Clinical Setting Comparative Analysis of Uropathogens and Antibiotic Resistance: A Retrospective Study Spanning the Coronavirus Disease 2019 Pandemic.

Background: Antimicrobial resistance (AMR) in uropathogens has been increasing in Australia. Many nations observed heightened AMR during the coronavirus disease 2019 (COVID-19) pandemic, but it is not known how this may vary across clinical settings and in nations with lower infection rates. Methods: We investigated the uropathogen composition and corresponding antibiotic resistance of 775 559 Australian isolates from the community, hospitals, and aged care facilities before (2016-2019) and during (2020-2022) the COVID-19 pandemic. A mathematical model was developed to predict the likelihood of resistance to currently recommended antibiotics for treating urinary tract infections (UTIs). Results: Among uropathogens originating from the community, hospitals, and aged care facilities, Escherichia coli accounted for 71.4%, 57.6%, and 65.2%, respectively. During the COVID-19 pandemic period, there was an increase in UTIs caused by E coli across all settings. Uropathogens from aged care and hospitals frequently showed higher resistance to antibiotics compared to those isolated from the community. Interestingly, AMR among uropathogens showed a declining trend during the COVID-19 pandemic. Based on the resistance patterns of the past 3 years, our modeling predicted that 30%, 42.6%, and 38.8% of UTIs in the community, hospitals, and aged care facilities, respectively, would exhibit resistance to trimethoprim treatment as empirical therapy. In contrast, resistance to nitrofurantoin was predicted to be 14.6%, 26%, and 24.1% from these 3 respective settings. Conclusions: Empirical therapy of UTIs in Australia with trimethoprim requires evaluation due to high rates of resistance observed across clinical settings.

Genomic Epidemiology and Multilevel Genome Typing of Australian Salmonella enterica Serovar Enteritidis.

Salmonella enterica serovar Enteritidis is one of the leading causes of salmonellosis in Australia. In this study, a total of 568 S. Enteritidis isolates from two Australian states across two consecutive years were analyzed and compared to international strains, using the S. Enteritidis multilevel genome typing (MGT) database, which contained 40,390 publicly available genomes from 99 countries. The Australian S. Enteritidis isolates were divided into three phylogenetic clades (A, B, and C). Clades A and C represented 16.4% and 3.5% of the total isolates, respectively, and were of local origin. Clade B accounted for 80.1% of the isolates which belonged to seven previously defined lineages but was dominated by the global epidemic lineage. At the MGT5 level, three out of five top sequence types (STs) in Australia were also top STs in Asia, suggesting that a fair proportion of Australian S. Enteritidis cases may be epidemiologically linked with Asian strains. In 2018, a large egg-associated local outbreak was caused by a recently defined clade B lineage prevalent in Europe and was closely related, but not directly linked, to three European isolates. Additionally, over half (54.8%) of predicted multidrug resistance (MDR) isolates belonged to 10 MDR-associated MGT-STs, which were also frequent in Asian S. Enteritidis . Overall, this study investigated the genomic epidemiology of S. Enteritidis in Australia, including the first large local outbreak, using MGT. The open MGT platform enables a standardized and sharable nomenclature that can be effectively applied to public health for unified surveillance of S. Enteritidis nationally and globally. IMPORTANCE Salmonella enterica serovar Enteritidis is a leading cause of foodborne infections. We previously developed a genomic typing database (MGTdb) for S. Enteritidis to facilitate global surveillance of this pathogen. In this study, we examined the genomic features of Australian S. Enteritidis using the MGTdb and found that Australian S. Enteritidis is mainly epidemiologically linked with Asian strains (especially strains carrying antimicrobial resistance genes), followed by European strains. The first large-scale egg-associated local outbreak in Australia was caused by a recently defined lineage prevalent in Europe, and three European isolates in the MGTdb were closely related but not directly linked to this outbreak. In summary, the S. Enteritidis MGTdb open platform is shown to be a potentially powerful tool for national and global public health surveillance of this pathogen.

MGTdb: a web service and database for studying the global and local genomic epidemiology of bacterial pathogens.

