Gasterosteus Aculeatus Research Articles

Environmentally independent selection for hybrids between divergent freshwater stickleback lineages in semi-natural ponds.

Hybridization following secondary contact of genetically divergent populations can influence the range expansion of invasive species, though specific outcomes depend on the environmental dependence of hybrid fitness. Here, using two genetically and ecologically divergent threespine stickleback lineages that differ in their history of freshwater colonization, we estimate fitness variation of parental lineages and hybrids in semi-natural freshwater ponds with contrasting histories of nutrient loading. In our experiment, we found that fish from the older freshwater lineage (Lake Geneva) and hybrids outperformed fish from the younger freshwater lineage (Lake Constance) in terms of both growth and survival, regardless of the environmental context of our ponds. Across all ponds, hybrids exhibited the highest survival. Although wild-caught adult populations differed in their functional and defence morphology, it is unclear which of these traits underlie the fitness differences observed among juveniles in our experiment. Overall, our work suggests that when hybrid fitness is insensitive to environmental conditions, as observed here, introgression may promote population expansion into unoccupied habitats and accelerate invasion success.

Origin matters: Using a local reference genome improves measures in population genomics.

Genome sequencing enables answering fundamental questions about the genetic basis of adaptation, population structure and epigenetic mechanisms. Yet, we usually need a suitable reference genome for mapping population-level resequencing data. In some model systems, multiple reference genomes are available, giving the challenging task of determining which reference genome best suits the data. Here, we compared the use of two different reference genomes for the three-spined stickleback (Gasterosteus aculeatus), one novel genome derived from a European gynogenetic individual and the published reference genome of a North American individual. Specifically, we investigated the impact of using a local reference versus one generated from a distinct lineage on several common population genomics analyses. Through mapping genome resequencing data of 60 sticklebacks from across Europe and North America, we demonstrate that genetic distance among samples and the reference genomes impacts downstream analyses. Using a local reference genome increased mapping efficiency and genotyping accuracy, effectively retaining more and better data. Despite comparable distributions of the metrics generated across the genome using SNP data (i.e. π, Tajima's D and FST ), window-based statistics using different references resulted in different outlier genes and enriched gene functions. A marker-based analysis of DNA methylation distributions had a comparably high overlap in outlier genes and functions, yet with distinct differences depending on the reference genome. Overall, our results highlight how using a local reference genome decreases reference bias to increase confidence in downstream analyses of the data. Such results have significant implications in all reference-genome-based population genomic analyses.

Exposure and hazard of bisphenol A, S and F: a multi-biomarker approach in three-spined stickleback.

Due to the estrogenic behavior of bisphenol (BP) A, industries have developed many substitutes, such as BPS and BPF. However, due to their structural similarities, adverse effects on reproduction are currently observed in various organisms, including fish. Even if new results have shown impacts of these bisphenols on many other physiological functions, their mode of action remains unclear. In this context, we proposed to better understand the impact of BPA, BPS, and BPF on immune responses (leucocyte sub-populations, cell death, respiratory burst, lysosomal presence, and phagocytic activity) and on biomarkers of metabolic detoxification (ethoxyresorufin-O-deethylase, EROD, and glutathione S-transferase, GST) and oxidative stress (glutathione peroxidase, GPx, and lipid peroxidation with thiobarbituric acid reactive substance method, TBARS) in an adult sentinel fish species, the three-spined stickleback. In order to enhance our understanding of how biomarkers change over time, it is essential to determine the internal concentration responsible for the observed responses. Therefore, it is necessary to explore the toxicokinetics of bisphenols. Thus, sticklebacks were exposed either to 100 μg/L of BPA, BPF or BPS for 21 days, or for seven days to 10 and 100 μg/L of BPA or BPS followed by seven days of depuration. Although BPS has very different TK, due to its lower bioaccumulation compared to BPA and BPF, BPS affect oxidative stress and phagocytic activity in the same way. For those reasons, the replacement of BPA by any substitute should be made carefully in terms of risk assessment on aquatic ecosystems.

Long-term environmental stability does not erode plasticity in nest building responses to changing ambient conditions.

