Draft genome of the honey bee ectoparasitic mite, Tropilaelaps mercedesae, is shaped by the parasitic life history
Background The number of managed honey bee colonies has considerably decreased in many developed countries in recent years and the ectoparasitic mites are considered as major threats to honey bee colonies and health. However, their general biology remains poorly understood. Results We sequenced the genome and transcriptomes of Tropilaelaps mercedesae, the prevalent ectoparasitic mite infesting honey bees in Asia. The de novo assembled genome sequence (353 Mb) represents 53% of the estimated genome size because of the compression of repetitive sequences; nevertheless, we predicted 15,190 protein-coding genes which were well supported by the mite transcriptomes and proteomic dataes. Although amino acid substitutions have been accelerated within the conserved core genes in of two mites, T. mercedesae and Metaseiulus occidentalis, T. mercedesae has undergone the least gene family expansion and contraction between the seven arthropods we tested. The number of sensory system genes has been dramatically reduced; meanwhile, T. mercedesae may have evolved a specialized cuticle and water homeostasis mechanisms, as well as epigenetic control of gene expression for ploidy compensation between males and females., and water homeostasis. T. mercedesae contains all gene sets required to detoxify xenobiotics, enabling it to be miticide resistant. T. mercedesae is closely associated with a symbiotic bacteriuma (Rickettsiella grylli-like) and DWVdeformed wing virus (DWV), the most prevalent honey bee virus. The presence of DWV in both adult male and female mites was also confirmed by the proteomic analysis. Conclusions T. mercedesae has a very specialized life history and habitat as the ectoparasitic mite strictly dependsing on the honey bee inside the a stable colony. Thus, comparison of the genome and transcriptome sequences with those of a tick and free-living mites and tick has revealed the specific features of the genome shaped by interaction with the honey bee and colony environment. T. mercedesae, as well as Varroa destructor, genome and transcriptome sequences not only provide insights into the mite biology, but may also help to develop measures to control the most serious pests of the honey bee.
Sample Processing Protocol
Proteomic analysis Pools of male or female Mites were lysed by sonication in 0.1 % (w/v) Rapigest (Waters MS technologies) in 50 mM ammonium bicarbonate. Samples were heated at 80 °C for 10 min, reduced with 3 mM DTT at 60 °C for 10 min, cooled, then alkylated with 9 mM iodoacetamide (Sigma) for 30 min (room temperature) protected from light; all steps were performed with intermittent vortex-mixing. Proteomic-grade trypsin (Sigma) was added at a protein:trypsin ratio of 50:1 and incubated at 37 °C overnight. Rapigest was removed by adding TFA to a final concentration of 1 % (v/v) and incubating at 37 ⁰C for 2 hours. Peptide samples were centrifuged at 12,000 x g for 60 min (4 ⁰C) to remove precipitated Rapigest. The peptide supernatant was desalted using C18 reverse-phase stage tips (Thermo Scientific) according to the manufacturer’s instructions. Samples were desalted and reduced to dryness as above and re-suspended in 3 % (v/v) acetonitrile, 0.1 % (v/v) TFA for analysis by MS. Peptides were analysed by on-line nanoflow LC using the nanoACQUITY-nLC system (Waters MS technologies) coupled with Q-Exactive mass spectrometer (Thermo Scientific). Samples were loaded on a 50cm Easy-Spray column with an internal diameter of 75 µm, packed with 2 µm C18 particles, fused to a silica nano-electrospray emitter (Thermo Scientific). The column was operated at a constant temperature of 35 °C. Chromatography was performed with a buffer system consisting of 0.1 % formic acid (buffer A) and 80 % acetonitrile in 0.1 % formic acid (buffer B). The peptides were separated by a linear gradient of 3.8 – 50 % buffer B over 90 minutes at a flow rate of 300 nl/min. The Q-Exactive was operated in data-dependent mode with survey scans acquired at a resolution of 70,000. Up to the top 10 most abundant isotope patterns with charge states +2, +3 and/or +4 from the survey scan were selected with an isolation window of 2.0Th and fragmented by higher energy collisional dissociation with normalized collision energies of 30. The maximum ion injection times for the survey scan and the MS/MS scans were 250 and 50 ms, respectively, and the ion target value was set to 1E6 for survey scans and 1E5 for the MS/MS scans. Repetitive sequencing of peptides was minimized through dynamic exclusion of the sequenced peptides for 20s.
Data Processing Protocol
Thermo RAW files were imported into Progenesis LC–MS (version 4.1, Nonlinear Dynamics). Runs were time aligned using default settings and using an auto selected run as reference. Peaks were picked by the software using default settings and filtered to include only peaks with a charge state between +2 and +7. Spectral data were converted into .mgf files with Progenesis LC–MS and exported for peptide identification using the Mascot (version 2.3.02, Matrix Science) search engine. Tandem MS data were searched against translated ORFs from T. mercedesae, Apis mellifera (OGSv3.2)  and Deformed Wing Virus (Uniprot 08 2016) (total; 30,666 sequences; 12,194,618 residues). The search parameters were as follows: precursor mass tolerance was set to 10 ppm and fragment mass tolerance was set as 0.01Da. Two missed tryptic cleavages were permitted. Carbamidomethylation (cysteine) was set as a fixed modification and oxidation (methionine) set as variable modification. Mascot search results were further validated using the machine learning algorithm Percolator embedded within Mascot. The Mascot decoy database function was utilised and the false discovery rate was < 1%, while individual percolator ion scores >13 indicated identity or extensive homology (P <0.05). Mascot search results were imported into Progenesis LC–MS as XML files. Peptide intensities were normalised against the reference run by Progenesis LC-MS and these intensities are used to highlight relative differences in protein expression between samples.
Stuart Armstrong, Infection Biology
Julian Hiscox, Chair in Infection and Global Health, Department of Infection Biology, Institute of Infection and Global Health, University of Liverpool, ic2 Building, Liverpool, L3 5RF, UK. ( lab head )
Dong X, Armstrong SD, Xia D, Makepeace BL, Darby AC, Kadowaki T. Draft genome of the honey bee ectoparasitic mite, Tropilaelaps mercedesae, is shaped by the parasitic life history. Gigascience. 2017 Feb 22 PubMed: 28327890