Multilevel genome typing (MGT) enables the genomic characterization of bacterial isolates and the relationships among them. The MGT system describes an isolate using multiple multilocus sequence typing (MLST) schemes, referred to as levels. Thus, for a new isolate, sequence types (STs) assigned at multiple precisely defined levels can be used to type isolates at multiple resolutions. The MGT designation for isolates is stable, and the assignment is faster than the existing approaches. MGT's utility has been demonstrated in multiple species. This paper presents a publicly accessible web service called MGTdb, which enables the assignment of MGT STs to isolates, along with their storage, retrieval and analysis. The MGTdb web service enables upload of genome data as sequence reads or alleles, which are processed and assigned MGT identifiers. Additionally, any newly sequenced isolates deposited in the National Center for Biotechnology Information's Sequence Read Archive are also regularly retrieved (currently daily), processed, assigned MGT identifiers and made publicly available in MGTdb. Interactive visualization tools are presented to assist analysis, along with capabilities to download publicly available isolates and assignments for use with external software. MGTdb is currently available for Salmonella enterica serovars Typhimurium and Enteritidis and Vibrio cholerae. We demonstrate the usability of MGTdb through three case studies - to study the long-term national surveillance of S. Typhimurium, the local epidemiology and outbreaks of S. Typhimurium, and the global epidemiology of V. cholerae. Thus, MGTdb enables epidemiological and microbiological investigations at multiple levels of resolution for all publicly available isolates of these pathogens. Database URL: https://mgtdb.unsw.edu.au.

Spatial dynamics of inflammation-causing and commensal bacteria in the gastrointestinal tract.

In recent years, new research programmes have been initiated to understand the role of gut bacteria in health and disease, enabled in large part by the emergence of high-throughput sequencing. As new genomic and other data emerge it will become important to explain observations in terms of underlying population mechanisms; for instance, it is of interest to understand how resident bacteria interact with their hosts and pathogens, and how they play a protective role. Connecting underlying processes with observed patterns is aided by the development of mathematical models. Here, we develop a spatial model of microbial populations in the gastrointestinal tract to explore conditions under which inflammation-causing bacteria can invade the gut and under which such pathogens become persistent. We find that pathogens invade both small and large intestine from even a relatively small inoculum size but are usually eliminated by the host response. When the immune response is weak, the pathogen is able to persist for a long period. Spatial structure affects these dynamics by creating moving refugia which facilitate bouts of pathogen resurgence and inflammation in persistent infections. Space also plays a role in repopulation by commensals after infection. We further find that the rate of decay of inflammation has a stronger effect on outcomes than the initiation of inflammation or other parameters. Finally, we explore the impact of partially inflammation-resistant commensals on these dynamics.

Surviving environmental change: when increasing population size can increase extinction risk.

Populations threatened by an abrupt environmental change-due to rapid climate change, pathogens or invasive competitors-may survive if they possess or generate genetic combinations adapted to the novel, challenging condition. If these genotypes are initially rare or non-existent, the emergence of lineages that allow a declining population to survive is known as 'evolutionary rescue'. By contrast, the genotypes required for survival could, by chance, be common before the environmental change. Here, considering both of these possibilities, we find that the risk of extinction can be lower in very small or very large populations, but peaks at intermediate population sizes. This pattern occurs when the survival genotype has a small deleterious effect before the environmental change. Since mildly deleterious mutations constitute a large fraction of empirically measured fitness effects, we suggest that this unexpected result-an intermediate size that puts a population at a greater risk of extinction-may not be unusual in the face of environmental change.

Genomic dissection of the microevolution of Australian epidemic Bordetella pertussis.

