Establishing the genetic basis of symbiosis in an insect host

Lead Research Organisation: University of Liverpool
Department Name: Institute of Integrative Biology

Abstract

An animal body is habitat for billions of bacteria, which live in the gut and on the skin. These bacteria were long regard as passengers we called commensals - using the animal as a host, but not strongly affecting the biology of the animal. In contrast, we now recognise that these microbes are an important and active component of the individual - how an organism develops, its immunity and resistance to infection all misbehave when the microbiome is absent or depleted. In insects, symbiosis ('living together') is commonly even better established - microbial partners exist within the body of the insect, not just within the gut and upon skin surfaces. Further, they may be heritable- transmitted from a female to her progeny. These microbial partners define very important properties of the insect -the ability to utilize a plant as a pest; whether the insect can transmit pathogens onward to plants and animals; whether the individual is susceptible to viral/parasitic infection. These properties are exploitable - in Cairns, Australia, mosquitoes carrying a symbiotic microbe that prevents the transmission of dengue are released en masse to protect the residents from infection by dengue.

We currently know little about how these symbionts work. One observation is that they commonly have degraded genomes - which predicts increased reliance on the genes remaining. In addition, the microbes have to deploy an array of genes to establish persistent symbiosis - a long life within the host. The first part of this project will examine how much of the genome is required for these functions. We will also ask if symbiosis is a 'redeployment' of pathogen systems, or whether novel mechanisms are involved. Beyond this, we will establish how symbiosis genes enable the microbe to complete their life cycle, and how they modify the biology of their host - in our case, showing male-limited pathogenesis (male-killing).

The reason we have not been able to answer this question before is simple: adaptation to life within a host makes these bacteria very hard to study outside of the host. In this project, we will exploit a symbiosis where the microbe can be grown in culture, where we can alter the genetic constitution of the microbe and can re-introduce strains easily to the insect. We will use this system to test which aspects of the microbe's genome determine its ability to be symbiotic. We have created 10,000 strains of the bacterium Arsenophonus nasoniae, each with a different gene 'knocked out'. We will reintroduce these into the host insect (the tiny parasitic jewel wasp). We are interested in the strains that fail to establish a symbiosis - these will be ones where the gene in question is necessary for the bacteria to live in symbiosis. We will then explore how these genes aid in establishing a symbiotic life style. Further to this, we will identify the genes responsible for male-killing.

In completing this analysis, we will achieve the first examination of the genes and systems that microbes require to live within an insect and modify its biology. In addition to the intrinsic scientific interest of the research, our findings will allow us to better exploit symbionts to improve human health and food supply. Our focal microbe is closely related to insect vectored plant pathogens that damage fruit plants, and other bacteria that are associated with poor honeybee health. More generally, the knowledge gained will allow us to better engineer novel host-symbiont combinations for pest and vector control, with the aim of improving agricultural yields and human health.

Technical Summary

Using the Aresnophonus nasoniae/Nasonia vitripennis model we will characterise the required genome for A. nasoniae, and determine genetic basis for symbiosis and male-killing as a model for symbioses between insects and gammaproteobacteria.

We will answer the following questions:
1a: What fraction of the A. nasoniae genome is required for in vitro growth?
We will establish the number of required genes for in vitro growth by chatacterising 50,000 Tn5 insert strains. The size and quality of the set of genes required for in vitro growth will be compared to other gammaproteobacteria a) to test the hypothesis that the number of in vitro-required genes is greater in symbionts and b) to establish the genes/pathways essential only in the symbiont.

1b: What fraction of the genome is required for establishment and persistence in the wasp host?
We will take 10,000 Tn5 insert strains and determine the number and nature of genes required for establishing symbiosis. In parallel, we will determine the part of the symbiont genome that is upregulated in vivo using Cappable-seq, and from this determine the fraction of symbiosis-expressed genes that are required for symbiosis.

2: What genes are required for maintaining symbiont invasion and establishment?
We will select 5-10 functional groups from objective 1a and use qRT PCR and microscopy to determine the nature of their requirement for symbiosis in terms of broad processes (initial establishment; spread; vertical transmission), and detailed mechanism (e.g. failure to penetrate gut wall, failure to evade immunity).

