Uncovering the Cultivable Microbial Diversity of Costa Rican Beetles and Its Ability to Break Down Plant Cell Wall Components

<div><p>Coleopterans are the most diverse insect order described to date. These organisms have acquired an array of survival mechanisms through their evolution, including highly efficient digestive systems. Therefore, the coleopteran intestinal microbiota constitutes an important source of novel plant cell wall-degrading enzymes with potential biotechnological applications. We isolated and described the cultivable fungi, actinomycetes and aerobic eubacteria associated with the gut of larvae and adults from six different beetle families colonizing decomposing logs in protected Costa Rican ecosystems. We obtained 611 isolates and performed phylogenetic analyses using the ITS region (fungi) and 16S rDNA (bacteria). The majority of fungal isolates belonged to the order Hypocreales (26% of 169 total), while the majority of actinomycetes belonged to the genus <i>Streptomyces</i> (86% of 241 total). Finally, we isolated 201 bacteria spanning 19 different families belonging into four phyla: Firmicutes, α, β and γ-proteobacteria. Subsequently, we focused on microbes isolated from Passalid beetles to test their ability to degrade plant cell wall polymers. Highest scores in these assays were achieved by a fungal isolate (<i>Anthostomella</i> sp.), two <i>Streptomyces</i> and one <i>Bacillus</i> bacterial isolates. Our study demonstrates that Costa Rican beetles harbor several types of cultivable microbes, some of which may be involved in symbiotic relationships that enable the insect to digest complex polymers such as lignocellulose.</p></div

Distribution of enzymatic activities of microbial isolates.

a<p>Lignin peroxidase activity in MEA-RBBR.</p>b<p>Cellulase activity in CMC agar.</p>c<p>Laccase activity on ABTS test.</p>d<p>Glucanase activity.</p>e<p>Xylanase activity.</p>f<p>Cellobiase activity.</p><p>*Taxonomic identification was not possible with the molecular markers available.</p><p>Distribution of enzymatic activities of microbial isolates.</p

Pattern of enzymatic degradation of plant cell wall components for the fungal and bacterial isolates obtained from Passalid beetles.

<p>Heat map of the enzymatic activity of fungi, eubacteria and actinomycetes by sample type (blue represents the lower activity whereas red represents higher activity on the assay). Numbers correspond to sample number. RBBR: Lignin peroxidase activity in MEA-RBBR. CMC: Cellulase activity in CMC agar. LAC: Laccase activity on ABTS test. G: Glucanase activity. X: Xylanase activity. C: Cellobiase activity.</p

Bayesian phylogenetic tree of the ITS region sequences of fungi isolated from the gut of Costa Rican beetles.

<p>Numbers above branches represent their Bayesian-calculated posterior probabilities (two million generations, chain temperature  = 0.2, standard deviation <0.02). Numbers between brackets represent the number of isolates that shared the same sequence. Compress branches are colored in green. Red diamonds indicates positive cellulolytic activity.</p

Bayesian phylogenetic tree of the rDNA 16S gene sequences of actinomycetes isolated from the gut of Costa Rican beetles.

<p>Numbers above branches represent their Bayesian-calculated posterior probabilities (two million generations, chain temperature  = 0.2, standard desviation <0.02). Compress branches are colored in green. Red diamonds indicates positive cellulolytic activity.</p

Distribution of microbial isolates according to Coleopteran families sampled.

<p>Distribution of microbial isolates according to Coleopteran families sampled.</p