The primary function of animal nests is to protect developing offspring from hostile and fluctuating environments. Animal builders have been shown to adjust nest construction in response to changes in their environment. However, the extent of this plasticity, and its dependence on an evolutionary history of environmental variability, is not well understood. To test whether an evolutionary history with flowing water impacts male ability to adjust nests in response to flow regime, we collected three-spined sticklebacks (Gasterosteus aculeatus) from three lakes and three rivers, and brought them into reproductive condition in controlled laboratory aquaria. Males were then allowed to nest under both flowing and static conditions. Nest building behaviour, nest structure and nest composition were all recorded. In comparison to males building nests under static conditions, males building in flowing water took longer to construct their nests and invested more in nesting behaviour. Moreover, nests built in flowing water contained less material, were smaller, more compact, neater and more elongated than nests built under static conditions. Whether males came from rivers or lakes had little impact on nesting activities, or male capacity to adjust behaviours in response to flow treatment. Our findings suggest that aquatic animals which have experienced a stable environment over a long period of time retain plasticity in nest-building behaviours that allow them to adjust nests to ambient flow conditions. This ability may prove crucial in coping with the increasingly unpredictable flow regimes found in anthropogenically altered waterways and those resulting from global climate change. This article is part of the theme issue 'The evolutionary ecology of nests: a cross-taxon approach'.

A Baltic pelagic fish community revisited: Indications of profound changes in species composition in the Stockholm Archipelago

While the central Baltic herring (Clupea harengus) stock biomass is estimated to have been reduced by ∼80 per cent since the 1970 s, the local effects of this decline remain unassessed. Coastal commercial fishermen have witnessed a widespread depletion at the spawning grounds and generally low herring abundance in inshore areas. We carried out hydroacoustic surveys, supported by midwater trawling in August 2021, to test whether herring abundance and recruitment have changed over time in the inshore areas. We used assessed recruitment success, measured as young-of-the-year (YOY) abundance, as a proxy for herring abundance in several areas of the southeast Stockholm Archipelago and compared the results with previous investigations in this area (hydroacoustics 2000–2001 and midwater trawling 2002–2004 monitoring). We found that the composition of the fish community had undergone significant changes. While acoustic backscattering (sA) representing the whole fish community were similar to previous estimates, species composition was significantly changed. YOY herring, previously dominant, had declined from 55% to 4% in numbers, whereas three-spined stickleback (Gasterosteus aculeatus) increased from no representation to 76%. The observed changes makes it imperative that further investigation concerning herring population structure, biomass, and recruitment are conducted.

Assessing the potential for successful translocation and co‐management of two endangered aquatic species

AbstractTranslocation programmes for endangered species typically focus on a single species, but in areas where little native habitat remains, it may be necessary to translocate multiple species to the same sites. Interactions between translocated species, such as predation and competition, are among the factors that need to be considered when planning multispecies translocations. Translocation sites for aquatic species are particularly scarce in southern California, where a limited number of sites exist for historically co‐occurring endangered mountain yellow‐legged frogs Rana muscosa and unarmoured three‐spine sticklebacks Gasterosteus aculeatus williamsoni. To determine how these species would interact if translocated to the same sites, we carried out experiments ex situ with R. muscosa tadpoles and a surrogate subspecies of stickleback (G. a. microcephalus). We found that (1) adult sticklebacks preyed on hatchling tadpoles but did not consume R. muscosa eggs or large tadpoles; (2) tadpoles did not consume stickleback eggs or disturb sticklebacks nests; and (3) both species' microhabitat use shifted slightly when the other was present. Our results suggest that these species can likely be co‐managed successfully, if measures are taken to curb stickleback predation on tadpoles until the R. muscosa population is well established. Using ex situ studies to evaluate species interactions prior to translocation is an approach that could prove useful in other species recovery programmes. Multispecies translocations could make better use of available resources when habitat is limited and promote ecosystem recovery by re‐establishing interactions among native species.

Repeated parallel differentiation of social learning differences in benthic and limnetic threespine stickleback fish.

Individuals can reduce sampling costs and increase foraging efficiency by using information provided by others. One simple form of social information use is delayed local enhancement or increased interest in a location because of the past presence of others. We tested for delayed local enhancement in two ecomorphs of stickleback fish, benthic and limnetic, from three different lakes with putative independent evolutionary origins. Two of these lakes have reproductively isolated ecomorphs (species-pairs), whereas in the third, a previously intact species-pair recently collapsed into a hybrid swarm. Benthic fish in both intact species-pair lakes were more likely to exhibit delayed local enhancement despite being more solitary than limnetic fish. Their behaviour and morphology suggest their current perceived risk and past evolutionary pressure from predation did not drive this difference. In the hybrid swarm lake, we found a reversal in patterns of social information use, with limnetic-looking fish showing delayed local enhancement rather than benthic-looking fish. Together, our results strongly support parallel differentiation of social learning differences in recently evolved fish species, although hybridization can apparently erode and possibly even reverse these differences.