3: Which gene causes the male-killing phenotype.
We will isolate potential male-killing defective stains and test function through complementation. Structural and bioinformatics analysis will then be completed to determine the likely functional basis of male-killing, in terms of the toxin itself or loss of capacity to secrete toxins.

Planned Impact

Heritable microbes represent important associates of pest and vector insects. The modifications they make to host biology make them a potent weapon in the fight against pest and vector borne disease. For instance, Wolbachia is now deployed as a front line defence against dengue transmission by mosquitoes, with release of symbiont infected mosquitoes breaking the dengue transmission cycle.

However, our poor understanding of the mechanistic basis of symbiosis represents a major impediment to the more widespread use of heritable microbes in pest/vector control. Our lack of understanding of the systems underlying symbiosis is a barrier to full exploitation.

This proposal will deliver the first screen for the factors required for symbiotic persistence of a microbe. This will pump prime application for two closely related microbes, one an insect vectored phytopathogen, one an associate of honey bees. It will additionally inform application for important pest and vector species, such as aphid, tse tse flies and lice.

As such the study informs research underpinning both human health and food security.

Publications

10 25 50
 
Description Microbe genomes carry most of their coding content on a single circular chromosome. To this are added 'extrachromosomal elements, which often contain important genes like antibiotic resistance. These elements can shuttle between microbes, that allows the flow of important traits between microbe species.

We have discovered our microbe has incredibly diverse extrachromosomal elements - 17 in total - that encode many important aspects permitting the microbe to colonise insects.

We have also made a fluorescent version of our microbe, that enables tracking of it in the insect host. This reveals a profound tendency for the microbe to aggregate in the ovipositor - which allows its onward transmission.

We have further isolated mutations that disrupt the symbiosis, in terms of overreplication mutations. These indicate the balanced nature of the symbiosis. The systems identified are interesting, as they are repurposed from pathogenesis. They indicate that symbiont are selected for control over titre so as not to damage their hosts. The overreplication mutant transmits better but is pathogenic.

Finally, we have identified genomic changes that occurred within the A. nasoniae clade that indicate systems important in this symbiosis. Key findings are that:
a) Genome complexity increases on first transition to vertical transmission, contrary to standard thinking.
b) GC content of symbiont does not decline until they are obligate symbionts.
c) Pseudogenization initiates rapidly on transition into vertical transmission, is highest after some evolutionary time before purging occurs.
d) Early changes in symbiosis involve loss of the CRISPR system, which initially is associated with phage accumulation, which are then purged.
e) The type VI secretion system (associated with bacterial warfare) is lost on entering symbiosis, but the range of type III effectors and toxins increases initially.
The present the first holistic picture of evolution on entry into symbiosis.

Further to this, we have shown that the Rcs system is modified in Arsenophonus to allow a balanced (and not overproliferating) symbiosis. Knockout of this system resulted in increased titre and harm to the host compared to wild type symbionts. This repurposing is the opposite of that found in pathogens where the system enables proliferation in the host context.

In addition, we have cultured, sequenced and described a related Arsenophonus, A. apicola that is a likely bee pathogen with worldwide incidence. We have demonstrated the Galleria model system is usable for this strain, providing a new model system for investigating insect-pathogen interactions.

Finally, we have cultured, sequenced and described a further gammaproteobcterium, Symbiopectobacterium purcelli, which is a symbiont related to Arsenophonus that infects planthoppers, which are important agricultural pests and vectors. This species has a less complex genome that is easier to work with, in terms of functional genomics, than Arsenophonus, so provides a promising forward avenue for research.
Exploitation Route It allows tracking of symbionts in vivo without the need to kill the insect host first. This will be very useful for understanding how microbes colonise insects.
It impacts on bee health and hive productivity.
It impacts on plant pest health and biology, and may be useful in mitigating these disease risks.
Sectors Agriculture

Food and Drink

Environment

 
Description Discovery of Arsenophonus apicola in bees has led to investigation of the importance of this microbe in honey bee health. Methods for Culturability of Arsenophonus isolates and transformation has been utilized in projects to develop means to reduce damage by the brown planthopper to rice in China through altering its symbiont.
First Year Of Impact 2022
Sector Agriculture, Food and Drink
Impact Types Economic