ABSTRACTWhooping cough (pertussis) is a highly contagious respiratory disease caused by the bacterium Bordetella pertussis. Despite high vaccine coverage, pertussis has re-emerged in many countries including Australia and caused two large epidemics in Australia since 2007. Here, we undertook a genomic and phylogeographic study of 385 Australian B. pertussis isolates collected from 2008 to 2017. The Australian B. pertussis population was found to be composed of mostly ptxP3 strains carrying different fim3 alleles, with ptxP3-fim3A genotype expanding far more than ptxP3-fim3B. Within the former, there were six co-circulating epidemic lineages (EL1 to EL6). The multiple ELs emerged, expanded, and then declined at different time points over the two epidemics. In population genetics terms, both hard and soft selective sweeps through vaccine selection pressures have determined the population dynamics of Australian B. pertussis. Relative risk estimation suggests that once a new B. pertussis lineage emerged, it was more likely to spread locally within the first 1.5 years. However, after 1.5 years, any new lineage was likely to expand to a wider region. Phylogenetic analysis revealed the expansion of ptxP3 strains was also associated with replacement of the type III secretion system allele bscI1 with bscI3. bscI3 is associated with decreased T3SS secretion and may allow B. pertussis to reduce immune recognition. This study advanced our understanding of the epidemic population structure and spatial and temporal dynamics of B. pertussis in a highly immunized population.

Hazardous Loss of Genetic Diversity through Selective Sweeps in Asexual Populations.

AbstractWith the twofold cost of sex, derived asexual organisms have an immediate reproductive advantage over their sexual sisters. Yet the "twiggy" phylogenetic distribution of asexual lineages implies that they become extinct relatively quickly over evolutionary time. Meanwhile, bacteria and archaea have persisted for billions of years without requiring sexual reproduction. A simple explanation for this difference is that prokaryotes have very large population sizes that are not subject to the accumulation of deleterious mutations, but this implies that drift and mutational meltdown dominate derived asexual populations. Here, we explore a different hazard, quantifying the degree to which genetic variation is lost in asexual populations experiencing selective sweeps. Even though large populations generate diversity by mutation during sweeps, we find that populations that are safe from mutational meltdown may still be reduced to dangerous effective population sizes by sweeps. Thus, ironically, adaptation itself reduces further adaptive potential and may predispose asexual populations to extinction. We derive a simple approximation for the effective population size after a hard sweep and explore the impact of recent sweeps on evolutionary rescue. These factors may help to explain the phylogenetic twigginess of asexuals, the maintenance of sex and recombination, and the evolutionary persistence of prokaryotes.

Gut mutualists can persist in host populations despite low fidelity of vertical transmission.

Humans harbour diverse microbial communities, and this interaction has fitness consequences for hosts and symbionts. Understanding the mechanisms that preserve host-symbiont association is an important step in studying co-evolution between humans and their mutualist microbial partners. This association is promoted by vertical transmission, which is known to be imperfect. It is unclear whether host-microbial associations can generally be maintained despite 'leaky' vertical transmission. Cultural practices of the host are expected to be important in bacterial transmission as they influence the host's interaction with other individuals and with the environment. There is a need to understand whether and how cultural practices affect host-microbial associations. Here, we develop a mathematical model to identify the conditions under which the mutualist can persist in a population where vertical transmission is imperfect. We show with this model that several factors compensate for imperfect vertical transmission, namely, a selective advantage to the host conferred by the mutualist, horizontal transmission of the mutualist through an environmental reservoir and transmission of a cultural practice that promotes microbial transmission. By making the host-microbe association more likely to persist in the face of leaky vertical transmission, these factors strengthen the association which in turn enables host-mutualist co-evolution.

The role of a programmatic immune response on the evolution of pathogen traits.

In modelling pathogen evolution during epidemics, it is important to understand the interactions between within-host infection dynamics and between-host pathogen transmission. Multiscale models often assume an immune response that is highly responsive to pathogen dynamics. Empirical evidence, however, suggests that the immune response in acute infections is triggered and programmatic. This leads to somewhat more predictable infection trajectories where transition times and, consequently, the infectious window are non-exponentially distributed. Here, we develop a within-host model where the immune response is triggered by pathogen growth but otherwise develops independently, and use this to obtain analytic expressions for the infectious period and peak pathogen load. This allows us to model the basic reproductive number in terms of explicit functional relationships among within-host traits including the growth rate of the pathogen. We find that the dependence of pathogen load and the infectious window on within-host parameters constrains the evolution of the pathogen growth rate. At low growth rate, selection favours a higher pathogen load and therefore increasing pathogen growth rate. At high growth rates, selection for a longer infectious window trades off against selection against the effects of virulence. At intermediate growth rates the basic reproductive number is relatively insensitive to changes in the growth rate. The resulting "flat" region of the pathogen fitness landscape is due to the stability of the programmatic immune response in clearing the infection.