Opening a can of worms: a test of the co-infection facilitation hypothesis

Parasitic infections are a global occurrence and impact the health of many species. Coinfections, where two or more species of parasite are present in a host, are a common phenomenon across species. Coinfecting parasites can interact directly or indirectly via their manipulation of (and susceptibility to) the immune system of their shared host. Helminths, such as the cestode Schistocephalus solidus, are well known to suppress immunity of their host (threespine stickleback, Gasterosteus aculeatus), potentially facilitating other parasite species. Yet, hosts can evolve a more robust immune response (as seen in some stickleback populations), potentially turning facilitation into inhibition. Using wild-caught stickleback from 20 populations with non-zero S. solidus prevalence, we tested an a priori hypothesis that S. solidus infection facilitates infection by other parasites. Consistent with this hypothesis, individuals with S. solidus infections have 18.6% higher richness of other parasites compared to S. solidus-uninfected individuals from the same lakes. This facilitation-like trend is stronger in lakes where S. solidus is particularly successful but is reversed in lakes with sparse and smaller cestodes (indicative of stronger host immunity). These results suggest that a geographic mosaic of host–parasite co-evolution might lead to a mosaic of between-parasite facilitation/inhibition effects.

Elemental content of a host-parasite relationship in the threespine stickleback.

Parasite infections are ubiquitous and their effects on hosts could play a role in ecosystem processes. Ecological stoichiometry provides a framework to study linkages between consumers and their resource, such as parasites and their host, and ecosystem process; however, the stoichiometric traits of host-parasite associations are rarely quantified. Specifically, it is unclear whether parasites' elemental ratios closely resemble those of their host or if infection is related to host stoichiometry, especially in vertebrate hosts. To answer such questions, we measured the elemental content (%C, %N, and %P) and molar ratios (C:N, C:P, and N:P) of parasitized and unparasitized Gasterosteus aculeatus (three-spined stickleback) and their cestode parasite, Schistocephalus solidus. Host and parasite elemental content were distinct from each other, and parasites were generally higher in %C and lower in %N and %P. Parasite infections were related to host C:N, with infected hosts being lower in C:N. Parasite elemental content was independent of their host, but parasite body mass and parasite density were important drivers of parasite stoichiometry. Overall, these potential effects of parasite infections on host stoichiometry along with parasites' distinct elemental compositions suggest parasites may further contribute to differences in how individual hosts store and recycle nutrients.

Repeatability of crossover rate in wild sticklebacks

AbstractCrossovers in meiosis have an important role in sexual reproduction in ensuring the segregation of chromosomes and shuffling of genetic material. Despite their profound importance, little is known about heritability and repeatability of the crossover rate in the wild. We studied crossover rate variation in outbred nine- (Pungitius pungitius) and three-spined (Gasterosteus aculeatus) stickleback populations to estimate its heritability (h2) and repeatability (R). Based on crossover rate interpreted from parent–F1-offspring linkage maps created from single nucleotide polymorphism data, the repeatability of crossover rate was moderate at best, but higher in females (R = 0.24 and R = 0.33) than in males (R = 0.017 and R = 0.11) in both species. Owing to the low degree and variance of parental relatedness and low sample sizes, heritabilities could not be estimated with confidence. However, given that R sets the upper limit to h2, the heritability of crossover rate is indicated to be low in males. A review and comparison with the previously reported repeatability and heritability estimates revealed that the repeatabilities in stickleback females were relatively high, whereas those in males were very low. Collectively, our results and review of earlier estimates suggest low evolvability of the crossover rate.

