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. 2022 Aug 29;17(8):e0273543.
doi: 10.1371/journal.pone.0273543. eCollection 2022.

Annotations of novel antennae-expressed genes in male Glossina morsitans morsitans tsetse flies

Affiliations

Annotations of novel antennae-expressed genes in male Glossina morsitans morsitans tsetse flies

Billiah K Bwana et al. PLoS One. .

Abstract

Tsetse flies use antennal expressed genes to navigate their environment. While most canonical genes associated with chemoreception are annotated, potential gaps with important antennal genes are uncharacterized in Glossina morsitans morsitans. We generated antennae-specific transcriptomes from adult male G. m. morsitans flies fed/unfed on bloodmeal and/or exposed to an attractant (ε-nonalactone), a repellant (δ-nonalactone) or paraffin diluent. Using bioinformatics approach, we mapped raw reads onto G. m. morsitans gene-set from VectorBase and collected un-mapped reads (constituting the gaps in annotation). We de novo assembled these reads (un-mapped) into transcript and identified corresponding genes of the transcripts in G. m. morsitans gene-set and protein homologs in UniProt protein database to further annotate the gaps. We predicted potential protein-coding gene regions associated with these transcripts in G. m. morsitans genome, annotated/curated these genes and identified their putative annotated orthologs/homologs in Drosophila melanogaster, Musca domestica or Anopheles gambiae genomes. We finally evaluated differential expression of the novel genes in relation to odor exposures relative to no-odor control (unfed flies). About 45.21% of the sequenced reads had no corresponding transcripts within G. m. morsitans gene-set, corresponding to the gap in existing annotation of the tsetse fly genome. The total reads assembled into 72,428 unique transcripts, most (74.43%) of which had no corresponding genes in the UniProt database. We annotated/curated 592 genes from these transcripts, among which 202 were novel while 390 were improvements of existing genes in the G. m. morsitans genome. Among the novel genes, 94 had orthologs in D. melanogaster, M. domestica or An. gambiae while 88 had homologs in UniProt. These orthologs were putatively associated with oxidative regulation, protein synthesis, transcriptional and/or translational regulation, detoxification and metal ion binding, thus providing insight into their specific roles in antennal physiological processes in male G. m. morsitans. A novel gene (GMOY014237.R1396) was differentially expressed in response to the attractant. We thus established significant gaps in G. m. morsitans genome annotation and identified novel male antennae-expressed genes in the genome, among which > 53% (108) are potentially G. m. morsitans specific.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Schematic flowchart diagram of processing of samples and bioinformatics analysis of RNA-Seq transcriptome data from male G. m. morsitans tsetse fly antennae.
Fig 2
Fig 2. Quality and mapping statistics of concatenated RNA-Seq library from male G. m. morsitans tsetse fly antennae.
Panel A. Quality assessment statistics of the libraries. We extracted total RNA from blood fed/unfed teneral male G. m. morsitans exposed/unexposed to attractant (ε-nonalactone) or repellent (δ-nonalactone) in two/three independent replicates. We sequenced the RNA on Illumina HiSeq 2500, concatenated the resultant reads into a single library and established quality of the library using FastQC v11.0 software package. We used the FastQC results to clean (trim) and remove low quality reads from respective transcriptomes using Trimmomatic software version 3.8 [39]. Panel B. Mapping statistics of the libraries. We mapped clean paired (concatenated) reads onto G. m. morsitans transcripts gene-set version 1.9 or genome version 1.0 from Vectorbase [28] using Bowtie 2 ultrafast short sequence reads aligning software version 2.3.5 [40] with settings that also isolated unmapped reads.
Fig 3
Fig 3. Quality assessment of read representation in de novo assembled antennal transcripts from male G. m. morsitans RNA-Seq antennal libraries.
We de novo assembled the unmapped reads into transcripts and assessed quality of the assembled transcripts using short reads Trinity de novo assembly software 2.10.0 [41] that mapped the reads onto their respective assembled transcripts to account for individual reads (incorporation).
Fig 4
Fig 4. Orthologs of novel annotated male G. m. morsitans antennae genes in D. melanogaster, M. domestica and An. gambiae genomes.

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Grants and funding

The author(s) received no specific funding for this work.