjoenia sp. are proposed to belong to the Methanobrevibacter subgroup XSAT1A.

Evaluation of primers and PCR conditions for the analysis of 16S rRNA genes from a natural environment

We investigated biases occurring in the polymerase chain reaction (PCR) amplification of 16S rRNA genes from an environmental sample, by comparing the clone libraries that we had previously prepared from the gut homogenate of the termite Reticulitermes speratus. We detected a significant increase in the expected number of phylotypes by lowering the annealing temperature, and a significant decrease in the proportion of clones belonging to the predominant group by raising the number of PCR cycles. We also found that the Bacteria-universal primer, 63F, introduced a seriously biased amplification caused by primer mismatches, in contrast to a previous report. These results, together with suggestions from previous studies using simplified model samples, will help us to recognize the limitations of PCR-based analysis.

Molecular analysis of bacterial microbiota in the gut of the termite Reticulitermes speratus (Isoptera; Rhinotermitidae)

The molecular diversity and community structure of bacteria from the gut of the termite Reticulitermes speratus were analyzed by the sequencing of near-full-length 16S rRNA genes, amplified by polymerase chain reaction. The results of the analysis of 1344 clones indicated a predominance of spirochetes in the gut. Spirochetal clones accounted for approximately half of the analyzed clones. The clones related to Bacteroides, Clostridia, and the candidate division Termite Group I each accounted for approximately 5–15% of the analyzed clones. The rest were comprised of Proteobacteria, Actinobacteria, Mycoplasma and others. Using the criterion of 97% sequence identity, the clones were sorted into 268 phylotypes, including 100 clostridial, 61 spirochetal and 31 Bacteroides-related phylotypes. More than 90% of the phylotypes were found for the first time, and some constituted monophyletic clusters with sequences recovered from the gut of other termite species.

Cooperative colony foundation by termite female pairs: altruism for survivorship in incipient colonies

To determine the costs and benefits of queen association in termites we examined for the first time female–female interactions in colonies founded by two unmated females. In the termite Reticulitermes speratus, females that fail to pair with males found colonies cooperatively with partner females and reproduce by parthenogenesis. We analysed the relationship between queen dominance and initial size ranking in two-queen colonies from the viewpoint of first worker brood production and weight gain. To assign parentage to offspring of two-queen colonies we used mtDNA restriction fragment length polymorphism (RFLP); the results suggested that the two queens produced first worker brood equally throughout colony foundation. Furthermore, initially smaller females gained significantly more weight than initially larger females. This may have resulted from altruistic behaviour of the larger females towards the smaller ones. A simple mathematical model, which considered resource allocation and survivorship, could explain why the larger females behave altruistically towards the smaller females. We also examined the responses of females when more than two females were placed in a petri dish in the presence or absence of a male. If a partner male was present, only one female survived in the colony. In the absence of a partner male, two females, but never more than two, founded a colony cooperatively. These results show that females need a partner to found, and retain, a colony. Copyright 2002 The Association for the Study of Animal Behaviour. Published by Elsevier Science Ltd. All rights reserved .

Mechanism, induction factors, and adaptive significance of dealation in the subterranean termite Reticulitermes speratus (Isoptera, Rhinotermitidae)

We studied the dealation of a subterranean termite Reticulitermes speratus Kolbe from three different angles; mechanism, induction factors, and adaptive significance. During nuptial flight alates prevent the wings from breaking off by holding the wings in a 3-dimensional configuration. In the process of dealation, the alates hold the wings in a 2-dimensional configuration, half spread the wings in a lowered position, and bend the abdomen back toward the wings, thereby exerting high pressure at the basal sutures. This separates the wings from the body. The primary factor determining the timing of dealation appears to be isolation from other alates: It induces alates to shed their wings, whereas pairing has no effect on dealation in this species. Alates of termites are especially vulnerable to ant predation during the period between flight and colony foundation. An experiment designed to compare the risk of alates and dealates to predation by a termite-hunting ant, Brachyponera chinensis Emery, showed that dealation reduced the predation risk during the period spent running on the ground.