PBTK-TD model of the phagocytosis activity in three-spined stickleback exposed to BPA

Due to the high production volume and persistence in the environment of bisphenol A (BPA) and its substitutes, realistic exposure scenarii were proposed in some species to better understand the relationship between external and internal concentrations. For example, a recent PBTK model has been developed and adapted to BPA ADME (Absorption, Distribution, Metabolization, and Excretion) processes in three-spined stickleback. These substances have an impact on organism physiology including reproductive and immune functions. In this context, physiologically-based toxicokinetic models coupled with toxicodynamics (PBTK-TD) have proven to be valuable tools to fill the knowledge gap between external exposure and effect dynamics. The aim of the current work was to explain the impact of BPA on the immune response by determining its temporality. In addition, the relationship between BPA dose and these responses was investigated using a PBTK-TD model. Two experiments were performed on stickleback to characterize their biomarker responses, (i) a short exposure (14 days) at 0, 10 and 100 µg/L, including a depuration phase (7 days), and (ii) a long exposure (21 days) at 100 µg/L to measure the immunomarker dynamic over a long period. The fish spleens were sampled to analyze immune responses of stickleback at various times of exposure and depuration: leucocyte distribution, phagocytic capacity and efficiency, lysosomal presence and leucocyte respiratory burst index. At the same date, blood, muscle, and liver were sampled to quantify BPA and their metabolites (BPA monoglucuronide and BPA monosulfate). All these data enabled the development of the indirect pharmacodynamic models (PBTK-TD) by implementing the responses of biomarkers in the existing BPA PBTK of stickleback. The results shown a high induction of phagocytosis activity by BPA in the two exposure conditions. Furthermore, the immunomarkers exhibit very different temporal dynamics. This study demonstrates the need of a thorough characterization of biomarker response for a further use in Environmental Biomonitoring.

Maternal effect senescence via reduced DNA repair ability in the three-spined stickleback.

Maternal effect senescence, a decline in offspring viability with maternal age, has been documented across diverse animals, but its mechanisms remain largely unknown. Here, we test maternal effect senescence and explore its possible molecular mechanisms in a fish. We compared the levels of maternal mRNA transcripts of DNA repair genes and mtDNA copies in eggs and the levels of DNA damage in somatic and germline tissues between young and old female sticklebacks. We also tested, in an in vitro fertilization experiment, whether maternal age and sperm DNA damage level interactively influence the expression of DNA repair genes in early embryos. Old females transferred less mRNA transcripts of DNA repair genes into their eggs than did young females, but maternal age did not influence egg mtDNA density. Despite a higher level of oxidative DNA damage in the skeletal muscle, old females had a similar level of damage in the gonad to young females, suggesting the prioritization for germline maintenance during ageing. The embryos of both old and young mothers increased the expression of DNA repair genes in response to an increased level of oxidative DNA damage in sperm used for their fertilization. The offspring of old mothers showed higher rates of hatching, morphological deformity and post-hatching mortality and had smaller body size at maturity. These results suggest that maternal effect senescence may be mediated by reduced capacity of eggs to detect and repair DNA damages, especially prior to the embryonic genomic activation.

Local adaptation and host specificity to copepod intermediate hosts by the tapeworm Schistocephalus solidus.

Host-parasite coevolution may lead to patterns of local adaptation in either the host or parasite. For parasites with complex multi-host life cycles, this coevolution may be more challenging as they must adapt to multiple geographically varying hosts. The tapeworm Schistocephalus solidus exhibits some local adaptation to its second intermediate host, threespine stickleback, to which the parasite is strictly specialized. However, the tapeworm's adaptation to its first intermediate host (any of a number of copepod species) is not documented. We investigated if there was local adaptation and host specify in the tapeworm Schistocephalus solidus to its copepod first intermediate hosts. We exposed copepods from five lakes in Vancouver Island (BC, Canada) to local (i.e. same lake) and foreign tapeworms in a reciprocal exposure experiment. Results indicate that the tapeworm is not locally adapted to the copepods. Instead, we observed moderate-effect host specificity, infection rates being higher in certain copepod species than in others. Infection rates also varied among cestode populations. These results show that although S. solidus infects multiple copepod genera, they are not equally competent hosts. Differences in S. solidus epidemiology among lakes is likely to be driven more by this partial specialization, than by local adaptation to first intermediate hosts.

HARNESSING MODELLING FOR ASSESSING THE POPULATION RELEVANCE OF EXPOSURE TO ENDOCRINE ACTIVE CHEMICALS.

