Osamu Kitade

Phylogenetic evidence for a single, ancestral origin of a ‘true’ worker caste in termites

Phylogenetic analysis based on sequence variation in mitochondrial large‐subunit rRNA and cytochrome oxidase II genes was used to investigate the evolutionary relationships among termite families. Maximum likelihood and parsimony analyses of a combined nucleotide data set yield a single well‐supported topology, which is: (((((Termitidae, Rhinotermitidae), Serritermitidae), Kalotermitidae), (Hodotermitidae, Termopsidae)), Mastotermitidae). Although some aspects of this topology are consistent with previous schemes, overall it differs from any published. Optimization of ‘true’ workers onto the tree suggests that this caste originated once, early in the history of the lineage and has been lost secondarily twice. This scenario differs from the more widely accepted notion that workers are derived and of polyphyletic origin and that extant pseudergates, or ‘false’ workers, are their developmentally unspecialized ancestor caste. Worker gains and losses covary directly in number and direction with shifts in ‘ecological life type’. A test for correlated evolution which takes phylogenetic structure into account indicates that this pattern is of biological significance and suggests that the variable occurrence of a worker caste in termites has ecological determinants, apparently linked to differences in feeding and nesting habits.

Endosymbiotic Bacteroidales Bacteria of the Flagellated Protist Pseudotrichonympha grassii in the Gut of the Termite Coptotermes formosanus

ABSTRACT A unique lineage of bacteria belonging to the order Bacteroidales was identified as an intracellular endosymbiont of the protist Pseudotrichonympha grassii (Parabasalia, Hypermastigea) in the gut of the termite Coptotermes formosanus. We identified the 16S rRNA, gyrB, elongation factor Tu, and groEL gene sequences in the endosymbiont and detected a very low level of sequence divergence (<0.9% of the nucleotides) in the endosymbiont population within and among protist cells. The Bacteroidales endosymbiont sequence was affiliated with a cluster comprising only sequences from termite gut bacteria and was not closely related to sequences identified for members of the Bacteroidales attached to the cell surfaces of other gut protists. Transmission electron microscopy showed that there were numerous rod-shaped bacteria in the cytoplasm of the host protist, and we detected the endosymbiont by fluorescence in situ hybridization (FISH) with an oligonucleotide probe specific for the 16S rRNA gene identified. Quantification of the abundance of the Bacteroidales endosymbiont by sequence-specific cleavage of rRNA with RNase H and FISH cell counting revealed, surprisingly, that the endosymbiont accounted for 82% of the total bacterial rRNA and 71% of the total bacterial cells in the gut community. The genetically nearly homogeneous endosymbionts of Pseudotrichonympha were very abundant in the gut symbiotic community of the termite.

Symbiotic Associations with Protists

Progress in understanding the symbiosis between protists and termites has not matched that between prokaryotes and termites. Methods are now available for the isolation of pure cultures of trichomonads and hypermastigids, although only a few have been cultivated. Sufficient molecular data are now available to construct tentative phylogenetic trees. Molecular data indicate that these organisms are amongst the most primitive eukaryotes lacking mitochondria; the trichomonads also use a prokaryote-like 70S ribosome. In metabolic terms, more is known about the cellulolytic protists but only in outline.

Molecular phylogeny of the Rhinotermitidae

Summary.Relationships among genera in the termite family Rhinotermitidae and their relationship to the families Termitidae and Serritermitidae were investigated based on analysis of three mitochondrial genes: COI, COII and 16S rDNA. Maximum Parsimony (MP) bootstrap analysis of each of these genes indicated a low level of phylogenetic incongruence between them, and thus they were combined and analysed by MP and Bayesian analysis. Six main lineages were clearly identified, however relationships among these were not well defined. Tentative support was found for the Rhinotermitid genera Coptotermes, Heterotermes and Reticulitermes being the sister group to the Termitidae, rendering the Rhinotermitidae paraphyletic. The species Serritermes serrifer and Glossotermes oculatus were found to group with strong support, in agreement with the recent transfer of the latter species to the family Serritermitidae based on morphological characteristics. No support was found for the Rhinotermitidae being paraphyletic with respect to the Serritermitidae. A number of disagreements were found between the molecular tree and traditional classifications of genera within subfamilies.

