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Genomics and metagenomics of bacterial endosymbionts of pest insect

Final Report Summary - GENOESYMBPESTINSECTS (Genomics and metagenomics of bacterial endosymbionts of pest insect)

Project objectives

The project dealt with the genomic analysis of bacterial endosymbionts of undesired or agricultural pests like cockroaches, white flies and the olive fly. All these insect groups harbour symbiotic bacteria as obligate or facultative partners, which play important functional roles for the host, e.g. via provision of essential amino acids or an involvement in nitrogen recycling. The impact of the project is a better understanding of the biology of endosymbionts of insects, especially of Blattabacterium sp., the symbiont of cockroaches.

The objectives, research focus and methods were different for each of the three studied symbioses:

1. full genome sequencing, assembly, annotation and comparative analysis of genomes of Blattabacterium sp., the cockroach symbiont;
2. the characterisation of the bacterial microbiota of white flies, consisting of one primary and several secondary symbionts;
3. determining the genome size of the olive fly symbiont, Cand. Erwinia dacicola.

Genome sequencing and comparative sequence analysis of exemplary members of these insect groups will yield new insights in the evolution of the symbionts and the biology of the partnerships.

Work performed

Regarding the Blattabacterium sp./cockroach symbiosis, an analysis of complete genomes of two blattabacteria clarified the role of the bacterial partner. Blattabacterium sp. is implicated in the nitrogen economy of the host, thereby fulfilling a double function: firstly providing essential amino acids to the hosts and secondly, being involved in recycling and excreting nitrogen via an urease. The latter activity explains the excretion of nitrogen in the form of ammonia, contrary to the excretion as uric acid in most other insects. In comparative genome analyses, blattabacteria showed to have a gene content that was most similar to members of the genus Blochmannia, symbionts of carpenter ants. The hosts of these two groups of symbionts share an omnivorous lifestyle. For the wood-feeding roach, Cryptocercus punctulatus, complete sequencing showed a further reduction of the Blattabacterium sp. genome. The C. punctulatus symbiont has, in particular, lost genes for the synthesis of amino acids, which leaves its capacity for urea degradation as its central symbiotic function. Besides Blattabacterium, wood roaches contain intestinal symbionts that are responsible for digesting wood, similar to termites. The change of lifestyle to xylophagy, however, occurred when blattabacteria were already long-established symbiotic partners. Co-evolution of Blattabacterium sp. and C. punctulatus resulted in the loss of biosynthetic pathways in the symbiont. Due to their distinct biology, wood roaches appear to have an intermediary position between common cockroaches and termites. Comparative data on the blattabacteria genomes supports this.

For the white fly, Bemisia tabaci, two strains originating from Spain and Israel and belonging to biotypes Q and B were fully characterised for their symbiont content. These two biotypes are known to have the highest negative impact of agricultural pests in the Mediterranean. Beside the common primary symbiont, Portiera aleyrodidarum, the two strains contained Hamiltonella as secondary symbionts and, in addition, the Israeli strain also contained Rickettsia and the Spanish strain Cardinium. A quantitative PCR (qPCR) assay was set up to measure the deoxyribonucleic acid (DNA) contents of the different symbionts more precisely. It was used routinely to determine the composition of the extracted DNA as a preparatory step for the metagenomic sequencing of the symbiotic communities.

For the olive fly symbiont, Candidatus Erwinia dacicola, a pulsed-field gel electrophoresis approach was used to determine the genome size. Using two different rare-cutting restriction enzymes, BlnI and I-CeuI, the total size of the chromosome of Cand. E. dacicola was estimated to be about 3.5 Mb.

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