Elucidation of global and national genomic epidemiology of Salmonella enterica serovar Enteritidis through multilevel genome typing.

Salmonella enterica serovar Enteritidis is a major cause of foodborne Salmonella infections and outbreaks in humans. Effective surveillance and timely outbreak detection are essential for public health control. Multilevel genome typing (MGT) with multiple levels of resolution has been previously demonstrated as a promising tool for this purpose. In this study, we developed MGT with nine levels for S. Enteritidis and characterised the genomic epidemiology of S. Enteritidis in detail. We examined 26 670 publicly available S. Enteritidis genome sequences from isolates spanning 101 years from 86 countries to reveal their spatial and temporal distributions. Using the lower resolution MGT levels, globally prevalent and regionally restricted sequence types (STs) were identified; avian associated MGT4-STs were found that were common in human cases in the USA; temporal trends were observed in the UK with MGT5-STs from 2014 to 2018 revealing both long lived endemic STs and the rapid expansion of new STs. Using MGT3 to MGT6, we identified multidrug resistance (MDR) associated STs at various MGT levels, which improves precision of detection and global tracking of MDR clones. We also found that the majority of the global S. Enteritidis population fell within two predominant lineages, which had significantly different propensity of causing large scale outbreaks. An online open MGT database has been established for unified international surveillance of S. Enteritidis. We demonstrated that MGT provides a flexible and high-resolution genome typing tool for S. Enteritidis surveillance and outbreak detection.

Modelling the effect of birth and feeding modes on the development of human gut microbiota.

The human gut microbiota is transmitted from mother to infant through vaginal birth and breastfeeding. Bifidobacterium, a genus that dominates the infants' gut, is adapted to breast milk in its ability to metabolize human milk oligosaccharides; it is regarded as a mutualist owing to its involvement in the development of the immune system. The composition of microbiota, including the abundance of Bifidobacteria, is highly variable between individuals and some microbial profiles are associated with diseases. However, whether and how birth and feeding practices contribute to such variation remains unclear. To understand how early events affect the establishment of microbiota, we develop a mathematical model of two types of Bifidobacteria and a generic compartment of commensal competitors. We show how early events affect competition between mutualists and commensals and microbe-host-immune interactions to cause long-term alterations in gut microbial profiles. Bifidobacteria associated with breast milk can trigger immune responses with lasting effects on the microbial community structure. Our model shows that, in response to a change in birth environment, competition alone can produce two distinct microbial profiles post-weaning. Adding immune regulation to our competition model allows for variations in microbial profiles in response to different feeding practices. This analysis highlights the importance of microbe-microbe and microbe-host interactions in shaping the gut populations following different birth and feeding modes.

Enhancing genomics-based outbreak detection of endemic Salmonella enterica serovar Typhimurium using dynamic thresholds.

Salmonella enterica serovar Typhimurium is the leading cause of salmonellosis in Australia, and the ability to identify outbreaks and their sources is vital to public health. Here, we examined the utility of whole-genome sequencing (WGS), including complete genome sequencing with Oxford Nanopore technologies, in examining 105 isolates from an endemic multi-locus variable number tandem repeat analysis (MLVA) type over 5 years. The MLVA type was very homogeneous, with 90 % of the isolates falling into groups with a five SNP cut-off. We developed a new two-step approach for outbreak detection using WGS. The first clustering at a zero single nucleotide polymorphism (SNP) cut-off was used to detect outbreak clusters that each occurred within a 4 week window and then a second clustering with dynamically increased SNP cut-offs were used to generate outbreak investigation clusters capable of identifying all outbreak cases. This approach offered optimal specificity and sensitivity for outbreak detection and investigation, in particular of those caused by endemic MLVA types or clones with low genetic diversity. We further showed that inclusion of complete genome sequences detected no additional mutational events for genomic outbreak surveillance. Phylogenetic analysis found that the MLVA type was likely to have been derived recently from a single source that persisted over 5 years, and seeded numerous sporadic infections and outbreaks. Our findings suggest that SNP cut-offs for outbreak cluster detection and public-health surveillance should be based on the local diversity of the relevant strains over time. These findings have general applicability to outbreak detection of bacterial pathogens.

