A Genomic Analysis and Transcriptomic Atlas of Gene Expression in Psoroptes Ovis Reveals Feeding- and Stage-Specific Patterns of Allergen Expression Stewart T
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Burgess et al. BMC Genomics (2019) 20:756 https://doi.org/10.1186/s12864-019-6082-6 RESEARCH ARTICLE Open Access A genomic analysis and transcriptomic atlas of gene expression in Psoroptes ovis reveals feeding- and stage-specific patterns of allergen expression Stewart T. G. Burgess1* , Edward J. Marr1, Kathryn Bartley1, Francesca G. Nunn1, Rachel E. Down2, Robert J. Weaver2, Jessica C. Prickett2, Jackie Dunn2, Stephane Rombauts3,4,5, Thomas Van Leeuwen6, Yves Van de Peer3,4,5,7 and Alasdair J. Nisbet1 Abstract Background: Psoroptic mange, caused by infestation with the ectoparasitic mite, Psoroptes ovis, is highly contagious, resulting in intense pruritus and represents a major welfare and economic concern for the livestock industry Worldwide. Control relies on injectable endectocides and organophosphate dips, but concerns over residues, environmental contamination, and the development of resistance threaten the sustainability of this approach, highlighting interest in alternative control methods. However, development of vaccines and identification of chemotherapeutic targets is hampered by the lack of P. ovis transcriptomic and genomic resources. Results: Building on the recent publication of the P. ovis draft genome, here we present a genomic analysis and transcriptomic atlas of gene expression in P. ovis revealing feeding- and stage-specific patterns of gene expression, including novel multigene families and allergens. Network-based clustering revealed 14 gene clusters demonstrating either single- or multi-stage specific gene expression patterns, with 3075 female-specific, 890 male- specific and 112, 217 and 526 transcripts showing larval, protonymph and tritonymph specific-expression, respectively. Detailed analysis of P. ovis allergens revealed stage-specific patterns of allergen gene expression, many of which were also enriched in “fed” mites and tritonymphs, highlighting an important feeding-related allergenicity in this developmental stage. Pair-wise analysis of differential expression between life-cycle stages identified patterns of sex-biased gene expression and also identified novel P. ovis multigene families including known allergens and novel genes with high levels of stage-specific expression. Conclusions: The genomic and transcriptomic atlas described here represents a unique resource for the acarid- research community, whilst the OrcAE platform makes this freely available, facilitating further community-led curation of the draft P. ovis genome. Keywords: Psoroptes ovis, Sheep scab, Transcriptome, Life-cycle, Development, Allergens * Correspondence: [email protected] 1Moredun Research Institute, Pentlands Science Park, Bush Loan, Edinburgh, Midlothian EH26 0PZ, UK Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Burgess et al. BMC Genomics (2019) 20:756 Page 2 of 23 Background mitesareabletosurviveforalimitedtime(15–16 days) Psoroptic mange, caused by the ectoparasitic mite Psor- off-host, enabling their transfer from animal to animal via optes ovis, is characterised by pruritus and skin irritation fomites [8]. Psoroptes ovis is a non-burrowing mite, which and is a major welfare and economic concern for the feeds at the skin surface consuming serous exudate, lymph livestock industry as the parasite infests both cattle and and red blood cells [9]. Mites survive on the surface of the sheep, causing the disease “sheep scab” in the latter [1, skin and their mouthparts, which are thought to abrade ra- 2]. In sheep, control relies on injectable macrocyclic ther than pierce the skin, do not penetrate beyond the lactone-based endectocides and organophosphate dips stratum corneum, the outermost layer of the skin [10]. As but concerns over residues, environmental contamination the mites move across the surface of the skin they secrete and the development of resistance threaten the sustainability and excrete allergens and other potent pro-inflammatory of this approach and have highlighted interest in develop- factors and this combination of mechanical skin abrasion, ing alternative control methods [3, 4]. However, the devel- allergen deposition and grooming behaviour by the host in opment of novel interventions (including vaccines and the response to the pruritus caused by the mites all contribute identification of potential chemotherapeutic targets) has to the subsequent cutaneous