Comparative analysis of microbiomes of wood feeding organisms Isabelle George, Univ. of Brussels Iris Uy, Philippine Genome Center, Univ. of the Philippines Jing Ge, China Research Center, Dupont Surya Saha, Cornell University

Hindgut Nasutitermes (Costa Rica) Fungus-growing Termite worker from South Africa The beetles bore through pine bark to eat and lay their eggs; the fungus spreads inside the bark and « pre-digests » the wood. SAMPLING: GALLERIES Mountain Pine Beetle from McBride, British Columbia, Canada Xyleborus affinis (Coleoptera, Curculionidae) from Bern, Switzerland, Dendroctonus frontalis (Coleoptera, Curculionidae) from Southern Mississipi Food: dead plant material (wood,fungi growing on dead leaves). SAMPLING:HINDGUT MICROFLORA ASSOCIATED WITH WOOD-FEEDING INSECTS METADATA NOT DETAILED ENOUGH

Our Questions ● Phylogenetic diversity ● Who are there? ● Is there a core set of taxa? ● Genes/Pathways of interest ● Cellulose degradation enzymes in action?? ● Nitrogen fixation genes in play?

NameProposal NameGenome SizeGene Count 5S rRNA Count 16S rRNA Count 23S rRNA Count Nasutitermes Costa Rica Gut microbiome of Costa Rica Nasutitermes termites from P3 luminal contents Fungus growing Termite South Africa Fungus-growing Termite Worker Mountain Pine Beetle British Columbia, CA Mountain Pine Beetle microbial communities from Grand Prairie, Alberta Dendroctonus frontalis Mississippi Dendroctonus frontalis Bacterial community Xyleborus affinis Switzerland Xyleborus affinis microbiome from Bern, Switzerland Selected Metagenomes : Some details

NameProposal NameGenome SizeGene Count 5S rRNA Count 16S rRNA Count 23S rRNA Count Nasutitermes Costa Rica Gut microbiome of Costa Rica Nasutitermes termites from P3 luminal contents Fungus growing Termite South Africa Fungus-growing Termite Worker Mountain Pine Beetle British Columbia, CA Mountain Pine Beetle microbial communities from Grand Prairie, Alberta Dendroctonus frontalis Mississippi Dendroctonus frontalis Bacterial community Xyleborus affinis Switzerland Xyleborus affinis microbiome from Bern, Switzerland Selected Metagenomes : Some details

NameProposal NameGenome SizeGene Count 5S rRNA Count 16S rRNA Count 23S rRNA Count Nasutitermes Costa Rica Gut microbiome of Costa Rica Nasutitermes termites from P3 luminal contents Fungus growing Termite South Africa Fungus-growing Termite Worker Mountain Pine Beetle British Columbia, CA Mountain Pine Beetle microbial communities from Grand Prairie, Alberta Dendroctonus frontalis Mississippi Dendroctonus frontalis Bacterial community Xyleborus affinis Switzerland Xyleborus affinis microbiome from Bern, Switzerland Selected Metagenomes : Some details

Functional annotation of selected metagenomes

Genome composition of selected metagenomes Dendroctonus frontalis Mississippi Fungus growing Termite South Africa Nasutitermes Costa Rica Mountain Pine Beetle British Columbia, CA Xyleborus affinis Switzerland Dendroctonus frontalis Mississippi Fungus growing Termite South Africa Nasutitermes Costa Rica Mountain Pine Beetle British Columbia, CA Xyleborus affinis Switzerland Pearson correlation matrix by Taxonomy (Family level)

Genome composition of selected metagenomes Dendroctonus frontalis Mississippi Fungus growing Termite South Africa Nasutitermes Costa Rica Mountain Pine Beetle British Columbia, CA Xyleborus affinis Switzerland Dendroctonus frontalis Mississippi Fungus growing Termite South Africa Nasutitermes Costa Rica Mountain Pine Beetle British Columbia, CA Xyleborus affinis Switzerland Pearson correlation matrix by Taxonomy (Family level)

Genome composition of selected metagenomes Dendroctonus frontalis Mississippi Fungus growing Termite South Africa Nasutitermes Costa Rica Mountain Pine Beetle British Columbia, CA Xyleborus affinis Switzerland Dendroctonus frontalis Mississippi Fungus growing Termite South Africa Nasutitermes Costa Rica Mountain Pine Beetle British Columbia, CA Xyleborus affinis Switzerland Pearson correlation matrix by PFAM profiles

