Culturing-Enriched Metabarcoding Analysis of the Oryctes rhinoceros Gut Microbiome.

Abstract

Simple SummaryThe coconut rhinoceros beetle is a pest of palm trees, which may have symbiotic gut microbes that help it digest its food. These microbes may produce enzymes like cellulase, which have uses in human industry. If the microbes are essential for the beetle’s survival, then finding ways to attack the microbes could help fight the pest. We sampled microbes from the guts of larval beetles collected in coconut trees in southern Taiwan, and identified the microbes both by culturing and with molecular biology methods. We found several species of bacteria and a yeast, Candida xylanolytica, with potential digestive functions for the beetle. Some of these microbes had been reported in these beetles before while others are new. Broader surveys of the beetle microbiome are needed to determine whether or not they have a conserved microbiome.Wood-feeding insects should have a source of enzymes like cellulases to digest their food. These enzymes can be produced by the insect, or by microbes living in the wood and/or inside the insect gut. The coconut rhinoceros beetle, Oryctes rhinoceros, is a pest whose digestive microbes are of considerable interest. This study describes the compartments of the O. rhinoceros gut and compares their microbiomes using culturing-enriched metabarcoding. Beetle larvae were collected from a coconut grove in southern Taiwan. Gut contents from the midgut and hindgut were plated on nutrient agar and selective carboxymethylcellulose agar plates. DNA was extracted from gut and fat body samples and 16S rDNA metabarcoding performed to identify unculturable bacteria. Cellulase activity tests were performed on gut fluids and microbe isolates. The midgut and hindgut both showed cellulolytic activity. Bacillus cereus, Citrobacter koseri, and the cellulolytic fungus Candida xylanilytica were cultured from both gut sections in most larvae. Metabarcoding did not find Bacillus cereus, and found that either Citrobacter koseri or Paracoccus sp. were the dominant gut microbes in any given larva. No significant differences were found between midgut and hindgut microbiomes. Bacillus cereus and Citrobacter koseri are common animal gut microbes frequently found in Oryctes rhinoceros studies while Candida xylanilytica and the uncultured Paracoccus sp. had not been identified in this insect before. Some or all of these may well have digestive functions for the beetle, and are most likely acquired from the diet, meaning they may be transient commensalists rather than obligate mutualists. Broader collection efforts and tests with antibiotics will resolve ambiguities in the beetle–microbe interactions.