 
Title A. nasoniae genome data 
Description The complete genome sequence for A. nasoniae, and analysis therein. 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? Yes  
Impact Complete genome resource for this model insect symbiont 
URL https://www.ncbi.nlm.nih.gov/bioproject/529362
 
Title ANI, AAI and dDDH analysis of A. apicola vs other Arsenophonus strains 
Description Average Nucleotide Identity (ANI), Average Amino acid identity (AAI) and Digital DNA-DNA Hybridization (dDDH) data for Arsenophonus apicola compared to other sequenced strains within the clade Arsenophonus, and with Providencia stuartii and Proteus mirabilis as outgroup genera. Sheet 1: Pairwise ANI between strains. Sheet 2: Pairwise AAI between strains. Sheet 3: dDDH of A. apicola vs other strains using three calculation methods. ANI and AAI Calculations were made using the enveomics toolkit (Rodriguez-R LM, Konstantinidis KT. The enveomics collection: a toolbox for specialized analyses of microbial genomes and metagenomes. PeerJ Preprints. 2016;4:e1900v1.). dDDH calculations were completed using the Genome-to-Genome Distance Calculator GGDC 3.0 from dsmz (https://www.dsmz.de/services/online-tools/genome-to-genome-distance-calculator-ggdc). 
Type Of Material Database/Collection of data 
Year Produced 2022 
Provided To Others? Yes  
Impact Standard for description of diversity within this important genus of insect associates. 
URL https://figshare.com/articles/dataset/ANI_AAI_and_dDDH_analysis_of_A_apicola_vs_other_Arsenophonus_s...
 
Title Accession numbers for markers used in phylogenetic analysis of Arsenophonus 
Description Accession numbers for DNA sequence of fbaA, ftsK and yaeT genes from Arsenophonus strains used for phylogenetic reconstruction. 
Type Of Material Database/Collection of data 
Year Produced 2022 
Provided To Others? Yes  
Impact Allows community to identify this potential honey bee pathogen. 
URL https://figshare.com/articles/dataset/Accession_numbers_for_markers_used_in_phylogenetic_analysis_of...
 
Title F1 P. vindemmiae progeny recovered from factorial crosses with Arsenophonus infected and uninfected mother and father wasps 
Description Female P. vindemmiae wasps with and without Arsenophonus were crossed to males with and without Arsenophonus in a full factorial design, and then permitted to lay on D. melanogaster pupae. The number of flies emerging were recorded alongside the sex and number of F1 wasp progeny. One or two wasps per replicate were then tested for Arsenophonus presence by PCR. In addition, Wolbachia presence was also tested. Column A: Treatment code (each colour represents a different cross type). Column B: Replicate within cross type. Column C: Cross type. First set is the female, second male. A+ = Arsenophonus +ve W+ = Wolbachia positive. A- =Arsenophonus -ve W- Wolbachia -ve. Column D: # flies emerging within 48 hours (measure of unparasitized fly pupae). Mother data: Column E: QC PCR outcome for mother. + = good DNA, - = poor DNA Column F: PCR assay for mother for Wolbachia outcome: += present, -= absent Column G: PCR assay for mother for Arsenophonus outcome test 1: += present, -= absent Column H: PCR assay for mother for Arsenophonus test 2 outcome (HS PCR): += present, -= absent Column I: Conclusion concerning mother Arsenophonus/Wolbachia status from data in columns E-H. A+ = Arsenophonus +ve W+ = Wolbachia positive. A- =Arsenophonus -ve W- Wolbachia -ve. Brood data: Column J: # wasp offspring emerging. Column K: # flies surviving Column L: # female wasps emerging Column M: #male wasps emerging. Column N: % male wasps in brood (males/total) Column O: # parasitized flies where wasp did not appear Column P: #Flies that died Column Q: fate unknown Individual F1 progeny infection status: Offspring 1: Column R: sex of wasp Column S: QC status of DNA (+ = pass, - = fail) Column T: PCR assay for Wolbachia (+= present, - = absent) Column U: PCR assay for Arsenophonus (+= present, - = absent) Column V: Conclusion for infection status of wasp one (A+ = Arsenophonus present, W+= Wolbachia present). Offspring 2 Column W: sex of wasp Column X: QC status of DNA (+ = pass, - = fail) Column Y: PCR assay for Wolbachia (+= present, - = absent) Column Z: PCR assay for Arsenophonus (+= present, - = absent) Column AA: Conclusion for infection status of wasp two (A+ = Arsenophonus present, W+= Wolbachia present). 
Type Of Material Database/Collection of data 
Year Produced 2022 
Provided To Others? Yes  
Impact Transmission efficiency through maternal and paternal lines of Arsenophonus in a wasp model. 
URL https://figshare.com/articles/dataset/F1_P_vindemmiae_progeny_recovered_from_factorial_crosses_with_...
 