Molecular Phylogeny of Orthopteroid Insects based on the Mitochondrial Cytochrome Oxidase II Gene

Abstract Phylogenetic relationships among 18 species of orthopteroid insects (Blattaria: cockroaches, Isoptera: termites, Mantodea: mantids, Grylloblattodea: grylloblattids, Phasmatodea: stick-insects, Orthoptera-Caerifera: locusts, Orthoptera-Ensifera: crickets, and Dermaptera: earwigs), were estimated based on DNA sequencing of the mitochondrial cytochrome oxidase II gene. Our results drew attention to the need for caution in using third codon positions for tree construction, since it was likely that base pair substitutions of third codon positions in the COII gene were saturated among taxa used in the present study. We also detected that there were many phylogenetically informative sites in first codon positions. Phylogenetic trees using first and second codon positions based on both the neighbor-joining method and parsimony analysis indicated that the topology was nearly identical to each other. The phylogenetic relationships among these taxa differ from the current classification based on morphological characters. The inferred trees showed that grylloblattids were not a primitive group, but closely related to the Dictyoptera. Stick-insects were closely related to the Dictyoptera and grylloblattids, not to crickets. Locusts and crickets formed a monophyletic group. Earwigs were only distantly related to the Dictyoptera. Within the Dictyoptera, cockroaches and termites constituted a monophyletic group, with mantids as a sister group to that complex.

Nitrogen recycling through proctodeal trophallaxis in the Japanese damp-wood termite Hodotermopsis japonica (Isoptera, Termopsidae)

Summary: Trophallaxis is one of a variety of nutritional tactics that enable termites to conserve and recycle nitrogenous compounds. In this study, we designed three experiments to reveal the function and importance of proctodeal trophallaxis in the Japanese damp-wood termite Hodotermopsis japonica. First, our observations showed that younger instars and soldiers tended to behave as recipients of proctodeal fluid, while older instars acted mainly as donors. Secondly, nitrogen-deficient groups of termite individuals were introduced to groups of nourished individuals; results indicate that the frequency of proctodeal trophallaxis was significantly higher than in control groups. Additionally, the nitrogen content in the proctodeal fluid of nitrogen-deficient individuals increased after trophallactic interactions with well-nourished individuals. Finally, we set up three groups reared with different concentrations of nitrogenous compounds (amino acids) in their diet, to investigate the correlation between the frequency of trophallactic behavior and the protein concentration of proctodeal fluid. As expected, proctodeal fluid of low protein content was frequently exchanged among individuals in nitrogen-poor conditions, while high-protein proctodeal fluid was transferred less frequently under nitrogen-rich conditions. These results suggest that termites have plasticity of trophallactic behavior in response to their nutritional conditions, and trophallaxis contributes to the nutritional homeostasis of colonies.

Novel Clade of Rickettsia spp. from Leeches

ABSTRACT Intracellular rickettsia-like structures were found in the tissues of a glossiphoniid leech, Torix tagoi, by transmission electron microscopy. Diagnostic PCR analysis using specific primers suggested that of the nine glossiphoniid species examined, two species, T. tagoi and Hemicrepsis marginata, harbored bacteria of the genus Rickettsia. A 1.5-kb eubacterial 16S rRNA gene segment obtained from each of these species was amplified by PCR, cloned, and sequenced. Phylogenetic analysis of the 16S rRNA gene demonstrated that the Rickettsia species found in the leeches constituted a novel clade that is distinct from the clade of arthropod-associated Rickettsia species. In natural populations, 97.7% (43 of 44) of T. tagoi leeches and 100% (9 of 9) of H. marginata leeches carried Rickettsia, suggesting that infection with Rickettsia is prevalent in these leeches. This is the first report of Rickettsia found in annelids.

