19-BBSRC-NSF/BIO Bacterial and host genetic factors contributing to microbiome acquisition and homeostatis in mosquitoes

Lead Research Organisation: Liverpool School of Tropical Medicine
Department Name: Vector Biology


Animals harbor gut microbial communities that are essential for their fitness. However, the mechanisms underlying how these communities assemble in the gut remain largely unexplored in most host systems. The proposed research builds on existing productive collaborations to expand our knowledge of how hosts control gut colonization by bacteria and, conversely, how bacteria adapt to the gut to form stable associations with hosts. To date, coevolution has almost exclusively been investigated in bacterial partners of highly stable, long-term associations such as obligate human pathogens or vertically transmitted obligate symbionts. Here, we hypothesize that host-microbiota coadaptation can also occur over repeated interactions spanning only very short time scales of the hosts' individual life span and is
likely mediated by genetic factors of both hosts and bacteria.

We will address our central hypothesis in the mosquito Aedes aegypti, an insect of interest because adult females can transmit Zika, dengue, and other arboviruses that cause severe disease in humans. Mosquitoes are an ideal system in which to test our hypothesis because they harbor simple gut communities containing bacterial taxa that can each be cultured and manipulated. Methods also exist to generate axenic (microbe-free) and gnotobiotic mosquitoes containing defined bacterial communities. Because both Ae. aegypti and its associated gut bacteria are genetically tractable, the proposed research provides a unique, in-depth mechanistic study of host-microbe gene interactions that are difficult to study in most systems.

Technical Summary

The long-term goal of this proposal is to improve understanding of how hosts mediate gut colonization by bacteria and, conversely, how gut bacteria form stable associations with their hosts. Specifically, we plan to examine host-microbiota coadaptation in transient environments such as the guts of holometabolous insects, which are shed and replaced during molting and remodeled during metamorphosis. Our central hypothesis is that host-microbiota coadaptation can occur over very short time scales and is likely mediated by genetic factors of both hosts and symbionts. Our system of choice is the mosquito Aedes aegypti, a holometabolous insect that has long been studied for its medical relevance as the primary vector of arboviruses that cause severe disease in humans. This makes mosquitoes an ideal system in which to test our hypothesis as they represent a widely studied group of insects of ecological relevance and in which evolution spanning several host generations can be readily observed given their short generation time. Furthermore, mosquitoes offer a tractable system in which to examine host-gut microbiota interactions. Methods exist to generate axenic (microbe-free) and gnotobiotic mosquitoes containing defined bacterial communities, and we have developed systems to genetically alter both the host and microbiota, enabling us to disentangle host-microbe and microbe-microbe interactions. The proposed research integrates high-throughput genomics and transcriptomics approaches with experimental studies of host and bacterial gene function and coevolution, and thus provides a unique, in-depth mechanistic study of host-symbiont interactions at maximum resolution.


10 25 50
Description Whilst the projects are mainly still ongoing, the work has already resulted in the publication of a software pipeline and a large dataset available online.
Exploitation Route The analysis pipeline as above, as well as the RNASeq analysis scripts (available here https://github.com/laura-brettell/microbiome_transplant_RNASeq), are freely available online for further exploration or use provided the source is appropriately cited.
Sectors Environment,Healthcare,Pharmaceuticals and Medical Biotechnology

