Imaging unimaginable symbiotic interactions - bacterial lineage splitting in a grain (pest) beetle.

Session

Poster Session

Authors

Dongik Chang (1), Julian Kiefer (2), Veit Grabe (3), Martin Kaltenpoth (1), Tobias Engl (1)

Affiliations

1. Department of Insect Symbiosis, Max Planck Institute for Chemical Ecology
2. Department of Symbiosis, Max planck Institute for Marine Microbiology,
3. Microscopic Imaging, Max Planck Institute for Chemical Ecology

Keywords

Insect symbiosis, Bostrichidae, shikimate pathway, FISH, super-resolution microscopy

Abstract text

The evolutionary success of many insects is attributed to mutualistic associations with microbes, which provide nutrition, defense compounds, and other natural products. Beetles are armored with a particularly thick and hard cuticle but not all diets provide sufficient amounts of the semi-essential amino acid tyrosine for cuticle formation. Thus, many beetles evolved symbiotic associations with symbiotic bacteria that complement dietary tyrosine via the shikimate pathway. Among grain pest beetles with tyrosine supplementary symbionts, Prostephanus truncatus harbors the ancient symbiont Shikimatogenerans bostrichidophilus that diverged into three lineages with complementary gene repertoires. Based on the symbiont genome assemblies, Fluorescence in situ hybridization (FISH) was used to localize bacterial lineage-specific DNA and RNA molecules. We resolved their 3D location within densely packed symbiont cells inside specialized insect organs via tissue clearing combined with highly resolved fluorescence microscopy (adaptive deconvolution and STED). Imaging fluorescence-lifetime (FLIM) allowed multiplexing of FISH probes and counter staining while differentiating autofluorescence of insect tissues. In addition, after manipulating beetle diet and symbiont load, we measured the cuticle thickness and melanization of beetles using µCT scanning and an AI-supported pixel classifier. The symbiont genomic analyses showed complementary gene distributions among three bacterial lineages and implied the obligate metabolic exchange among symbionts. Various FISH results demonstrated life-stage-dependent dynamics of symbiont distribution and presumable interactions among symbiont cells via connections and transport. Lastly, experimental manipulation of symbionts resulted in thinner and brighter beetle cuticles, which verified the functional integrity of metabolically fragmented symbiont genomes. The results suggest that Prostephanus necessarily needs to maintain and transmit three lineages of symbionts, which is widely believed to be non-adaptive. Applying advanced imaging techniques, we depicted potential mechanisms of interactions among the interdependent bacteria. Quantification of the often co-localized signals and imaging of the presumably exchanged metabolites will be necessary to understand biochemical dynamics. Further, more studies on the biological dynamics of the symbiotic interactions and distribution in different beetle life stages are needed to elucidate the evolutionary driver of symbiont lineage splitting and their maintenance, which may be an overlooked phenomenon in other symbiosis systems due to the genomic similarities.