Nonsynonymous Polymorphism Counts in Bacterial Genomes: a Comparative Examination.

Genomic data reveal single-nucleotide polymorphisms (SNPs) that may carry information about the evolutionary history of bacteria. However, it remains unclear what inferences about selection can be made from genomic SNP data. Bacterial species are often sampled during epidemic outbreaks or within hosts during the course of chronic infections. SNPs obtained from genomic analysis of these data are not necessarily fixed. Treating them as fixed during analysis by using measures such as the ratio of nonsynonymous to synonymous evolutionary changes (dN/dS) may lead to incorrect inferences about the strength and direction of selection. In this study, we consider data from a range of whole-genome sequencing studies of bacterial pathogens and explore patterns of nonsynonymous variation to assess whether evidence of selection can be identified by investigating SNP counts alone across multiple WGS studies. We visualize these SNP data in ways that highlight their relationship to neutral baseline expectations. These neutral expectations are based on a simple model of mutation, from which we simulate SNP accumulation to investigate how SNP counts are distributed under alternative assumptions about positive and negative selection. We compare these patterns with empirical SNP data and illustrate the general difficulty of detecting positive selection from SNP data. Finally, we consider whether SNP counts observed at the between-host population level differ from those observed at the within-host level and find some evidence that suggests that dynamics across these two scales are driven by different underlying processes.IMPORTANCE Identifying selection from SNP data obtained from whole-genome sequencing studies is challenging. Some current measures used to identify and quantify selection acting on genomes rely on fixed differences; thus, these are inappropriate for SNP data where variants are not fixed. With the increase in whole-genome sequencing studies, it is important to consider SNP data in the context of evolutionary processes. How SNPs are counted and analyzed can help in understanding mutation accumulation and trajectories of strains. We developed a tool for identifying possible evidence of selection and for comparative analysis with other SNP data. We propose a model that provides a rule-of-thumb guideline and two new visualization techniques that can be used to interpret and compare SNP data. We quantify the expected proportion of nonsynonymous SNPs in coding regions under neutrality and demonstrate its use in identifying evidence of positive and negative selection from simulations and empirical data.

Emergence of pertactin-deficient pertussis strains in Australia can be explained by models of vaccine escape.

A number of developed countries including Australia have experienced significant pertussis outbreaks in recent years despite consistent high levels of vaccine coverage. Evolutionary changes in Bordetella pertussis carrying variants of the gene encoding pertactin, and the emergence of pertactin-deficient strains (PRN-) in the recent epidemics suggest a possible role of vaccine-driven evolution. In this study, we considered a deterministic 2-strain compartmental model to characterize the relative fitness of PRN- strains and vaccine efficacy against PRN- infection in comparison to the wild-type pertactin-expressing (PRN+) strains. We first showed that the model's equilibrium behavior allows for replacement and co-existence, depending on key parameters related to transmission, vaccine efficacy and durations of immunity. We then fitted the model to epidemiological and pathogen PRN data from the state of New South Wales, Australia. Fitted model parameters showed that the changes in pertussis epidemiology have been governed by a vaccine-escape B. pertussis strain (PRN-) having a basic reproduction number ∼ 1/2 of the wild-type strain which was in circulation prior to April 2009, against which the vaccine was estimated to have substantially reduced efficacy. While not causal, our results suggest that selective pressure from acellular pertussis vaccination is consistent with the changing epidemiology observed in Australia over the analysis period.

Multilevel genome typing: genomics-guided scalable resolution typing of microbial pathogens.

