Culturing-Enriched Metabarcoding Analysis of the Oryctes rhinoceros Gut Microbiome

Abstract

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.

Chryseobacterium oryctis sp. nov., isolated from the gut of the beetle Oryctes rhinoceros, and Chryseobacterium kimseyorum sp. nov., isolated from a stick insect rearing cage

“…The strain, identified with the code 09-1422, was stored in glycerol at −80 °C in the Phaff Yeast Culture Collection of the University of California Department of Food Science and Technology. In 2022, a sample from this stored strain was sent to National Taiwan University and PCR used to re-amplify the 16S rRNA and rpoB housekeeping genes, using the respective primer pairs 27F−1492R and rpoB1206−rpoBR3202 [13], as described previously [19]. Its closest relatives were Chryseobacterium aquaticum subsp.…”

“…Plates were incubated aerobically at 30 °C. Microbes with distinct morphologies were isolated into pure cultures on tryptone soy agar (TSA; HiMedia) plates and identified using PCR to amplify the 16S gene and rpoB housekeeping genes using the respective primer pairs 27F-1492R and rpoB1206-rpoBR3202 [13], as described previously [19].…”

“…On 17 August, 2020, Oryctes rhinoceros beetle larva were collected from a rotting Areca catechu palm in the rural Wanluan township, Pingtung County, Taiwan (22.589360° N 120.577031° E), and transported in palm pulp to the laboratory of the Department of Entomology, National Taiwan University. As part of a study on the microbiology of these beetles [19], larvae were killed by freezing, surface-sterilized in 50 % bleach, rinsed twice in RO-water, and dissected with sterile tools under a laminar flow hood. Their midgut and hindgut contents were separately plated onto nutrient agar (NA; HiMedia) plates with the following composition: 0.5 % peptic digest of animal tissue, 0.5 % sodium chloride, 0.13 % beef extract, 0.15 % yeast extract and 1.5 % agar.…”

Pseudomonas schmalbachii sp. nov., isolated from the gut of a millipede (Trigoniulus corallinus) from a coconut tree

“…These were identified by sequencing of the 16S rRNA and rpoB housekeeping genes, using the respective primer pairs 27 F-1492R and rpoB1206-rpoBR3202 [13]. PCR conditions were as previously described [11]. While three of the strains were positively identified with 99.5–100% sequence similarities to those of published type specimens in GenBank using blast n [14], Milli4 T was not and had closest similarities to Pseudomonas knackmussi B13 T (98.7 % 16S sequence identity) and Pseudomonas humi CCA1 T (92.6% rpoB sequence identity).…”

“…As part of a larger study on the microbes associated with rotting palm trees in Taiwan and the saprophytic arthropods that consume them [11], we isolated a strain of Pseudomonas named Milli4 T from the digestive tract of the common Asian or rusty millipede, Trigoniulus corallinus (Spirobolida: Trigoniulidae), taken from a decaying coconut tree, Cocos nucifera (Arecaecae). The strain differs from extant Pseudomonas species in its genomics and chemotaxonomy, and so we propose to establish for this strain the species name Pseudomonas schmalbachii sp.…”