Establishing the genetic basis of symbiosis in an insect host

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.

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.

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.