BackgroundBoth long- and short-term epidemiology are fundamental to disease control and require accurate bacterial typing. Genomic data resulting from implementation of whole genome sequencing in many public health laboratories can potentially provide highly sensitive and accurate descriptions of strain relatedness. Previous typing efforts using these data have mainly focussed on outbreak detection.AimWe aimed to develop multilevel genome typing (MGT), using consecutive multilocus sequence typing (MLST) schemes of increasing sizes, stepping up from seven-gene MLST to core genome MLST, to allow examination of genetic relatedness at multiple resolution levels.MethodsThe system was applied to Salmonellaenterica serovar Typhimurium. The MLST scheme used at each step (MGT level), defined a given MGT-level specific sequence type (ST). The list of STs generated from all of these increasing MGT levels, was named a genome type (GT). Using MGT, we typed 9,096 previously characterised isolates with publicly available data.ResultsOur approach could identify previously described S. Typhimurium populations, such as the DT104 multidrug resistance lineage (GT 19-2-11) and two invasive lineages of African isolates (GT 313-2-3 and 313-2-752). Further, we showed that MGT-derived clusters can accurately distinguish five outbreaks from each other and five background isolates.ConclusionMGT provides a universal and stable nomenclature at multiple resolutions for S. Typhimurium strains and could be implemented as an internationally standardised strain identification system. While established so far only for S. Typhimurium, the results here suggest that MGT could form the basis for typing systems in other similar microorganisms.

Lifting the veil on arid-to-hyperarid Antarctic soil microbiomes: a tale of two oases.

BACKGROUND: Resident soil microbiota play key roles in sustaining the core ecosystem processes of terrestrial Antarctica, often involving unique taxa with novel functional traits. However, the full scope of biodiversity and the niche-neutral processes underlying these communities remain unclear. In this study, we combine multivariate analyses, co-occurrence networks and fitted species abundance distributions on an extensive set of bacterial, micro-eukaryote and archaeal amplicon sequencing data to unravel soil microbiome patterns of nine sites across two east Antarctic regions, the Vestfold Hills and Windmill Islands. To our knowledge, this is the first microbial biodiversity report on the hyperarid Vestfold Hills soil environment. RESULTS: Our findings reveal distinct regional differences in phylogenetic composition, abundance and richness amongst microbial taxa. Actinobacteria dominated soils in both regions, yet Bacteroidetes were more abundant in the Vestfold Hills compared to the Windmill Islands, which contained a high abundance of novel phyla. However, intra-region comparisons demonstrate greater homogeneity of soil microbial communities and measured environmental parameters between sites at the Vestfold Hills. Community richness is largely driven by a variable suite of parameters but robust associations between co-existing members highlight potential interactions and sharing of niche space by diverse taxa from all three microbial domains of life examined. Overall, non-neutral processes appear to structure the polar soil microbiomes studied here, with niche partitioning being particularly strong for bacterial communities at the Windmill Islands. Eukaryotic and archaeal communities reveal weaker niche-driven signatures accompanied by multimodality, suggesting the emergence of neutrality. CONCLUSION: We provide new information on assemblage patterns, environmental drivers and non-random occurrences for Antarctic soil microbiomes, particularly the Vestfold Hills, where basic diversity, ecology and life history strategies of resident microbiota are largely unknown. Greater understanding of these basic ecological concepts is a pivotal step towards effective conservation management.

Evolution of the Stator Elements of Rotary Prokaryote Motors.

The bacterial flagellar motor is driven by an ion flux that is converted to torque by motor-attendant complexes known as stators. The dynamics of stator assembly around the motor in response to external stimuli have been the subject of much recent research, but less is known about the evolutionary origins of stator complexes and how they select for specific ions. Here, we review the latest structural and biochemical data for the stator complexes and compare these with other ion transporters and microbial motors to examine possible evolutionary origins of the stator complex.

MERCAT: Visualising molecular epidemiology data combining genetic markers and drug resistance profiles.

Molecular epidemiology uses genetic information from bacterial isolates to shed light on the population structure and dynamics of pathogens. Bacterial pathogens can now be studied by whole genome sequencing, but for some well-studied pathogens such as Mycobacterium tuberculosis a wealth of information is also available from other sources such as spoligotyping and multi-locus variable-number-tandem-repeats (VNTR). Isolates are also frequently tested for susceptibility to antibiotics. Methods of analysis are available for each type of data but it would be informative to combine multiple sources of information into a single analysis or visualisation. Here, we propose and implement a simple way to visualise genotypes along with drug resistance profiles for multiple drugs. We also present a way to combine information from different markers to aid in visualising relationships among isolates. These methods help to reveal the origins and spread of multi-drug resistant lineages of pathogens. We introduce a new computational package, MERCAT (Molecular Epidemiology Researcher's Collection of Analytical Tools), for analysing genotypic data from bacterial isolates. The software is available as an open source package in the statistical language R with a user-friendly interface using R Shiny. Although we focus on tuberculosis and the major molecular markers used to understand tuberculosis transmission - multilocus VNTR-typing (MLVA or MIRU) and spoligotyping - the methods and tools can be applied to other bacteria and can be easily tailored to other genetic markers such as SNP data from whole genome sequencing.

