Microbiome composition and pathogen genetic diversity in arthropod vectors

Abstract

SUMMARY In the current world, mosquitoes and ticks are the most important vectors of a wide variety of pathogens that affect humans and animals. Microbes are part of the hologenome of vectors, which participate in many biological processes implicated in eco-epidemiological dynamics of the vector. Microbial communities might confer attributes to their host, firstly, affecting the ability of vectors to adapt to new environments and vertebrate hosts; and secondly the capacity to acquire, maintain and transmit pathogens. Providing3 answers to central questions regarding vector-pathogen-microbiota interactions is a challenge for current research, ie: who is there, what biological processes are occurring, and where are these processes happening?. In this sense, metaomics approaches applied to the study of the vector microbiome (ticks and mosquitoes), have the power to integrate network-based tools metagenomics, transcriptomics and proteomics to describe the complexity of the microbiota and the biological processes involved in host/vector-pathogen and host/vector-microbiota interactions. The success of the combining proteomics and genomics methods implemented for the identification and characterization of microbiota and tick-borne pathogens paves the way for future comparative genomic studies that will help to identify differences in the proteome/genome that could be involved in the tropism by the vector. Also, it will let us know if those differences are related to the functionality and therefore the pathogenesis and virulence. Furthermore, on the base of meatomics approaches a key priority is to identify microbial targets and to generate solid genomic annotations for better design of prevention strategies against vector-borne diseases. Ultimately, the database collection of microorganisms that compose the microbiome of vectors, in combination with genome editing, reconstruction and identification of vector-borne pathogens genomic traits, are the most promising directions for the surveillance and control of vector-borne diseases. Chapter I. General introduction Content: Chapter I is structured as a review paper [Bonnet, S. I., Binetruy, F., Hernández-Jarguín, A. M., & Duron, O. (2017). The tick microbiome: why non-pathogenic microorganisms matter in tick biology and pathogen transmission. Frontiers in Cellular and Infection Microbiology, 7, 236]. This paper contributes to the introduction of the current thesis. In this publication, four fundamental biological aspects are reviewed to better understand the composition and complexity of the vector-microbiome interactions. First, the role of commensal microorganisms and symbionts in tick's biology; second, the effect of tick microbiota in the acquisition and transmission of pathogens; third, the microbial interactions, especially those between symbionts and closely related pathogens, and finally, the role of symbionts as potential vertebrate pathogens. In addition, this review includes a brief description of a wide diversity of microbial communities associated with different families and species of ticks with complex effects on tick's biology. A special emphasis on the effect of symbiotic microorganisms on survival, reproduction and their impact on the colonization and transmission of pathogens was also reviewed. Nevertheless, factors that shape the composition of the microbiome are still under investigation and is clear that host genetics, the environment and geography, and pathogens eco-epidemiological cycle are important to shape the diversity of tick microbial communities. Chapter II. Microbiome composition and pathogen genetic diversity in ticks using omics approaches Content: In Chapter II we addressed the identification of the microbiota composition of Ixodes ricinus and Ixodes ventalloi, tick vector-borne arthropods within the family Ixodidae. To exhaustively explore vector-microbiota-pathogen interactions, a first fundamental requirement is to include a methodological approach that yields accurate microbial taxonomic profiles. Hence, we explored the bacterial microbiota of the laboratory-reared I. ricinus using a metaomic analysis strategy combining metaproteomic and metatranscriptomic analysis. The workflow for the study, was designed in order to reuse the available data of RNA-seq, and the construction of a target bacterial database for the metaomic analysis. An important contribution of this approach was the validation at the protein level of the bacterial sequences identified by RNA-seq and the identification of metabolically active bacterial communities that provided the metaproteomics. One promising result of this approach was to generate further knowledge on the mechanisms occurring between pathogenic bacteria and tick gut microbiota that facilitate infection and proliferation [Hernández-Jarguín, A., Díaz-Sánchez, S., Villar, M., de la Fuente, J. (2018). Integrated metatranscriptomics and metaproteomics for the characterization of bacterial microbiota in unfed Ixodes ricinus. Ticks Tick Borne Diseases. 