Genome composition of selected metagenomes Dendroctonus frontalis Mississippi Fungus growing Termite South Africa Nasutitermes Costa Rica Mountain Pine Beetle British Columbia, CA Xyleborus affinis Switzerland Dendroctonus frontalis Mississippi Fungus growing Termite South Africa Nasutitermes Costa Rica Mountain Pine Beetle British Columbia, CA Xyleborus affinis Switzerland Pearson correlation matrix by PFAM profiles

Genome composition of selected metagenomes PCA clustering by COG function

Genome composition of selected metagenomes Nasutitermes Costa Rica PCA clustering by COG function Fungus growing Termite South Africa Xyleborus affinis Switzerland Mountain Pine Beetle BC, CA Dendroctonus frontalis Mississippi

Genome composition of selected metagenomes Nasutitermes Costa Rica PCA clustering by COG function Fungus growing Termite South Africa Xyleborus affinis Switzerland Mountain Pine Beetle BC, CA Dendroctonus frontalis Mississippi

Our Questions ● Phylogenetic diversity ● Who is there? ● Is there a core set of taxa? ● Genes/Pathways of interest ● Cellulose degradation enzymes in action?? ● Nitrogen fixation genes in play?

Phylogenetic analysis based on % identity: 30-59% estimated gene copies that fall within each category

-> only Coleoptera genome in the database ;-) 55% genes match with Coleoptera, Tenebrionidae, Tribolium castaneum = not the right bug!! Very little eukaryotic DNA contamination

0.54% = Methanomicrobia (Methanocellales, Methanomicrobiales, Methanosarcinales 0.16% = Methanomicrobia (Methanocellales, Methanomicrobiales, Methanosarcinales

uncl. Spirochaeta and Treponema, N 2 -fixing organisms, acetogens (CO 2, H 2 -> acetate) All Fibrobacter succinogenes: cellulolytic anaerobic bacterium (not in other termite) 13.4%= anaerobic, fermentative bacteria (Clostridium sp., Acetivibrio cellulolyticus, Lachnospiraceae, Ruminococcaceae etc.) Bacteroidiia (Bacteroides sp. = carbohydrate fermenters) and Sphingobacteriia (Mucilaginibacter= facultative aerobic, xylan, pectin-degrading) Sphingobacteriia (Sphingobacterium spiritivorum = facultative aerobe, alcohol-oxidizing <- HMP)

Desulfo-xx (not in beetle samples) Comamonadaceae + Burkholderiaceae (beetles) Termites:1) Enterobacteriales(E.coli, Klebsiella, Salmonella sp.) – 2) Pseudomonadales (Pseudomonas spp.) + Nasutitermes: Alteromonadales (Shewanella sp.) ? Beetles : 1) Pseudomonadales (Pseudomonas spp.)- 2) Enterobacteriales(E.coli, Klebsiella, Salmonella sp.) ?

Our Questions ● Phylogenetic diversity ● Who is there? ● Is there a core set of taxa? ● Genes/Pathways of interest ● Cellulose degradation enzymes in action?? ● Nitrogen fixation genes in play?

Comparison of PFAM GH5

Genome Name No. of GH5 (pfam00151) Gene count Mountain Pine Beetle microbial communities from McBride, British Columbia, Canada, sample from Lodgepole pine (Lodgepole pine) Xyleborus affinis microbiome from Bern, Switzerland, sample of adult community (Ambrosia beetle adult) Hindgut microbiome of Nasutitermes sp. (Costa Rica) (version 3) Fungus-growing Termite worker microbial community from South Africa, sample from Oerleman's Farm Dendroctonus frontalis bacterial community182571

Nitrogen Fixation-related Genes

● In the termite hindgut (yellow), most are contributions from ● Gammaproteobacteria ● Actinobacteria ● Spirochaetes ● Unclassified ● K03737 (putative pyruvate-flavodoxin oxidoreductase) is present in all metagenomes

Summary - perspectives ● Phylogenetic diversity ● Consistent with host lifestyle ● Is there a core set of taxa? TBD (+ phylogenetic COG markers?) ● Genes/Pathways of interest ● PFAMs for cellulose degradation enzymes were found ● Nitrogen fixation genes present ● Suggestions ● More meta data!! ● Compatibility of file system datasets (Bankia) ● More time for data analysis (great collective exercise, but 1 day is not enough)

Thank you!!