Biology subjects

Bolnick, D. I.

Publications and source records attributed to Bolnick, D. I..

At least 19 recordsLinked to original sources

bioRxiv2026

Heritable morphology-environment correlations among lake populations of threespine stickleback

Phenotypic differences among populations can arise through heritable genetic divergence, phenotypic plasticity, or both, making it difficult to determine whether trait-environment correlations observed in nature reflect adaptive evolution. Within threespine stickleback (Gasterosteus aculeatus) studies, numerous document morphological differences among allopatric-, parapatric-, and even sympatric populations. These phenotypic differences among populations are often correlated with diet and lake habitat (e.g., lake size), suggesting an adaptive value to the population differences. However, many studies of ecomorphological divergence in stickleback use wild-caught stickleback, which may differ due to evolution or plasticity. Although common garden experiments have confirmed that population differences can be heritable, such experiments typically entail small numbers of populations. Consequently, we still do not know to what extent well-known trait-environment correlations in stickleback are a result of evolution. To address this gap, we reared stickleback embryos from 27 lake populations on Vancouver Island, in a laboratory environment. Morphological differences among populations persist in common-garden fish, confirming a large role for divergent evolution. These heritable differences were associated with environmental variation among lakes, implying an adaptive value. However, some well-known trait-environment relationships in stickleback did not persist in common-garden fish and may be primarily plastic.

evolutionary biology↗

bioRxiv2026

Confounding effects of inferring gene co-expression networks from pooled data from different biological populations

Weighted Gene Co-expression Network Analysis (WGCNA) is routinely applied to pooled datasets from multiple biological populations, genotypes, or treatment groups, implicitly assuming a shared module structure across groups. While the distortion of pairwise correlations by pooling heterogeneous groups is well established statistically, three aspects of this problem have received little systematic attention in the context of co-expression network analysis: the extent to which pooling disrupts the discrete module-level community structure inferred by WGCNA; whether this disruption is detectable from the global topology metrics researchers routinely report; and how prevalent the pooling practice is in published multi-group WGCNA studies. Using analytical toy examples and a four-scenario simulation framework, we address all three questions. Module preservation Zsummary scores declined progressively with between-population divergence, from full preservation under identical populations (mean median Zsummary = 25.2 {+/-} 3.3, 95% interval 19.0-30.7 across 20 simulation replicates) to substantial disruption when both network structure and mean expression differed (mean median Zsummary = 11.9 {+/-} 1.0, 95% interval 10.2-13.5). This disruption was undetectable from global topology metrics: modularity and clustering coefficient remained stable across all scenarios, while edge density was sensitive but non-specific. These findings were corroborated in an empirical reanalysis of divergent lake and stream stickleback transcriptomes, where merged analysis collapsed 26 lake-specific and 59 stream-specific modules into only 19 merged modules. A survey of 100 publications found that 78.7% (95% CI 69.4-87.9%) of multi-group WGCNA studies with sufficient methodological reporting used a single merged analysis. Results were robust across network sizes of 250-1,000 genes and rewiring rates of 10-50%. We provide concrete recommendations including module preservation testing in both directions, population-specific baseline networks, and consensus WGCNA as a principled alternative.

bioinformatics↗

bioRxiv2026

Immune variation and host ontogeny constrain pathogen virulence and transmission in a helminth parasite

Host immune strategies vary widely, yet how this variation shapes parasite virulence and transmission in natural systems remains unresolved. We tested whether host immune strategy governs macroparasite transmission timing and magnitude in Schistocephalus solidus, a cestode with a three-host life cycle. Over 13 months, we surveyed infection and immune activation (fibrosis) in second-intermediate host populations (threespine stickleback) from eight lakes. Host immune strategy (ranging from resistance to tolerance) strongly influenced whether and when parasites reached transmissible sizes, and shifted the synchrony between transmissible-stage peaks and definitive host availability. Across populations, transmission potential peaked at intermediate parasite burdens, consistent with virulence-transmission trade-off theory. These findings demonstrate that host immune phenotype alters epidemiological outcomes in complex life cycle parasites, by shifting both the magnitude and phenology of parasite growth to structure the effective transmission window.