Title Frequency of A. nasoniae in calliphorid fly pupae collected from European bird's nests 
Description Nasonia vitripennis commonly oviposits in calliphorid 'filth fly' pupae found in birds' nests. Nests of blue tit (Cyanistes caeruleus), great tit (Parus major) and other passerine birds were collected shortly following fledging from the nest and placed in bags. Collections were made in June/July 2012 from the area surrounding Harjavalta Town, Finland; from Lausanne, Switzerland; from Kraslava, Latvia; and from Marbury, Cheshire in the UK. Each nest was given an ID number, was manually inspected for calliphorid/protocalliphorid pupae, and these pupae removed individually to a tube for any parasitising wasps to emerge. Where more than one pupa was collected from the same nest this was noted, as pupae from the same nest may be parasitised by the same individual wasp. The pupae, which were monitored daily, were placed at 25 °C for 14 days to allow for N. vitripennis wasps to emerge. Arsenophonus nasoniae presence in N. vitripennis was then assayed. DNA was prepared using the Chelex method. Template quality was assessed based on COI amplification using primers HCO/LCO (Folmer et al, (1994), with 30 PCR cycles of 93°C for 15s, 47°C for 1m, 72°C for 1m and an expected amplicon size of. c. 710bp. Templates passing this quality control were then tested for A. nasoniae using primers amplifying the metalloprotease gene (M1f GGGTCACATACCTATTTT, M1r GTAGTCGCCTGGGTGGG), with 30 cycles of 93°C for 15s, 55°C for 45s, 72°C for 30s and an expected amplicon size of 594bp . Sheet 1: Pupa ID: Unique identifier for fly pupa collected from birds nest. Note, the initial number represents the nest ID (column B) and a suffix letter indicates independent pupae from that nest. Nest ID: Unique identified for the birds nest the pupa was collected from. Arse +/-: Presence of Arsenophonus (+) in Nasonia emerging from that wasp as ascertained through PCR assay. Null value means Arsenophonus not detected. Location: Place the nest/pupa was located. Sheet 2: Summary data. 
Type Of Material Database/Collection of data 
Year Produced 2023 
Provided To Others? Yes  
Impact European survey data for wasps attacking filth flies from across Europe. 
URL https://figshare.com/articles/dataset/Frequency_of_A_nasoniae_in_calliphorid_fly_pupae_collected_fro...
 
Title Genome sequence for Arsenophonus apicola 
Description Assembled genome sequence and raw reads for Arsenophonus apicola. 
Type Of Material Database/Collection of data 
Year Produced 2021 
Provided To Others? Yes  
Impact Aid in bee health research 
URL https://www.ncbi.nlm.nih.gov/bioproject/PRJNA766690
 
Title Genome sequence for Symbiopectobacterium purcelli 
Description The assembled genomes and reads underpinning completion of the genome. 
Type Of Material Database/Collection of data 
Year Produced 2021 
Provided To Others? Yes  
Impact Genome for associate of plant pest species. 
URL https://www.ncbi.nlm.nih.gov/nuccore/CP081864
 
