Kodamaea ohmeri is a symbiont of the small hive beetle (SHB), which is a scavenger of honey bee colonies. The SHB causes absconding of the economically important honey bee (Apis mellifera) and deposits K. ohmeri in the honeycomb. We describe long-read sequencing and further analyses of the K. ohmeri genome.
ABSTRACT
Kodamaea ohmeri is a symbiont of the small hive beetle (SHB), which is a scavenger of honey bee colonies. The SHB causes absconding of the economically important honey bee (Apis mellifera) and deposits K. ohmeri in the honeycomb. We describe long-read sequencing and further analyses of the K. ohmeri genome.
ANNOUNCEMENT
The small hive beetle (SHB), Aethina tumida, deposits its symbiotic yeast (Kodamaea ohmeri, Saccharomycotina) when infesting honey bee (Apis mellifera) colonies (1, 2). K. ohmeri ferments honey, creating an odor and slimy appearance used for SHB diagnostics (3), as well as volatiles that mimic honey bee pheromones, which may attract more SHBs to the hive (2). K. ohmeri is also a human pathogen (4) and produces ethanol and food flavors (5).
Macerated midguts of bees from a SHB-infested honey bee colony were plated on yeast-peptone-dextrose agar (Sigma) with antibiotics and incubated at 25°C. A single colony was picked to produce an overnight culture, which was pelleted for genomic DNA (gDNA) extraction using the MagAttract high-molecular-weight (HMW) DNA kit (Qiagen). Primers ITS1_F, ITS4_R, NL1_F, and NL4_R were used in Phusion (NEB) reactions for identification.
A whole-genome sequence (WGS) library was constructed using the SMRTbell express template prep kit v1, size selected on a BluePippin system with a 15-kb cutoff, and sequenced on a PacBio Sequel system. One single-molecule real-time (SMRT) cell produced 6,057,833,952 bp in 835,386 reads for >475× coverage. PacBio data were corrected, trimmed, and assembled into 27 contigs with Canu v1.7.1 (6) using the parameters genomeSize = 12.16m and correctedErrorRate = 0.105. One contig flagged as circular was identified as the mitochondrion by BLAST. MUMmer v4.0.0beta2 (7) confirmed the overlapping ends. Circularization and removal of contigs contained within another contig were performed with Circlator v1.5.0 (–b2r_length_cutoff 60000 –split_all_reads [8]). Three iterations of Arrow (SMRT Link v5.1.0) polishing produced a 20-contig assembly (QUAST [9] and Galaxy v4.6.3 [10]), with 12,583,843 bp, 42.75% GC content, an N75 value of 1,584,252 bp, an L75 value of 5, and a largest contig of 2,525,569 bp. BUSCO v3 (11) assessment using the Fungi odb9 and Saccharomycetales odb9 data sets indicated 99% and 93.8% completeness, respectively. MAKER 3.01.02-beta (12) with AUGUSTUS v3.3.2 (13) and trained Candida guilliermondii, the previous species before reassignment and closest relative available (14), predicted 5,239 genes. Gene descriptions were assigned via BLASTP (BLAST+ 2.8 [15]) against the Reviewed (Swiss-Prot) database following support protocol 3 (16). BlastKOALA v2.1 (17), eggNOG-mapper (DIAMOND v4.5.1) (18), and GO FEAT v1.0 (19) assigned 58.9% proteins with KEGG orthology identifiers and 4,574 proteins with Clusters of Orthologous Groups. Twenty-eight percent of the annotatable predicted proteins are involved in cellular processes/signaling, 25% in information storage/processing, and 28% in metabolism. antiSMASH v5-beta (fungi) (20) identified a squalene synthase but no prominent natural product gene clusters for secondary metabolism. Because the SHB genome possesses no invertase for sucrose metabolism (21), we searched the proteome of K. ohmeri for genes that might assist in gut metabolism and identified an invertase and a hexose transporter. Identification of transaldolase and xylulose kinase genes corroborates the production of ethanol by xylose-fermenting K. ohmeri strains (5). To identify K. ohmeri sequences in the SHB genome assembly as speculated based on BLASTN, the K. ohmeri assembly was fragmented (500 bp) using BBMap v38 (22) and mapped to the SHB genome (assembly number GCA_001937115) using HISAT 2.1.0 (23), followed by SAMtools and BLASTN. Ribosomal DNA genes were mainly identified among the 1.14% of the K. ohmeri assembly that aligned to SHB (0.64% once and 0.50% more than once) using BLASTN. NCBI’s SRA taxonomy analysis found 1.6% alignment.
This genome will help unravel bee-SHB-yeast tripartite interactions, improve next-generation sequencing SHB studies, and advance clinical and industrial efforts.
Data availability.
Data were deposited at GenBank under BioProject number PRJNA525764 (BioSample number SAMN11074024, SRA number SRR8889280, and assembly number SKFK00000000). The strain was deposited at CBS-KNAW Collections–Westerdijk Fungal Biodiversity Institute as CBS 15370.
ACKNOWLEDGMENTS
James P. Tauber was supported by an appointment to the Agricultural Research Service (ARS) Research Participation Program administered by the Oak Ridge Institute for Science and Education (ORISE) through an interagency agreement between the U.S. Department of Energy (DOE) and the U.S. Department of Agriculture (USDA). ORISE is managed by ORAU under DOE contract number DE-SC0014664. James P. Tauber was also supported by the ARS Research Associate Program (Class of 2018).
We declare no competing interests. All opinions expressed in this paper are those of the authors and do not necessarily reflect the policies and views of the USDA, ARS, DOE, or ORAU/ORISE. The mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.
This research used resources provided by the SCINet project of the USDA Agricultural Research Service, ARS project number 0500-00093-001-00-D. Additional analyses were performed in an Xfce Desktop Environment (v4.10) on a high-performance computer (HPC) provided at the BAM Institute, Berlin, Germany, with the help of Dino McMahon. We thank Dawn Lopez for her technical expertise, and Francisco Posada-Florez and the entire BRL crew for maintaining the colonies used for this research. We also thank the Genomics Resource Center at the University of Maryland for sequencing.
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Data Availability Statement
Data were deposited at GenBank under BioProject number PRJNA525764 (BioSample number SAMN11074024, SRA number SRR8889280, and assembly number SKFK00000000). The strain was deposited at CBS-KNAW Collections–Westerdijk Fungal Biodiversity Institute as CBS 15370.