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Abstract:
A characterization of the bacterial community of the hindgut wall of two larval and the adult stages of the forest cockchafer (Melolontha hippocastani) was carried
out using amplicon sequencing of the 16S rRNA gene fragment. We found that,
in second-instar larvae, Caulobacteraceae and Pseudomonadaceae showed the
highest relative abundances, while in third-instar larvae, the dominant families were
Porphyromonadaceae and Bacteroidales-related. In adults, an increase of the relative
abundance of Bacteroidetes, Proteobacteria (g- and d- classes) and the family
Enterococcaceae (Firmicutes) was observed. This suggests that the composition of the
hindgut wall community may depend on the insect’s life stage. Additionally, specialized
bacterial niches hitherto very poorly described in the literature were spotted at both
sides of the distal part of the hindgut chamber. We named these structures “pockets.”
Amplicon sequencing of the 16S rRNA gene fragment revealed that the pockets
contained a different bacterial community than the surrounding hindgut wall, dominated
by Alcaligenaceae and Micrococcaceae-related families. Poly-b-hydroxybutyrate (PHB)
accumulation in the pocket was suggested in isolated Achromobacter sp. by Nile
Blue staining, and confirmed by gas chromatography–mass spectrometry analysis (GCMS)
on cultured bacterial mass and whole pocket tissue. Raman micro-spectroscopy
allowed to visualize the spatial distribution of PHB accumulating bacteria within the
pocket tissue. The presence of this polymer might play a role in the colonization of
these specialized niches.