Genomic and transcriptomic analyses of Wohlfahrtia magnifica, a myiasis-causing flesh fly, reveal key targets for potential control programs

Zhipeng Jia

doi:10.34876/x34z-n671

Title (eng)

Genomic and transcriptomic analyses of Wohlfahrtia magnifica, a myiasis-causing flesh fly, reveal key targets for potential control programs

Author

Zhipeng Jia

Degree supervisor

Pamela A. Burger

Claus Vogl

Surong Hasi

Description (eng)

PhD thesis - University of Veterinary Medicine Vienna - 2024

Abstract (eng)

Background Wohlfahrtia magnifica, a flesh fly, is one of the most important obligatory traumatic myiasis-causing flies, affecting a range of mammals in several European, African, and Asian countries. The infestation of W. magnifica can lead to significant health and welfare problems and substantial economic losses in the field of animal husbandry. However, research on W. magnifica is quite limited, especially at the molecular level. Aims of the thesis In light of this limited information, the aims of this thesis revolved around filling the existing knowledge gaps in W. magnifica. First, I aimed to sequence, assemble, and annotate the genome of W. magnifica. This new genome resource provides an essential starting point for many future fundamental and applied research areas. Second, I aimed to investigate the complex dynamics of gene expression throughout the life cycle of W. magnifica to understand how parasitic larvae regulate specific gene expressions to effectively parasitize their hosts. This part of my research will guide the selection of potential targets for vaccines or insecticides aimed at disrupting the establishment of the larvae on or in hosts. Third, I aimed to identify and characterize the critical components for the development of genetically modified strains in W. magnifica. This represents a significant step in the future establishment of genetic control programs designed to address the infestation of W. magnifica. Results In our first article, we adopted the strategy of low DNA input library preparation and successfully sequenced the genome of W. magnifica using a single adult specimen as the genetic source. The assembled genome spans 753.99 Mb, with a N50 length of 5.00 Mb, and contains 59.71% repeat elements. The overall RNA-seq alignment rate reached 93.62%, and 98.8% of complete BUSCOs (Benchmarking Universal Single-Copy Orthologs) were identified, indicating the completeness and high quality of the genome. The genome annotation process predicted 16,718 genes and 20,017 mRNA sequences in the genome, with 64.98% of genes functionally annotated using the UniProt/Swiss-Prot database. The phylogenetic analysis demonstrated that W. magnifica has the closest relationship to Sarcophaga bullata, another member of the Sarcophagidae family, followed by Lucilia cuprina, another myiasis-causing fly. The analysis of gene family expansion revealed that the function of the expanded gene families is associated with immunity, insecticide resistance, responses to heat stress, and cuticle development. Furthermore, using the comparative method 45 positively selected genes were identified exhibiting diverse functions. In the second article, we identified a set of 2049 excretory/secretory (ES) proteins in W. magnifica, which play critical roles in parasite-host interactions. Functional annotation indicated that these ES proteins are primarily associated with processes such as cuticle development, peptidase activity, immune responses, and metabolic activities. With the larval samples collected from wounds of the infested host species, Bactrian camel, and the following pupal and adult stages, the analysis of gene expression indicated that the functions of upregulated genes were distributed in cuticle development, proteolysis, and RNA transcription and translation in second stage larvae; peptidase inhibitor activity and nutrient reservoir activity in third stage larvae; cell and tissue morphogenesis and cell and tissue development in pupae; signal perception (a lot of genes implicated in light perception) and behaviors such as feeding, mating, and locomotion in adult flies. The analysis of gene expression related to parasitism revealed that 88 out of 480 peptidase genes, 110 out of 215 cuticle protein genes and 21 heat shock protein (hsp) genes showed a significant upregulation in the parasitic larval stages. Among peptidases, serine peptidases are actively involved in tissue degradation and nutrient acquisition during the parasitic processes of myiasis-causing larvae. Our investigation showed that up to 22.93% (47/205) of serine peptidase genes were highly expressed in the parasitic larval stages, but only 4.39% (9/205) in the pupal stage and 8.78% (18/205) in the adult stage. Interestingly, the expression of 2 antimicrobial peptide (AMP) genes, including 1 defensin and 1 diptericin upregulated in the parasitic second-stage larvae and 10 genes in the GO term "nutrient reservoir activity" is dominantly high in the parasitic third-stage larvae. In the third article, we successfully isolated and characterized two pivotal sex-determination genes in W. magnifica, namely Wmtra (W. magnifica transformer) and Wmtra2 (W. magnifica transformer2), whose orthologs have been utilized to develop genetic control approaches in several insect pests. The splicing of Wmtra transcripts exhibited a sex-specific pattern, with the female variant encoding a fully functional protein and the male counterpart yielding a truncated, non-functional polypeptide, which is similar to blow flies such as Lucilia cuprina and Cochliomyia hominivorax. Furthermore, we identified the elements essential for the construction of a Cas9-based homing gene drive in W. magnifica. From sex-biased gene sets, a range of useful resources for the introduction site of the Cas9-gRNA cassette were provided. In addition, an examination of the set of genes exhibiting a bias toward females led to the identification of five potential candidate genes: vasa (vas), nanos (nanos), bicoid (bcd), Bicaudal C (BicC), and innexin5 (inx5). The promoters of these genes can be used to drive Cas9 germline expression. Our further analysis suggested that the promoter from nanos is better suited than others due to its simple structure and successful use for this purpose in other Diptera. We have also identified six U6 RNA genes in W. magnifica; their promoters can serve as potential candidates for regulating gRNA transcription in Cas9-based homing gene drive. Conclusions In the first article, we successfully sequenced the genome of W. magnifica from a single fly specimen using the strategy of low DNA input library preparation. This approach holds promise for its applicability to small Diptera flies that are difficult to rear in a laboratory or are amenable to laboratory rearing but present difficulties in inbreeding. The genome of W. magnifica serves as a valuable resource for fundamental research in areas such as molecular biology, biochemistry, and genetics of W. magnifica. Moreover, it opens doors to numerous applications in comparative genomics, transcriptomics, functional genomics, population genetics, and notably, the development of novel control strategies. The second article offers an in-depth investigation into the dynamics of gene expression across the life cycle of W. magnifica, illuminating how the parasitic larvae upregulate specific gene expression to effectively parasitize their hosts. This study not only advances our understanding of the molecular-level parasitic life cycle of W. magnifica but also paves the way for innovative approaches to disrupt larval parasitism in the host. In the third article, we isolated and characterized essential elements for the construction of genetically modified strains in W. magnifica. This represents an important step towards the development of genetic control programs in W. magnifica. In summary, the thesis has shed light on the biology of W. magnifica, contributing to the development of novel control measures against the traumatic myiasis-causing flesh fly, W. magnifica.

Description (deu)

PhD Arbeit - Veterinärmedizinische Universität Wien - 2024

Type (eng)

Dissertation

Language

[eng]

Persistent identifier

https://phaidra.vetmeduni.ac.at/o:4702

AC number

AC17667309

DOI

10.34876/x34z-n671