The presence of endocrine active chemicals (EACs) in the environment continues to cause concern for wildlife given their potential for adverse effects on organisms. However, there is a significant lack of understanding on the potential effects of EACs on populations. This has real-world limitations for EAC management and regulation, where the aim in environmental risk assessment is to protect populations. Here we propose a methodological approach for the application of modelling in addressing the population relevance of EAC exposure in fish. We provide a case study with the fungicide prochloraz to illustrate how this approach could be applied. Two population models, one for brown trout (Salmo trutta, inSTREAM) and the other for three-spined stickleback (Gasterosteus aculeatus) that met regulatory requirements for development and validation were used in this study. Effects data extracted from the literature were combined with environmentally realistic exposure profiles generated with the FOCUS SW software. Population-level effects for prochloraz were observed in some modelling scenarios (hazard-threshold) but not others (dose-response), demonstrating the repercussions of making different decisions on implementation of exposure and effects. The population responses, defined through changes in abundance and biomass, of both trout and stickleback exposed to prochloraz were similar, indicating that the use of conservative effects/exposure decisions in model parameterisation may be of greater significance in determining population-level adverse effects to EAC exposure than life-history characteristics. Our study supports the use of models as an effective approach to evaluate the adverse effects of EACs on fish populations. In particular, our hazard-threshold parameterisation is proposed for the use of population modelling in a regulatory context in accordance with Commission Regulation (EU) 2018/605.

Northeast Pacific eelgrass fish communities characterized by environmental DNA represent local diversity and show habitat specificity

AbstractFish biodiversity is an important indicator of ecosystem health and a priority for the National Park Service in Drakes Estero, a shallow estuary within Point Reyes National Seashore, Marin County, California. However, fish diversity has yet to be described following the removal of oyster aquaculture infrastructure within Drakes Estero from 2016 to 2017. We used environmental DNA (eDNA) to characterize fish biodiversity within Drakes Estero. We amplified fish eDNA with MiFish primers and classified sequences with a 12S rRNA reference database. We identified 110 unique operational taxonomic units (OTUs, at 97% similarity) within the estuary from 40 samples across 4 sites. From these 110 OTUs, we identified 9 species and 13 taxonomic groups at the genus, family, order, or class level within the estuary. Species‐level assignments are limited by a lack of representative sequences targeted by the MiFish primers for 42% of eelgrass fishes in our region that we identified from a literature review in the Northeast Pacific (NEP) from Elkhorn Slough to Humboldt Bay. Despite this limitation, we identified some common Drakes Estero fishes with our eDNA surveys, including the three‐spined stickleback (Gasterosteus aculeatus), Pacific staghorn sculpin (Leptocottus armatus), surfperches (Embiotocidae), gobies (Gobiidae), and a hound shark (Triakidae). We also compared fish biodiversity within the estuary with that from nearby Limantour Beach, a coastal site. Limantour beach differed in community composition from Drakes Estero and was characterized by high relative abundances of anchovy (Engraulis sp.) and herring (Clupea sp.). Thus, we can distinguish estuarine and non‐estuarine sites (<10 km away) with eDNA surveys. Further, eDNA surveys accounted for greater fish diversity than seine surveys conducted at one site within the estuary. Environmental DNA surveys will likely be a useful tool to monitor fish biodiversity across eelgrass estuaries in the Northeast Pacific, especially as reference databases become better populated with regional species.

Molecular mechanisms of Eda-mediated adaptation to freshwater in threespine stickleback.

A main goal of evolutionary biology is to understand the genetic basis of adaptive evolution. Although the genes that underlie some adaptive phenotypes are now known, the molecular pathways and regulatory mechanisms mediating the phenotypic effects of those genes often remain a black box. Unveiling this black box is necessary to fully understand the genetic basis of adaptive phenotypes, and to understand why particular genes might be used during phenotypic evolution. Here, we investigated which genes and regulatory mechanisms are mediating the phenotypic effects of the Eda haplotype, a locus responsible for the loss of lateral plates and changes in the sensory lateral line of freshwater threespine stickleback (Gasterosteus aculeatus) populations. Using a combination of RNAseq and a cross design that isolated the Eda haplotype on a fixed genomic background, we found that the Eda haplotype affects both gene expression and alternative splicing of genes related to bone development, neuronal development and immunity. These include genes in conserved pathways, like the BMP, netrin and bradykinin signalling pathways, known to play a role in these biological processes. Furthermore, we found that differentially expressed and differentially spliced genes had different levels of connectivity and expression, suggesting that these factors might influence which regulatory mechanisms are used during phenotypic evolution. Taken together, these results provide a better understanding of the mechanisms mediating the effects of an important adaptive locus in stickleback and suggest that alternative splicing could be an important regulatory mechanism mediating adaptive phenotypes.