9(5):1241-1251]. A contrasting approach, termed whole-genome shotgun-metagenomic sequencing consisted in the use of supervised clustering to group shotgun reads into bacterial gene families. We used this method to first describe the microbiota composition of the wild-caught I. ventalloi microbiota. The major microbiota was structured by members of the genus Anaplasma, Borrelia and Rickettsia. Other cohabiting members such as symbionts, endosymbiont, ubiquitous and some pathogenic genera for animals and humans, were identified at low rates as well. [Díaz-Sánchez, S., Hernández-Jarguín, A., Torina, A., Fernández de Mera, I. G., Blanda, V., Caracappa, S., Gortázar, C., de la Fuente, J. (2019). Characterization of the bacterial microbiota in wild-caught Ixodes ventalloi. Ticks and Tick-Borne Diseases. 10(2):336-343.]. In summary, characterization of the microbiota of laboratory and wild-caught tick populations is necessary to enhance our knowledge of putative microbiota of specific tick species. The exploration of both biological contexts would allow to speculate with the functional roles of microbial putative members and their biological implications in the eco-epidemiological role of this vectors. Additionally, we characterized virus and bacteria vector-borne pathogens using different –omic analysis. Conventional amplification-based assays used to target pathogens remain difficult and unspecific, due to the low level of infection and/or the presence of closely related species. Herein, we proposed a metaproteomic pipeline that provide support and validate at protein level the identification of the Crimean-Congo hemorrhagic fever virus (CCHFV) in ticks by RT-PCR. Following this pipeline, we demonstrated that including a metaproteomic analysis the identification of CCHFV pipeline is more accurate as it is possible to obtain the genotypic differentiation between CCHFV virus and other Nairovirus. [Fernández de Mera, I. G., Chaligiannis, I., Hernández-Jarguín, A., Villar, M., Mateos-Hernández, L., Papa, A., de la Fuente, J. et al. (2017). Combination of RT-PCR and proteomics for the identification of CCHV virus in ticks. Heliyon, 3(7)]. Based on the implantation of genomics and the rapid progression of genome editing and mapping tools, we reconstructed the draft genome of different isolates of the tick-borne bacteria pathogen Anaplasma spp, including the strains A. phagocytophilum NY18, A. marginale Oklahoma-2, and the first report of A. ovis Idaho, using whole-genome sequencing and de novo assembly. [Díaz-Sánchez, S., Hernández-Jarguín, A., Fernández de Mera, I. G., Alberdi, P., Zweygarth, E., Gortázar, C., de la Fuente, J. (2018). Draft genome sequences of Anaplasma phagocytophilum, A. marginale, and A. ovis isolates from different hosts. Genome Announcements, 6:5]. In this study, SNPs, substitutions, insertions and deletions in different regions of the genome of these isolates were identified. In the future, genomic data available from different vector-borne pathogens will allow the design of comprehensive comparative genomic studies to answer central questions for the control of tick-borne diseases. Chapter III. Effect of abiotic and biotic factors in mosquito microbiome composition Content: Chapter III is a contribution to the study of the microbiota composition of wild-caught Culicoides imicola biting midges [Díaz-Sánchez, S., Hernández-Jarguín, A., Torina, A., Fernández de Mera, IG, Estrada-Peña, A., Villar, M., La Russa, F., Blanda, V., Vicente, J., Caracappa, S., Gortazar, C., de la Fuente, J. (2018). Biotic and abiotic factors shape the microbiota of wild populations of the arbovirus Culicoides imicola vector. Insect Molecular Biology 27(6):847-861]. To study the insect-associated microbiome we utilized a de novo whole-genome shotgun metagenomic sequencing pipeline. In addition, we included in the study the identification of the preferred host-feeding source (biotic factors) of C. imicola, using together a proteomic assay, and DNA-analysis based on the amplification of the cytochrome b gene. In parallel, we collected data related to the temperature and soil moisture for each wildcaught mosquito collection site (abiotic factors). Then, the combined effect of the biotic and abiotic factors over the microbiota composition of C. imicola was also reported. A core microbiome together with unique microbial taxa among C. imicola populations composed of virus, bacteria and fungus was observed. Interestingly, the combined effect of biotic and abiotic features composing the mosquito habitat could contribute to those differences observed in the microbiome composition. To this end, this research, apart from contributing to characterize the microbiome of C. imicola, has found unique microbial features implicated in C. imicola adaption and expansion to new environments. Importantly, it will be valuable data for comprehensive eco-epidemiological studies and successful control of vector-borne diseases strategies.