ecology↗

bioRxiv2026

Linking land-use change, water quality, and host-parasite dynamics with droplet digital PCR and Bayesian path analyses

Global changes in land use and nutrient cycling are transforming ecosystems at unprecedented rates, with significant consequences for infectious disease dynamics. Aquatic environments are particularly vulnerable because the interplay of habitat modification, nutrient enrichment, and biodiversity loss can drive pronounced changes in the community composition of food webs, including hosts and parasites. Yet, despite well-documented effects of habitat modification on aquatic communities and food webs, the mechanisms through which these changes influence infectious disease dynamics remain poorly resolved. This gap arises, in part, because it remains challenging to disentangle how multiple stressors interact to shape disease outcomes and quantify parasite levels and host densities from field-collected samples. Here, we illustrate two tools that might help address these challenges. First, highly sensitive droplet digital PCR can quantify infection loads even when the signal:noise ratio is low. Second, stepwise Bayesian path analyses can identify the direct and indirect pathways connecting land-use changes to infectious disease dynamics. As a case study, we examined cyclopoid copepods and their helminth parasite, Schistocephalus solidus, across 47 freshwater lakes on Vancouver Island, a region strongly shaped by commercial logging, including widespread clear-cutting of old-growth forests. Our results reveal a positive correlation between copepod density and deforestation, potentially mediated by associated changes in water quality and calanoid copepods, key competitors of the focal host. ddPCR enabled sensitive detection of extremely low parasite signals in field-collected copepods. We detected positive infections in only 19.5% of the lakes surveyed, highlighting the difficulty of assessing disease dynamics in natural populations. Nonetheless, this study highlights the challenges of linking land-use change to disease outcomes, while also demonstrating that sensitive molecular and statistical tools offer new ways to reveal these hidden connections.

ecology↗

bioRxiv2026

A Multi-Scale Ecological Approach to Assessing Antimicrobial Resistance in a Freshwater Fish

Antimicrobial resistance (AMR) genes are increasingly recognized as an emerging environmental contaminant. Yet, the ecological mechanisms shaping their distribution across natural landscapes remain poorly understood. Here, we quantified AMR gene abundances in microbial communities sampled from wild fish from eight freshwater lakes on Vancouver Island and paired these gene-level measurements with fine-scale limnological and land-use data. Using droplet digital PCR, field surveys, and an iterative spatial forecasting framework that integrates Random Forest models with regression kriging, we explored how watershed-scale processes relate to variation in AMR genes across lakes. Our analyses reveal potential associations between elevated AMR gene levels, changes in water quality, deforestation, and geographic proximity to salmon aquaculture. By integrating data across biological and spatial scales, from genes within microbial communities to lake-level conditions and landscape patterns, this study illustrates the value of combining quantitative molecular measurements with geospatial modeling to identify environmental factors that may promote antimicrobial resistance in natural systems. Our approach provides a proof-of-concept and a general predictive framework for generating hypotheses and informing future monitoring efforts aimed at understanding, managing, and forecasting environmental reservoirs of resistance. SignificanceAntimicrobial resistance (AMR) genes are ancient components of environmental microbiomes. Yet, the mechanisms that generate modern hotspots of resistance across natural landscapes remain unclear. Here, we reveal how watershed-scale environmental change, including water quality metrics linked with deforestation and proximity to salmon aquaculture, predicts elevated AMR gene levels in the microbiomes of wild fish populations. By combining quantitative droplet digital PCR with ecological data and geospatial modeling, we move beyond isolated surveillance data to identify ecological mechanisms that promote antimicrobial resistance in freshwater ecosystems. This integrative approach provides mechanistic insight into why certain habitats, and the organisms within them, become reservoirs of resistance while others do not. Our findings highlight the importance of ecological context in understanding resistance evolution and offer a predictive tool for informing proactive monitoring and management strategies.