Novel insights into endogenous RNA viral elements in Ixodes scapularis and other arbovirus vector genomes.

Many emerging arboviruses are not transmitted by traditional mosquito vectors, but by lesser-studied arthropods such as ticks, midges, and sand flies. Small RNA (sRNA) silencing pathways are the main antiviral defence mechanism for arthropods, which lack adaptive immunity. Non-retroviral integrated RNA virus sequences (NIRVS) are one potential source of sRNAs which comprise these pathways. NIRVS are remnants of past germline RNA viral infections, where viral cDNA integrates into the host genome and is vertically transmitted. In Aedes mosquitoes, NIRVS are widespread and produce PIWI-interacting RNAs (piRNAs). These are hypothesised to target incoming viral transcripts to modulate viral titre, perhaps rendering the organism a more efficient arbovirus vector. To explore the NIRVS landscape in alternative arbovirus vectors, we validated the NIRVS landscape in Aedes spp. and then identified novel NIRVS in six medically relevant arthropods and also in Drosophila melanogaster. We identified novel NIRVS in Phlebotomus papatasi, Culicoides sonorensis, Rhipicephalus microplus, Anopheles gambiae, Culex quinquefasciatus, and Ixodes scapularis. Due to their unexpected abundance, we further characterised NIRVS in the blacklegged tick I. scapularis (n = 143). Interestingly, NIRVS are not enriched in R. microplus, another hard tick, suggesting this is an Ixodes-specific adaptation. I. scapularis NIRVS are enriched in bunya- and orthomyxo-like sequences, reflecting that ticks are a dominant host for these virus groups. Unlike in mosquitoes, I. scapularis NIRVS are more commonly derived from the non-structural region (replicase) of negative-sense viruses, as opposed to structural regions (e.g. glycoprotein). Like other arthropods, I. scapularis NIRVS preferentially integrate into genomic piRNA clusters, and serve as a template for primary piRNA production in the commonly used embryonic I. scapularis ISE6 cell line. Interestingly, we identified a two-fold enrichment of non-long terminal repeat (non-LTR) retrotransposons, in genomic proximity to NIRVS, contrasting with studeis in Ae. aegypti, where LTR retrotransposons are instead associated with NIRVS formation. We characterised NIRVS phylogeny and integration patterns in the important vector, I. scapularis, revealing they are distinct from those in Aedes spp. Future studies will explore the possible antiviral mechanism conferred by NIRVS to I. scapularis,which may help the transmission of pathogenic arboviruses. Finally, this study explored NIRVS as an untapped wealth of viral diversity in arthropods.

The Role of Pleiotropy in the Evolutionary Maintenance of Positive Niche Construction.

Organisms often modify their environments to their advantage through a process of niche construction. Environments that are improved through positive niche construction can be viewed as a public good. If free riders appear that do not contribute to the shared resource and therefore do not incur any associated costs, the constructed niche may become degraded, resulting in a tragedy of the commons and the extinction of niche constructors. Niche construction can persist if free riders are excluded, for example, if niche constructors monopolize the resource they produce to a sufficient degree. We suggest, however, that the problem of free riders remains because it is possible that nonniche constructors with an enhanced ability to access the resource appear and invade a population of constructors. Using mathematical models we show that positive niche construction can be maintained if it is inextricably linked to a mechanism that makes free riding costly, such as a trait that confers a benefit to only niche constructors. We discuss this finding in terms of genetic interactions and illustrate the principle with a two-locus model. We conclude that positive niche construction can both evolve and be maintained when it has other beneficial effects via pleiotropy. This situation may apply generally to the evolutionary maintenance of cooperation.