Title Growth of A.apicola at different temperatures 
Description Growth of Arsenophonus apicola strain ArsBeeUS in BHI (brain heart infusion) media at different temperatures (30, 33, 35, 37 C). Data given is colony forming units (cfu/microlitre) after 48 hours growth determined through plating across a dilution series. Initial inoculum is calculated and given below these data. 
Type Of Material Database/Collection of data 
Year Produced 2022 
Provided To Others? Yes  
Impact Allows community better ability to grow and culture this emerging bee pathogen. 
URL https://figshare.com/articles/dataset/Growth_of_A_apicola_at_different_temperatures/19771705
 
Title Presence of Nasonia wasps and the bacterium Arsenophonus nasoniae in calliphorid fly pupae collected from great tit nests in Coimbra, Portugal. 
Description During the breeding season of 2021, we sampled pupae from great tit nests at Mata Nacional do Choupal (40º13'N, 8º27'W), a mixed woodland on the periphery of the city of Coimbra, Portugal (more details of the study area in (Norte et al., 2009). Old nest material from all nest boxes had been previously removed at the end of the previous breeding season to avoid the accumulation of overwintering Protocalliphora spp. adults (Gold and Dahlsten, 1983). Once all fledglings had left their nests in May/June, the nests were collected in plastic bags and transported to the laboratory. Blowfly puparia were sorted manually from the nest material and were stored in 1.5 mL tubes with absolute ethanol for further genetic analysis. To determine the presence of N. vitripennis and its endosymbiont A. nasoniae, up to five pupae collected from each nest were surface sterilised with 70% ethanol, added to a 2 ml Eppendorf tube, crushed and homogenised using a sterile pestle. Fly pupal shells were removed with the aid of a sterile forceps and DNA was extracted from the homogenised sample using a Zymoresearch Quick DNA TM Miniprep plus kit (Zymoresearch, USA). These were then subject to diagnostic QC PCR of the COI gene with HCO/LCO primers described above (this amplifies fly DNA in the absence of other parties, and establishes template quality). Then, PCR assays for wasp and microbe were performed. Amplicons for the A. nasoniae PCR assay were sequenced using the Sanger method to confirm identity. Column A: Nest ID Column B: Collection date for fly pupae Column C: Number of fly pupae combined for testing. Column D: Presence of Nasonia (- = not present += present) Column E: Presence of A. nasoniae (- = not present += present) 
Type Of Material Database/Collection of data 
Year Produced 2023 
Provided To Others? Yes  
Impact New method for detection of Arsenophonus in wild collected samples without recourse to breeding/transfer of live material across national boundaries 
URL https://figshare.com/articles/dataset/Presence_of_Nasonia_wasps_and_the_bacterium_Arsenophonus_nason...
 
Title Sex ratio produced by Arsenophonus infected vs uninfected Nasonia vitripennis from the UK and Switzerland 
Description Mated Individual Female N. vitripennis that emerged from field collected pupae were allowed to oviposit in Sarcophaga pupae. Female N. vitripennis were then categorised as carrying or not carrying Arsenophonus nasoniae by PCR assay. Emerging progeny were scored for sex. In this analysis, individuals producing no female offspring were discounted, as this is commonly the result of failure of the female wasp to mate and fertilize eggs. Column A: Female ID Column B: Source population (UK or Switzerland) Column C: Results of PCR assay for A. nasoniae (+ = present, - = absent). Column D: #Female wasp progeny emerging Column E: #Male wasp progeny emerging Column F: Proportion of progeny that were female (Column D/(Column D + Column E)) 
Type Of Material Database/Collection of data 
Year Produced 2023 
Provided To Others? Yes  
Impact Indicate Arsenophonus symbionts are widespread in Europe and act as male-killers; First survey of European populations. 
URL https://figshare.com/articles/dataset/Sex_ratio_produced_by_Arsenophonus_infected_vs_uninfected_Naso...
 
