Host genotype and genetic diversity shape the evolution of a novel bacterial infection

Emerging infectious diseases pose significant threats to wildlife populations, as exemplified by various documented cases. These emergence events can stem from reservoir host spill-overs, interspecies jumps, the evolution of new pathogen traits, or invasion of new environments. Pathogen adaptation is a key factor in emergence, and understanding the evolution of novel pathogens within their new host populations is crucial for predicting and managing the consequences. Established host-pathogen relationships demonstrate that pathogen evolution is influenced by factors such as host genetic diversity, spatial structure, and gene flow. Host genotype and genetic diversity can affect pathogen evolution through various mechanisms, including variation in immune resistance, pathogen avoidance, and starvation responses. Genetically homogeneous populations, while uncommon in the wild, are prevalent in conservation efforts focused on declining populations and in agricultural settings. The question arises whether these homogeneous populations are more susceptible to increasingly damaging emerging infections. In established interactions, high host genetic diversity often limits disease spread, virulence evolution, and evolutionary rates, sometimes leading to host range expansion. However, in newly introduced pathogens, it's unclear whether host genetic diversity impacts pathogen evolution early on, especially when most hosts are susceptible. This study aimed to investigate the influence of host genotype and genetic diversity on the evolution of virulence, infectivity, and host range of a newly introduced pathogen.

The introduction extensively reviews existing literature on emerging infectious diseases and their impact on wildlife. It highlights the roles of host genetic diversity, spatial structure, and gene flow in shaping pathogen evolution within established host-pathogen relationships. The review also addresses the existing knowledge gap regarding the impact of host genetic diversity on novel pathogen evolution, particularly during the early stages of association when most hosts are susceptible. The authors cite numerous studies demonstrating the devastating effects of emerging infectious diseases in various wildlife populations and the importance of understanding the evolutionary dynamics of these novel infections.

The study used *Staphylococcus aureus* MSSA476 as the pathogen and 24 natural isolates of *Caenorhabditis elegans* as the host, including the lab-adapted N2 strain. Nematodes were maintained on nematode growth medium (NGM) plates seeded with *E. coli* OP50. To synchronize life stages, a NaCIO and sodium hydroxide solution was used. The pathogen was passaged through ten host generations using two experimental setups: (i) 24 monoculture populations (each with a different *C. elegans* genotype) and (ii) six polyculture populations containing all 24 genotypes. Six no-host control replicates were also included. Passaging was done in 24-well plates containing viscous media to mitigate nematode avoidance behaviors. After 24h of pathogen exposure, nematodes were harvested, and the pathogen was extracted using a beadbeater. Approximately 100 colonies per replicate were picked and grown for the next passage. Host mortality and infection load (measured as colony-forming units, CFU) were assessed for both ancestral and evolved pathogen populations across host genotypes in monoculture and polyculture. To test for specialization, a subset of evolved populations was tested on a novel host genotype (CB4857). Genome sequencing of 40 clones per replicate (ancestor and passage 10) was conducted using Illumina HiSeq4000. Variant calling and filtering were performed using standard bioinformatics pipelines. Statistical analyses included binomial general linear models, ANOVA, Kendall's rank correlation, Spearman's rank correlation, split-plot ANOVAs, Wilcoxon-rank sum tests, and Mantel tests.

At the introduction point, no significant host genetic variation in resistance to *S. aureus* was observed in terms of host mortality or infection load. After ten passages, pathogen virulence significantly varied across host genotypes. Gene ontology (GO) analysis of genes differing in sequence between host genotypes showed that in most comparisons, host genotypes favoring divergent virulence levels differed in functions relating to metal ion binding. Infection load did not evolve to vary across host genotypes, but ancestral and evolved infection loads within sympatric host genotypes were positively associated, suggesting escalation of infectivity. No correlation was found between evolved infection load and pathogen-induced host mortality, indicating independent evolution of these traits. Pathogen genetic distances were positively correlated with corresponding nematode genetic distances, suggesting that host genotype influences pathogen evolution. In polycultures, pathogens evolved higher virulence but reduced infectivity compared to monocultures.

The findings indicate that host genotype significantly shapes the evolution of pathogen virulence, potentially through mechanisms involving metal ion acquisition. The lack of initial host genetic variation in resistance to *S. aureus* might be due to the novelty of this interaction. The contrasting evolutionary trajectories of virulence and infectivity, coupled with the positive association between ancestral and evolved infection loads within sympatric genotypes, suggest distinct selective forces acting on these traits. The positive correlation between pathogen and host genetic distances highlights the impact of host genetic background on pathogen evolution. The observation that polycultures select for higher virulence but constrained infectivity suggests complex interactions between pathogen adaptation and host diversity.

This study provides empirical evidence for the significant impact of host genotype and genetic diversity on the evolution of a novel bacterial infection. The findings highlight the importance of considering host genetic variation when predicting and managing the emergence and spread of infectious diseases. Further research could explore the specific mechanisms underlying the observed patterns, especially the roles of metal ion acquisition and the interaction between virulence and infectivity. Investigating other fitness traits of the host and broader host ranges is also warranted.

The study's experimental design, while robust, has certain limitations. The use of viscous media to mitigate nematode behavior might have influenced the results. Additionally, the study focused on mortality and infection load as measures of virulence and infectivity, neglecting other potential fitness traits of the host or pathogen. Finally, the study was conducted under controlled laboratory conditions, and results might not fully generalize to natural settings.