URL https://github.com/aidanfoo96/MINUUR
Description Expanding the scope of mosquito-gut microbiome research to incorporate insect-specific viruses
Amount £3,000 (GBP)
Funding ID BB/X010929/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2022 
End 08/2022
Title MInUUR - Microbial INsight Using Unmapped Reads 
Description MInUUR is a snakemake pipeline to extract unmapped whole genome shotgun sequencing reads and utilise a range of metagenomic analyses to characterise host-associated microbes. Orginally, MInUUR was intended to be used for the extraction of mosquito-associated bacterial symbionts, however, its application can be applied to other host-associated WGS data. 
Type Of Material Computer model/algorithm 
Year Produced 2022 
Provided To Others? Yes  
Impact MInUUR aims to leverage pre-existing WGS data to 'scavenge' for microbial information pertaining to host associated microbiomes - the key advantage being metagenomic reads as inputs to produce genus & species level classifications, functional inference and assembly of metagenome assembled genomes (MAGs). 
URL https://github.com/aidanfoo96/MINUUR
Title microbiome_transplant_RNASeq 
Description Analysis of transcriptome data to understand how a mosquito responds to a microbiome transplant R markdown file, which takes a raw counts file from HiSat2 and sample metadata and from this conducts Differential expression analysis using DESeq2 and generates associated plots. 
Type Of Material Database/Collection of data 
Year Produced 2023 
Provided To Others? Yes  
Impact Transparently sharing code used for analyses, enabling reproduction of results. 
URL https://github.com/laura-brettell/microbiome_transplant_RNASeq
Description Collaboration with A/Prof Ian Patterson on ISVs 
Organisation Brock University
Country Canada 
Sector Academic/University 
PI Contribution Mosquitoes are the major vector for many medically important human pathogens across the world. Their microbiomes play vital roles in development, fitness and ability to transmit these pathogens to humans. Therefore, manipulating the microbiome has the potential as a tool to control these pathogens. Using BBSRC funding we are working to gain a deeper understanding of the host and bacterial factors that affect the ability of bacteria to colonize the mosquito gut. Insect-specific viruses are an understudied factor that also has the potential to affect how bacteria and the mosquito host interact. With this ITAS funding, Drs Eva Heinz and Laura Brettell conducted a research visit to Brock University, Canada to draw upon the virology expertise of Dr Edward Patterson to better understand how we may be able to incorporate ISVs into our ongoing work.
Collaborator Contribution The BBSRC Travel Award enabled EH and LB to visit Brock University for one week, have extensive discussions and for LB to get insight into the lab work. During the visit, LB gained theoretical knowledge and practical training in many aspects of virology relevant for the study of insect-specific viruses. EP shared knowledge of how to design and create ISV molecular clones with fluorescent markers for conducting infection assays and visualising the results. LB received training in the required laboratory techniques, including insect cell culture, experimental infections and microscopy. Under the supervision of Anneliese Von Eicken (PhD student working with EP), LB successfully infected a mosquito-derived cell line (C7/10) using an infectious clone of Piura virus labelled with ZsGreen, visualised the resulting virus infected cells and harvested and stored the infective virus for future work.
Impact With these new skills and expertise, the team at Liverpool School of Tropical Medicine is now equipped to incorporate ISVs into their ongoing work. Research plans include determining how a prior infection with an ISV affects the ability of bacterial isolates of interest to colonize the mosquito gut. Further, using fluorescently labelled ISVs we can determine ISV localisation in the mosquito host and investigate whether virus infections in gut tissues specifically affect host-microbe interactions. The research visit also provided the opportunity to initiate new collaborations and project proposals. The team met with Prof Fiona Hunter and discussed new projects that may utilize all the team's complementary expertise and a valuable mosquito collection curated by Prof Hunter. LB gained valuable input from EP for a fellowship application currently being prepared which will involve investigating the potential of ISVs as novel arbovirus control tools and will include EP as a collaborator and mentor.
Start Year 2022
Description Capstone Project - University of Wisconsin 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact A capstone project at Univ. of Wisconsin, designed by Dr. Coon and Dr. Heinz, engages undergraduate students who isolate bacteria from mosquito samples, and learn about mosquito microbiomes and bacterial diversity.
Year(s) Of Engagement Activity 2022
Description Discussion with the Argentinian Ministry of Health to inform them about novel vector control strategies and research at LSTM. 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact The Argentinian Ministry of Health asked to visit the LSTM and hear about on-going research activity, particular in relation to novel vector control opportunities. I presented research undertaken in the laboratory, which included genetic and symbiotic vector control strategies which may be suitable for implementation in Argentina.
Year(s) Of Engagement Activity 2022