Characterization of the colonising bacterial populations of hindgut lobes from neonate larvae of Costelytra zealandica (Coleoptera: Scarabaeidae)

Larvae of the New Zealand grass grub, Costelytra zealandica (Coleoptera: Scarabaeidae) live in the soil where they feed on plant roots and organic matter of low nutritive value. They are characterised by modification of the hindgut into an expanded, microbe-rich, chamber lined with distinctive lobes. In order to assess the process of bacterial colonisation of the lobes, we examined hindgut samples from neonate and later stage larvae by fluorescence and electron microscopy, anaerobic cultivation and denaturing gradient gel electrophoresis (DGGE). Microscopy confirmed the absence of bacteria from the developing lobes of neonate larvae and the rapid colonisation of these lobes by bacteria. Electron microscopy and anaerobic cultivation from washed lobes confirmed the presence of spore forming bacteria. Characteristic isolates were identified by partial sequencing of the 16S rRNA gene. Sequences were affiliated to Carnobacterium sp. and Clostridium frigidicarnis which is known as an amoeba-resisting bacterium. Bacterial 16S rDNA clone libraries were generated from excised DGGE bands. Clones were characterized by amplified ribosomal DNA restriction analysis (ARDRA) and 16S rDNA sequencing.The results indicate that the bacterial community of the hindgut comprises of a stable population of spore forming bacteria that is established within 24 hours after larval emergence from the egg shell. The genus Clostridium has been identified as providing typical and numerically significant members of the hindgut microbiota of Costelytra zealandica.