inflammatory response [11– previously been hampered by a lack of detailed transcrip- 13]. However, the role of the different developmental stages tomic and genomic resources for P. ovis. of P. ovis in eliciting the pathology associated with the host The integration of newly-available transcriptomic and pro-inflammatory response, and subsequent semi- genomicdatawithcurrentknowledgeofthebasicbiology protective immunity, is currently unknown and would be of the mite is pivotal in the development of such novel in- greatly improved with knowledge of the individual life-cycle terventions: The basic biology of the obligate ectoparasitic stage transcriptomes. mite, P. ovis, on sheep is well understood, with the life- Existing transcriptomic tools and resources for P. ovis cycle taking place entirely on the ovine host and lasting are limited and include an expressed sequence tag (EST) from 11 to 19 days from egg hatch to egg production by survey of ~ 500 P. ovis cDNAs [14], a subtractive suppres- the adult [5]. The life-cycle progresses from egg through sive hybridisation (SSH) based comparison of gene expres- four developmental stages (larvae → protonymph → trito- sion between “fed” and “starved” P. ovis mites [15]anda nymph → adult (male/female)) (Fig. 1). Adult female mites cDNA microarray based on ~ 1000 P. ovis ESTs [16]. can survive on the host for up to 42 days and during this More recently a preliminary transcriptomic analysis of P. time they may deposit up to 80 eggs [1, 6, 7]. Psoroptes ovis ovis var. cuniculi across a limited number of developmental Fig. 1 Psoroptes ovis life-cycle. Image demonstrates progression from egg, through larvae (L), nymph stages (protonymph (P) and tritonymph (T)) and onto adult male (AM) and adult female (AF). Image adapted from “Diagram of the life-cycle of Psoroptes ovis parasitic mite of sheep and cattle” (commons.wikimedia.org/wiki/File:Life-cycle-psoroptes-ovis-mite-diagram.jpg) under Creative Commons License (CC-BY-SA-3.0) Burgess et al. BMC Genomics (2019) 20:756 Page 3 of 23 stage comparisons using Illumina RNA-seq was described function, GO terms, Pfam protein domains, protein homo- [17]. The recent generation of the P. ovis genome, which in- logues and significant BLAST hit data, gene structure, cod- cluded the prediction and annotation of the P. ovis tran- ing sequence, protein sequence and, where available, scriptome [18] has substantially improved the resources transcript evidence based on associated ESTs/cDNA data available and enables more detailed genomic and transcrip- (Fig. 3)[23]. Each gene was assigned a unique loci identifier tomic analyses of P. ovis. At 63.2 Mb, the draft genome as- with the following format: psoviXXgYYYYY, where XX de- sembly demonstrated that P. ovis has one of the smallest fines the scaffold ID and YYYYY denotes the specific loca- arthropod genomes sequenced to date, smaller than the tion within the scaffold. As with most large-scale genome genome of the two-spotted spider mite (Tetranychus projects, the P. ovis genome relied upon a computational urticae (90 Mb)) but comparable in size with the closely re- gene prediction and annotation pipeline and although we lated house dust mite (HDM) genomes (Dermatophagoides also incorporated additional RNA-seq data as transcript farinae (53.5 Mb) and D. pteronyssinus (70.76 Mb)) and the evidence in this process it is likely that some errors will re- ectoparasitic scabies mite (Sarcoptes scabiei (56.2 Mb)) [18– main. As such, continual manual curation of gene predic- 22]. Herein, using the recently described P. ovis genome tion and annotation remains a critical step in assessing and [18], we described the detailed annotation of the genome to improving gene prediction accuracy and overall confidence Gene Ontology (GO) level along with a quantitative tran- in the genome [24]. To facilitate this, the OrcAE platform scriptomic analysis of P. ovis gene expression across mul- is unique in that it also provides the tools and information tiple life-cycle stages, providing for the first time a complete for community-led manual validation of gene annotations. transcriptomic atlas of stage-specific and feeding-related gene expression in this economically-important ectopara- site of livestock. P. ovis collection, life-cycle staging and preparation of “fed” and “starved” mite populations Results and discussion Staging of P. ovis into individual life-cycle stages (Fig. 1)is Functional annotation of the P. ovis predicted challenging and time-consuming but can be performed transcriptome derived from the draft