Plate reduction in southern Japanese freshwater populations of threespine stickleback (Gasterosteus aculeatus).

Adaptation to similar environments can lead to the evolution of similar phenotypes in phylogenetically independent lineages. However, the extent of parallel evolution often varies. Because such variations can be due to environmental heterogeneity among seemingly similar habitats, identification of the environmental factors that cause non-parallel patterns can provide valuable insight into the ecological factors associated with phenotypic diversification. Armor plate reduction in replicate freshwater populations of the threespine stickleback (Gasterosteus aculeatus) represents a well-known example of parallel evolution. Many freshwater populations in multiple regions of the Northern Hemisphere have reduced plate numbers, but not all freshwater populations exhibit plate reduction. In this study, we characterized plate number variation in Japanese freshwater populations and investigated the association between plate number and several abiotic environmental factors. We found that most freshwater populations have not reduced plate numbers in Japan. Plate reduction tends to occur in habitats with warmer winter temperatures at lower latitudes in Japan. In contrast, low dissolved calcium levels or water turbidity had no significant effects on plate reduction, although these were reported to be associated with plate reduction in Europe. Although our data are consistent with the hypothesis that winter temperatures are associated with plate reduction, further studies on the relationship between temperatures and fitness using sticklebacks with varying plate numbers are necessary to confirm this hypothesis and understand the factors causing variations in the extent of parallel evolution.

Searching for intralocus sexual conflicts in the three-spined stickleback (Gasterosteus aculeatus) genome.

Differences between sexes in trait fitness optima can generate intralocus sexual conflicts that have the potential to maintain genetic diversity through balancing selection. However, these differences are unlikely to be associated with strong selective coefficients and are challenging to detect. Additionally, recent studies have highlighted that duplications on sexual chromosomes can create artifactual signals of intralocus sexual conflicts. Thus, testing the relationship between intralocus sexual conflicts and balancing selection requires stringent filtering of duplicated regions, and dedicated methods to detect loci with low levels of intersex differentiation. In this study, we investigated intralocus sexual conflicts in the three-spined stickleback using whole-genome sequencing (mean coverage = 12×) of 50 females and 49 males from an anadromous population in the St. Lawrence River, Québec, Canada. After stringent filtering of duplications from the sex chromosomes, we compared three methods to detect intralocus sexual conflicts. We found only two genomic regions under potential intralocus sexual conflict that also showed signals of balancing selection. Overall, our results suggest that most intralocus sexual conflicts do not drive long-term balancing selection and are most likely transient.

Exploring the limits to our understanding of whether fish feel pain.

Exploring the limits to our understanding of whether fish feel pain.

Single-Cell RNA Sequencing Reveals Microevolution of the Stickleback Immune System.

The risk and severity of pathogen infections in humans, livestock, or wild organisms depend on host immune function, which can vary between closely related host populations or even among individuals. This immune variation can entail between-population differences in immune gene coding sequences, copy number, or expression. In recent years, many studies have focused on population divergence in immunity using whole-tissue transcriptomics. But, whole-tissue transcriptomics cannot distinguish between evolved differences in gene regulation within cells, versus changes in cell composition within the focal tissue. Here, we leverage single-cell transcriptomic approaches to document signatures of microevolution of immune system structure in a natural system, the three-spined stickleback (Gasterosteus aculeatus). We sampled nine adult fish from three populations with variability in resistance to a cestode parasite, Schistocephalus solidus, to create the first comprehensive immune cell atlas for G. aculeatus. Eight broad immune cell types, corresponding to major vertebrate immune cells, were identified. We were also able to document significant variation in both abundance and expression profiles of the individual immune cell types among the three populations of fish. Furthermore, we demonstrate that identified cell type markers can be used to reinterpret traditional transcriptomic data: we reevaluate previously published whole-tissue transcriptome data from a quantitative genetic experimental infection study to gain better resolution relating infection outcomes to inferred cell type variation. Our combined study demonstrates the power of single-cell sequencing to not only document evolutionary phenomena (i.e., microevolution of immune cells) but also increase the power of traditional transcriptomic data sets.