Colony-level stabilization of soldier head width for head-plug defense in the termite Reticulitermes speratus (Isoptera: Rhinotermitidae)

Termite colonies invest in the production and maintenance of soldiers, which specialize in defense and must be fed by workers. In Reticulitermes termites, however, the role of soldiers as colony defenders has been questioned. I found that Reticulitermes speratus Kolbe soldiers use phragmotic defenses (blocking openings with their heads) that prevent enemies from invading an undamaged part of the nest. In experimental nest-like conditions, Reticulitermes soldiers effectively blocked nest access of the termite-hunter ant Brachyponera chinensis Emery, using a combination of head-plug defense and mandibular threats. Phragmotic defense requires that soldiers have heads wide enough to block the small nest openings. This suggests that stabilizing selection should act on soldier head width. Comparison of the coefficient of variation of head width with that of other body parts showed that head width is a stable character. Analysis of the relative growth of head width and head length suggested that stabilizing selection on soldier head width operates at the colony level. In contrast, soldier head width was as variable as other body parts in non-phragmotic Coptotermes formosanus Shiraki and Nasutitermes takasagoensis Shiraki soldiers. Within-colony uniformity of soldier head width is likely an adaptation of Reticulitermes soldiers for phragmotic defense.

Diversification of the microtubule system in the early stage of eukaryote evolution: elongation factor 1 alpha and alpha-tubulin protein phylogeny of termite symbiotic oxymonad and hypermastigote protists.

The symbiotic protists of the lower termite have been regarded as a model of early-branched eukaryotes because of their simple cellular systems and morphological features. However, cultivation of these symbiotic protists is very difficult. For this reason, these interesting protists have not been well characterized in terms of their molecular biology. In research on these organisms which have not yet been cultivated, we developed a method for retrieving specific genes from a small number of cells, through micromanipulation without axenic cultivation, and we obtained EF-1 alpha and alpha-tubulin genes from members of the Hypermastigida--the parabasalid protist Trichonympha agilis and the oxymonad protists Pyrsonympha grandis and Dinenympha exilis--from the termite Reticulitermes speratus gut community. Results of phylogenetic analysis of the amino acid sequences of both proteins, EF-1 alpha and alpha-tubulin, indicate that the hypermastigid, parabasalid, and oxymonad protists do not share a close common ancestor. In addition, although the EF-1 alpha phylogeny indicates that these two groups of protists branched at an early stage of eukaryotic evolution, the alpha-tubulin phylogeny indicates that these protists can be assigned to two diversified clades. As shown in a recent investigation of alpha-tubulin phylogeny, eukaryotic organisms can be divided into three classes: an animal--parabasalids clade, a plant--protists clade, and the diplomonads. In this study, we show that parabasalids, including hypermastigids, can be classified as belonging to the animal--parabasalids clade and the early-branching eukaryote oxymonads can be classified as belonging to the plant--protists clade. Our findings suggest that these protists have a cellular microtubule system that has diverged considerably, and it seems that such divergence of the microtubule system occurred in the earliest stage of eukaryotic evolution.

Diverse genes of cellulase homologues of glycosyl hydrolase family 45 from the symbiotic protists in the hindgut of the termite Reticulitermes speratus.

Diverse genes encoding cellulase homologues belonging to glycosyl hydrolase family 45 were identified from the symbiotic protists in the hindgut of the termite Reticulitermes speratus through the use of consensus PCR and the screening of a cDNA library. Fifteen full-length cDNA clones were isolated and sequenced, which encoded polypeptides consisting of 218-221 amino acid residues showing up to 63% identity to known family 45 cellulases. The cellulase sequences of the termite symbiotic protists were phylogenetically monophyletic, showing more than 75% amino acid identity with each other. These enzymes consist of a single catalytic domain, lacking the ancillary domains found in most microbial cellulases. By whole-cell in situ hybridization using oligonucleotide probes specific for regions conserved in some of the sequences, the origin of the genes was identified as symbiotic hypermastigote protists. The presence of diverse cellulase homologues suggests that symbiotic protists of termites may be rich reservoirs of novel cellulase sequences.