ecology↗

bioRxiv2026

Disassortative mating in replicated secondary contact experiments in nature

Ecological speciation is now regarded as one of the primary processes by which new species are generated. Adaptive divergence in allopatry begins this process, but it is often unclear when and how mechanisms that promote reproductive isolation, such as assortative mating and selection against hybrids, evolve. Here, we test for evidence of these mechanisms across replicated secondary contact experiments in natural settings. We introduced four to eight allopatric populations of threespine stickleback (Gasterosteus aculeatus), in both single-ecotype and mixed-ecotype treatments, into nine natural lakes, after which we inferred mating patterns by genotyping the resulting F1 generation. Contrary to expectations from the literature, we found no evidence of assortative mating or partial reproductive isolation among the introduced source populations. Instead, we detected evidence of disassortative mating by source population in three lakes and some evidence of disassortative mating by ecotype in one lake. These mating patterns were both context-dependent and population-dependent, varying substantially across lakes receiving the same source populations, and with some source populations generally displaying greater tendencies for disassortment. The absence of positive assortative mating in any replicate demonstrates that adaptive divergence in allopatry alone might be insufficient to generateassortative mating in many cases, while the possibility of disassortative mating in these contexts poses an additional hurdle on the path toward speciation.

evolutionary biology↗

bioRxiv2026

Constitutive and inducible fibrosis explain immune variation among threespine stickleback populations

Understanding how immune variation arises in natural populations requires disentangling the relative contributions of host genetic differences, environmental variation, and parasite effects, which is rarely possible in wild systems. Threespine stickleback populations vary in their use of intraperitoneal fibrosis as a defense against the helminth parasite Schistocephalus solidus, providing a natural system to study the genetic and ecological drivers of immune variation. We combined a 46-lake field survey with common garden experiments on 20 representative populations exposed to multiple parasite genotypes to test whether population differences in fibrosis persist under controlled conditions and whether they depend on parasite genotype or lake ecology. Fibrosis variation was strongly heritable, with both constitutive and inducible components persisting under common garden conditions. In contrast, parasite genotype had only a weak effect on fibrosis responses. Moreover, inducible fibrosis covaried with lake environmental conditions, with populations from more eutrophic-like lakes exhibiting stronger responses than those from more oligotrophic-like lakes. Together, these results reveal ecologically structured divergence in heritable immune responses among natural populations.

evolutionary biology↗

bioRxiv2026

Gene network centrality affects parallel evolution and local adaptation in wild yeast

The predictability of evolution remains a fundamental question in biology. While parallel evolution has been observed across taxa, we lack a mechanistic framework for predicting when evolution will be repeatable versus contingent. Here, we test whether gene network architecture predicts evolutionary repeatability and local (mal)adaptation in natural populations. We sequenced 38 wild Hanseniaspora uvarum yeast strains from two replicated apple varieties across four Connecticut orchards, yielding 29,489 high-quality SNPs. Population genomic analyses suggested that genetic differentiation was structured primarily by orchard environment. Reciprocal transplant experiments across all orchard-variety combinations demonstrated local (mal)adaptation at multiple ecological scales, with strongest (mal)adaptive responses at the orchard-variety level. By mapping H. uvarum genes to the comprehensive Saccharomyces cerevisiae genetic interaction network, we found that network centrality affects evolutionary outcomes. Highly connected genes occupying bow-tie network positions evolved in parallel across replicated apple varieties and central genes harbored alleles with adaptive fitness effects. In contrast, peripheral genes with fewer interactions facilitated rapid, non-parallel evolution to orchard-variety interactions and were associated with local maladaptation. Analysis of 150-gene sets evolving to apple variety within each orchard revealed greater-than-random similarity at genetic, functional, and network levels, with network-level predictability significantly exceeding gene-level predictability. These results suggest that gene network architecture could provide a mechanistic foundation for determining which evolutionary changes will be repeatable, potentially enabling evolutionary predictions even when specific genes cannot be identified.

