Ultrastructural and molecular characterization of endosymbionts of the reed beetle genus Macroplea (Chrysomelidae, Donaciinae), and proposal of "Candidatus Macropleicola appendiculatae" and "Candidatus Macropleicola muticae".

Intracellular bacterial symbionts are known from various insect groups, particularly from those feeding on unbalanced diets, where the bacteria provide essential nutrients to the host. In the case of reed beetles (Coleoptera: Chrysomelidae, Donaciinae), however, the endosymbionts appear to be associated with specialized "glands" that secrete a material used for the beetles' unusual water-tight cocoon. These glands were discovered over a century ago, but the bacteria they contain have yet to be characterized and placed in a phylogenetic context. Here, we describe the ultrastructure of two endosymbiotic species ("Candidatus Macropleicola appendiculatae" and "Candidatus Macropleicola muticae") that reside in cells of the Malpighian tubules of the reed beetle species Macroplea appendiculata and Macroplea mutica, respectively. Fluorescent in situ hybridization using oligonucleotides targeting the 16S rRNA gene specific to Macroplea symbionts verified the localization of the symbionts in these organs. Phylogenetic analysis of 16S rRNA placed "Candidatus Macropleicola" in a clade of typically endosymbiotic Enterobacteriaceae (gamma-proteobacteria). Finally, we discuss the evidence available for the hypothesis that the beetle larvae use a secretion produced by the bacteria for the formation of an underwater cocoon.