Symbiosis of a termite and a sclerotium‐forming fungus: Sclerotia mimic termite eggs

Brown balls, of a similar size but different shape to termite eggs, were found frequently in the piles of eggs of the subterranean termite Reticulitermes speratus. rDNA analysis identified the ball as the sclerotia of the fungus, Fibularhizoctonia sp. nov, which is phylogenetically closest to decay fungi, Athelia spp. Laboratory observation showed that the workers gathered the eggs and the sclerotia indiscriminately, even if they were widely scattered in a Petri dish, and piled them up in a specific place for egg care. We compared the morphology of the eggs with that of sclerotia of Fibularhizoctonia spp. and Athelia spp. in relation to egg carrying behaviour, and found that the workers could only carry the Fibularhizoctonia spp. sclerotia whose diameters were similar to the short diameter of the eggs. We also conducted a bioassay using termite eggs and dummy eggs (glass beads and sea sand) of two diameter‐classes, coated with or without the egg‐derived chemicals. The workers recognized the eggs based on a combination of the size, shape, and chemical cues. All the results suggested that the sclerotia mimic the eggs both morphologically and chemically. Finally, we found that the workers suppressed germination of sclerotia, and termite egg survival increased in the presence of sclerotia only if they were tended by the workers. If the workers were removed experimentally, the sclerotia germinated and grew by exploiting termite eggs. These results suggest that the sclerotia protect termite eggs from putative pathogens.

Foraging Populations and Territories of Reticulitermes speratus (Isoptera: Rhinotermitidae)

Three field colonies of the subterranean termite Reticulitermes speratus (Kolbe) on the Uji campus of Kyoto University, Uji, Kyoto, Japan, were surveyed to estimate foraging populations and territories using a mark—release–recapture method in 1994–1996. The 3 foraging populations ranged from 109,400 to 466,400 termites per colony. Although the data must be interpreted carefully because the assumptions that support the efficacy of the procedure may have been violated, these estimated foraging populations were much larger than those reported previously for the species. The longest linear distance of foraging movement was 10 m and foraging territories ranged from 6.0 to 56.6 m2 per colony during the period of survey.

Phylogenetic identification of the symbiotic hypermastigote Trichonympha agilis in the hindgut of the termite Reticulitermes speratus based on small-subunit rRNA sequence.

The phylogeny of a symbiotic hypermastigote Trichonympha agilis (class Parabasalia; order Hypermastigida) in the hindgut of the lower termite Reticulitermes speratus was examined by a strategy that does not rely on cultivation. From mixed-population DNA obtained from the termite gut, small subunit (16S-like) ribosomal RNA sequences were directly amplified by the polymerase chain reaction method using primers specific for eukaryotes. Comparative sequence analysis of the clones revealed two kinds of sequences, one from the termite itself and the other from a symbiotic protist. A fluorescent-labeled oligonucleotide probe for the latter sequence was designed and used in whole-cell hybridization experiments to provide direct visual evidence that the sequence originated from a larger hypermastigote in the termite hindgut, Trichonympha agilis. According to the phylogenetic trees constructed, the hypermastigote represented one of the deepest branches of eukaryotes. The hypermastigote along with members of the order Trichomonadida formed a monophyletic lineage, indicating that this hypermastigote and trichomonads shared a recent common ancestry.

Phylogenetic position of symbiotic protist Dinenympha [correction of Dinemympha] exilis in the hindgut of the termite Reticulitermes speratus inferred from the protein phylogeny of elongation factor 1 alpha.

The phylogenetic position of the symbiotic oxymonad Dinenympha exilis, found in the hindgut of the lower termite Reticulitermes speratus, was determined by analysis of translation elongation factor 1 alpha (EF-1 alpha). cDNA corresponding to a major part of the amino acid coding region of EF-1 alpha mRNA was amplified by the reverse transcription polymerase chain reaction (RT-PCR) method from total mRNA of termite hindgut microorganisms without cultivation. The product was cloned into a plasmid vector, pGEM-T, and the clones were isolated and sequenced. One of the EF-1 alpha clones isolated was assigned to the protist D. exilis by whole-cell in-situ hybridization using a specific oligonucleotide probe with enzymatic signal amplification. The deduced amino acid sequence was aligned with those of other eukaryotic and archaeabacterial EF-1 alpha s, and the phylogenetic relationships among early branching eukaryotes were inferred by using the distance matrix method and the maximum parsimony method. The phylogenetic analysis indicated that the D. exilis offshoot occurred before mitochondria-containing organisms and D. exilis branched out after the diplomonads clade. These results indicate that the oxymonad D. exilis is one of the early branching organisms and suggest that the oxymonads form a lineage independent of other early branching organisms.