Endosymbiotic Microbiota of the Bamboo PseudococcidAntonina crawii(Insecta, Homoptera)

We characterized the intracellular symbiotic microbiota of the bamboo pseudococcidAntonina crawii by performing a molecular phylogenetic analysis in combination with in situ hybridization. Almost the entire length of the bacterial 16S rRNA gene was amplified and cloned fromA. crawii whole DNA. Restriction fragment length polymorphism analysis revealed that the clones obtained included three distinct types of sequences. Nucleotide sequences of the three types were determined and subjected to a molecular phylogenetic analysis. The first sequence was a member of the γ subdivision of the divisionProteobacteria (γ-Proteobacteria ) to which no sequences in the database were closely related, although the sequences of endosymbionts of other homopterans, such as psyllids and aphids, were distantly related. The second sequence was a β-Proteobacteria sequence and formed a monophyletic group with the sequences of endosymbionts from other pseudococcids. The third sequence exhibited a high level of similarity to sequences ofSpiroplasma spp. from ladybird beetles and a tick. Localization of the endosymbionts was determined by using tissue sections ofA. crawii and in situ hybridization with specific oligonucleotide probes. The γ- and β-Proteobacteria symbionts were packed in the cytoplasm of the same mycetocytes (or bacteriocytes) and formed a large mycetome (or bacteriome) in the abdomen. The spiroplasma symbionts were also present intracellularly in various tissues at a low density. We observed that the anterior poles of developing eggs in the ovaries were infected by the γ- and β-Proteobacteria symbionts in a systematic way, which ensured vertical transmission. Five representative pseudococcids were examined by performing diagnostic PCR experiments with specific primers; the β-Proteobacteria symbiont was detected in all five pseudococcids, the γ-Proteobacteria symbiont was found in three, and the spiroplasma symbiont was detected only inA. crawii .