Phylogenetic Comparison of Endosymbionts with Their Host Ants Based on Molecular Evidence

Abstract Some Formicid ants have symbiotic intracellular bacteria in the epithelial cells of their midgut. These endosymbionts are believed to be derived from a common ancestor. A recent study revealed that endosymbionts of the ant genus Camponotus are closely related to Enterobacteriaceae, but their relationship to endosymbionts of other genera of ants is unknown. In this study, the nucleotide sequences of 16S ribosomal RNA (rRNA) of endosymbionts and mitochondrial cytochrome oxidase subunit I (COI) of their host were determined in five genera of the subfamily Formicinae (Hymenoptera: Formicidae). Based on these molecular data, we constructed phylogenetic trees in order to characterize the systematic position of the symbionts and to estimate the relationship of symbionts and hosts. The analysis showed that the endosymbionts were all connected with the Enterobacteriaceae but did not constitute a monophylitic group, while the three genera belonging to the tribe Camponotini, the endosymbionts and their hosts made a clade. The topologies of these trees were identical for the most part. These results suggest that the endosymbionts of ants have plural origins, and that in the Camponotini, ancestral symbionts have coevolved with their host ants, which are so divergent to several genera as to construct one tribe.

Molecular Phylogeny and Evolution of Parabasalia with Improved Taxon Sampling and New Protein Markers of Actin and Elongation Factor-1α

Background Inferring the evolutionary history of phylogenetically isolated, deep-branching groups of taxa—in particular determining the root—is often extraordinarily difficult because their close relatives are unavailable as suitable outgroups. One of these taxonomic groups is the phylum Parabasalia, which comprises morphologically diverse species of flagellated protists of ecological, medical, and evolutionary significance. Indeed, previous molecular phylogenetic analyses of members of this phylum have yielded conflicting and possibly erroneous inferences. Furthermore, many species of Parabasalia are symbionts in the gut of termites and cockroaches or parasites and therefore formidably difficult to cultivate, rendering available data insufficient. Increasing the numbers of examined taxa and informative characters (e.g., genes) is likely to produce more reliable inferences. Principal Findings Actin and elongation factor-1α genes were identified newly from 22 species of termite-gut symbionts through careful manipulations and seven cultured species, which covered major lineages of Parabasalia. Their protein sequences were concatenated and analyzed with sequences of previously and newly identified glyceraldehyde-3-phosphate dehydrogenase and the small-subunit rRNA gene. This concatenated dataset provided more robust phylogenetic relationships among major groups of Parabasalia and a more plausible new root position than those previously reported. Conclusions/Significance We conclude that increasing the number of sampled taxa as well as the addition of new sequences greatly improves the accuracy and robustness of the phylogenetic inference. A morphologically simple cell is likely the ancient form in Parabasalia as opposed to a cell with elaborate flagellar and cytoskeletal structures, which was defined as most basal in previous inferences. Nevertheless, the evolution of Parabasalia is complex owing to several independent multiplication and simplification events in these structures. Therefore, systematics based solely on morphology does not reflect the evolutionary history of parabasalids.

Parthenogenetic reproduction in neotenics of the subterranean termite Reticulitermes speratus (Isoptera: Rhinotermitidae)

Experiments with artificial colonies consisting of only female workers and those of female nymphs and workers of a subterranean termite, Reticulitermes speratus, showed that neotenics (ergatoids and nymphoids) can reproduce parthenogenetically. The larvae hatched from the eggs produced partheno‐genetically by the neotenics were all female and did not have any aberrant external morphology. They had 42 chromosomes and were diploid. The analysis of genotypes at microsatellite loci revealed that the eggs produced parthenogenetically had only one of the two alleles that the mother neotenics possessed, indicating that the eggs were produced by automixis.