Investigating the lignocellulolytic gut microbiome of huhu grubs

Abstract

New Zealand’s endemic longhorned beetle (Prionoplus reticularis) larvae, huhu grub, are xylophagous and feed on a broad range of dead trees. Since insects are not believed to have the ability to fully degrade lignocellulose, it is likely that microbes present in the gut assist in degrading lignocellulose to release energy rich sugars. This process is considered the rate limiting step in the utilisation of woody material in biofuel production. To better understand the gut consortium present while feeding on the lignocellulose and cellulose components, huhu grubs were initially collected and reared on non-degraded pine blocks (lignocellulose) or cotton (cellulose). The gut fungal and bacterial community of huhu grubs was investigated using high-throughput sequencing and analysis techniques. Furthermore, the effects of a dietary switch on both fungal and bacterial communities was explored by switching huhu grubs initially reared on pine to cotton, and vice versa. Cellulose- and lignocellulose-reared huhu grubs were expected to host a wide range of bacteria and fungi in their gut, some of which produce lignocellulose degrading enzymes. Assessment of the gut fungal community composition confirmed that cellulose-reared huhu grubs were significantly distinct from lignocellulose-reared grubs. The fungal community of lignocellulose-fed huhu grubs was more functionally diverse, with several yeasts, soft-rot fungi and potential hemicellulose and cellulose degraders present. Whereas, the fungal community of cellulose-reared grubs was mostly abundant in yeasts and potential cellulose degraders. When cotton-reared grubs where switched to pine (lignocellulose), lignocellulose was observed to have a ‘bottleneck effect’ or selective pressure on the fungal gut community. The gut bacterial community of huhu grubs reared on cellulose or lignocellulose diets seemed to be unaffected by the selective pressure of the diet, and no significant change in the bacterial community was observed following dietary switch. Therefore it was concluded that the bacterial community while still important is playing a secondary role to the fungi. Finally, bioprospecting studies conducted using the frass (excretia) of these huhu grubs resulted in the isolation of a bacterium, Acinetobacter H23, capable of producing an extracellular laccase. Overall, the increased understanding of structure and composition of the gut microbiome of huhu grubs reared on cellulose or lignocellulose diets, along with isolation of novel lignocellulolytic isolates could prove to be significant for the biofuel industry.