The gut of wood-feeding lower termites is rich in symbiotic protozoa that play important roles in cellulose degradation. Previously the diverse hindgut symbiotic protozoa in wood-feeding lower termites were identified mainly based on morphological characters observed under light microscopy. Due to incomplete descriptions such as the shortage of images of many flagellate species, the identification of symbiotic flagellate species in unknown termite species becomes difficult and uncertain. With the development of molecular techniques, it has become possible to identify certain flagellate species via cloning 18S rRNA genes from mixed flagellate populations, in combination with the whole-cell in situ hybridization that facilitates determination of the origin of amplified genes. Reticulitermes chinensis Snyder is a wood-feeding lower termite widely distributes in China. However, the diversity of its gut symbiotic flagellates has not been reported. To analyze the phylogenetic diversity of the symbiotic Oxymonad flagellates in the gut of R. chinensis, the 18S rRNA genes of the symbiotic Oxymonads were amplified from the DNA extracts of the whole gut using Oxymonad specific primers. A rDNA library was established and the 18S rRNA genes of the Oxymonad species were analyzed. Eleven Oxymonad 18S rRNA genes with the similarity between 86.9% and 99.3% were obtained. Seuqnence DataBank searches showed that the sequence of rDNA Clone Rc52, Clone Rc23 and Clone RC3 had a high similarity to the SSU rDNA sequence of Dinenympha exilis, Dinenympha sp. and Dinenympha parva obtained from Reticulitermes speratus with the similarity of 97%, 97% and 98%, respectively. Phylogenetic analysis showed that these eleven Oxymonad rDNAs formed five distinct clusters. Eight of the eleven 18S rRNA genes formed four separate Dinenympha clusters. Among them three Dinenympha genes (rDNA Clone Rc52, Rc23, Rc3) each formed a distinct cluster, Cluster I, II, III, respectively. rDNA Clones Rc4, Rc7, Rc8, Rc19 and Rc35 with the sequence similarity between 98.5% and 99.3% from each other formed Dinenympha Cluster IV. The other three rDNA clones, Clone Rc13, Rc5 and Rc42 together with several Pyrsonympha SSU rDNA sequences obtained from R. flavipes and R. hesperus formed the fifth Cluster, Pyrsonympha Cluster.
To assign the 18S rRNA genes to their corresponding Oxymonad flagellates, whole-cell fluorescence in situ hybridization with sequence-specific fluorescently labeled probes was conducted. The results showed that three morphologically different oxymonads can be successfully identified with this technique. Probe Rc_Oxy1 that was specific to 18S rDNA of clone Rc3 (Cluster III) detected a flagellate similar in morphology to Dinenympha parva observed in R. speratus with an average length of 20μm. In situ hybridization with probe Rc_Oxy2 that was specific to clone Rc52 (Cluster I) showed that the targeted flagellate was Dinenympha exilis with an average length of 40μm. Using probe Rc_Oxy3 that was specific to 18S rDNA of clone Rc5 (Cluster V), a pear formed protist with an average length of 65μm gave a strong fluorescent signal. This protist showed typical morphology of the genus Pyrsonympha as observed in other wood-feeding lower termites. The primary studies showed that symbiotic Oxymonad flagellates inhabited the hindgut of Reticulitermes chinensis Snyder were morphologically and phylogenetically diverse and all of the identified Oxymonads were affiliated with the genera Dinenympha and Pyrsonympha.