Title Transmission of Arsenophonus from single infected females vs mixed infected/uninfected groups 
Description Two treatments: a) Single Pachycrepoideus vindemmiae females infected with Arsenophonus were allowed to oviposit on D.melanogaster pupae within a vial. F1 progeny were then tested for Arsenophonus presence via PCR (with QC on insect to verify amplifiability). b) Groups of 2 infected and 2 uninfected P. vindemmiae females were allowed to oviposit on D.melanogaster pupae within a vial. F1 progeny were then tested for Arsenophonus presence via PCR (with QC on insect to verify amplifiability). Column A: Sample # Colimn B: Treatment (single female or group lay as defined above) Column C: Vial replicate number Column D: QC PCR results on insect DNA (1 = amplifiable 0= fail) Column E: PCR assay results for Arsenophonus presence (1 = present, 0 = absent) Column F: PCR plate/well Column G: Notes, e.g. if sample was rerrun when first assay was ambiguous. 
Type Of Material Database/Collection of data 
Year Produced 2022 
Provided To Others? Yes  
Impact Data indicating no infectious transmission of Arsenophonus in P. vindemmiae. 
URL https://figshare.com/articles/dataset/Transmission_of_Arsenophonus_from_single_infected_females_vs_m...
 
Description Arsenophonus from Australian bees 
Organisation Deakin University
Country Australia 
Sector Academic/University 
PI Contribution Sequencing of genome of Arsenophonus associated with Australian pollinators
Collaborator Contribution Isolation and culture of microbe
Impact Complete genome sequence of Arsenophonus apicola strain aAm_Au
Start Year 2022
 
Description Arsenophonus from bees 
Organisation U.S. Department of Agriculture USDA
Department Agricultural Research Service
Country United States 
Sector Public 
PI Contribution Nanopore sequencing of bee Arsenophonus strains
Collaborator Contribution Isolation to pure culture of bee Arsenophonus; illumina sequencing
Impact Will form part of comparative genomics paper and formal description of the symbiont
Start Year 2020
 
Description Arsenophonus from bees 
Organisation University of Wisconsin–Stout
Country United States 
Sector Academic/University 
PI Contribution Nanopore sequencing of bee Arsenophonus strains
Collaborator Contribution Isolation to pure culture of bee Arsenophonus; illumina sequencing
Impact Will form part of comparative genomics paper and formal description of the symbiont
Start Year 2020
 
Description Arsenophonus from planthopper pests 
Organisation Huazhong Agricultural University
Country China 
Sector Academic/University 
PI Contribution Advice on the culture and transformation of Arsenophonus symbionts of the rice plant hopper
Collaborator Contribution Experimental analysis of the above
Impact Succesful culture and transformation of symbiont to enable genome sequencing and additionally tracking of symbiosis in vivo in this important plant pest species.
Start Year 2022
 
Description Arsenophonus from tick symbionts 
Organisation National Institute for Public Health and Environment (RIVM)
Country Netherlands 
Sector Academic/University 
PI Contribution We have obtained long read sequence and assembled/analysed the genome of Arsenophonus symbionts of tick parasites.
Collaborator Contribution Isolating the symbiont to pure culture; Illumina sequencing
Impact Will contribute to paper on comparative genomics of Arsenophonus
Start Year 2020
 
Description Genome sequencing of Arsenophonus from tick parasites 
Organisation National Institute for Public Health and Environment (RIVM)
Country Netherlands 
Sector Academic/University 
PI Contribution Sequencing of Arsenophonus from tick parasite
Collaborator Contribution Isolation of Arsenophonus from tick parasite
Impact Publication in preparation - comparative genomics in the genus Arsenophonus
Start Year 2021
 
Description Phlomobacter 
Organisation University of Milan
Country Italy 
Sector Academic/University 
PI Contribution We supplied GFP labelled Arsenophonus and advised on experiments investigating the capacity of this bacterium to propagate through plants
Collaborator Contribution Conducted the main experiment.
Impact Potential paper led by collaborating group - ongoing.
Start Year 2019
 
Description Art activity for children centred on project 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact 120 children aged 3-15 attended art workshops based on our butterfly, and learnt about insect life cycles. We taught the parents about microbes and how they impact the health of insects and humans.
Year(s) Of Engagement Activity 2019
URL https://blogandlog.wordpress.com/2019/07/08/butterfly-crafternoon-at-the-williamson/
 
Description Interview on local news 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Media (as a channel to the public)
Results and Impact 10 minute interview on Radio Merseyside on insect related issues, viz ladybird sightings and mosquito biting activity in the local area.
Year(s) Of Engagement Activity 2023