evolutionary biology↗

bioRxiv2026

Testing vivo-morpholino mediated gene knockdown in threespine stickleback

Antisense vivo-morpholino oligonucleotides (vivo-MOs) allow transient gene knockdown in adult organisms with high specificity and low toxicity. Vivo-MOs are used in cell culture and in many established model organisms, but a method for their use has not been described in threepsine stickleback (Gasterosteus aculeatus (Linnaeus, 1758)). Stickleback are an emerging model system used in evolutionary and ecological genetic studies. While genomic techniques are commonly used in stickleback research, there are few studies and tools available to assess gene function in-vivo, especially for genes that may be difficult to knock out by CRISPR (e.g., lethal knock-outs). Here, we test the use of splice-blocking vivo-MOs for gene knockdown in stickleback using intraperitoneal injection of vivo-MOs targeting three candidate genes. Gene expression was assessed in the liver, spleen, and intestine. Successful knockdown of Spi1b was observed in the spleen, however, we observed no other significant knockdown at either timepoint tested. Injection of a fluorescently labeled control vivo-MO confirmed delivery to each target organ, validating this approach, but delivery was variable which may explain inconsistent effects. These results indicate that vivo-MOs have potential as a tool for in-vivo gene knockdown in stickleback. Optimizing delivery methods could improve reproducibility and knockdown efficiency in future studies.

molecular biology↗

bioRxiv2026

Intrinsic fitness differences outweigh environmental matching in shaping introduction outcomes in nature

Species introductions and transplants offer powerful contexts to understand evolutionary patterns and processes, and they are increasingly critical for conservation. However, introduction success varies widely, and predicting outcomes remains challenging. Introducing multiple source populations should increase the chance of success, while also providing an opportunity to explore the factors that predict success of individual source populations in the same environment. We used replicated, mixed-population introductions of >12,000 threespine stickleback (Gasterosteus aculeatus) to test whether source population success could be predicted by environmental matching between source and recipient environments and/or by intrinsic source population characteristics. We introduced four to eight source populations of stickleback into each of nine natural lakes and tracked their relative success over the following two years (up to two generations). Source population success was largely consistent across lakes, despite divergent environmental conditions. These results point to the importance of intrinsic source population characteristics rather than environmental matching in predicting introduction success in natural settings. Source populations that were consistently successful tended to have greater stress tolerance (mortality rate during translocation) and higher genetic diversity, though these relationships were not conclusive. Our study highlights the value of considering factors that generate fitness differences independent of environmental contexts in predicting ecological and evolutionary dynamics and planning conservation programs. Significance StatementPredicting which populations will succeed when introduced to new environments is a central challenge in ecology, evolution, and conservation. Environmental conditions are often assumed to be crucial in determining which populations succeed, given the expectation that populations preadapted to local conditions should perform best. We test this assumption by introducing >12,000 threespine stickleback fish from up to eight source populations into nine natural lakes spanning diverse environmental conditions. We show that the relative success of individual source populations was remarkably consistent across lakes and environmental conditions, indicating that some populations are intrinsically better suited to introductions. Our findings underscore the importance of considering intrinsic population characteristics alongside environmental conditions when predicting ecological and evolutionary outcomes and guiding conservation efforts.

evolutionary biology↗

bioRxiv2025

Lake size shapes the relationship between body mass and gut microbiota in threespine stickleback (Gasterosteus aculeatus)

Host-microbe interactions are shaped by both host and environmental factors. However, little is known about how host-microbe interactions vary across populations within a species. Here, we characterized the gut microbiota of 191 wild threespine stickleback fish (Gasterosteus aculeatus) from six populations from Alaskan lakes spanning a gradient of surface area. We tested how environmental context (lake size and ecotype) and host traits (sex, body mass, gravidity, Schistocephalus solidus (S. solidus) infection, and fibrosis) influence stickleback gut microbial composition using 16S rRNA gene sequencing. We found that the lake surface area strongly predicted fish gut microbial alpha diversity. Fish from intermediate-sized lakes harbored significantly more diverse microbiota than those from small and large lakes, independent of ecotype. Body mass was associated with gut microbial diversity. Model-predicted marginal effects from the mass and lake surface area interaction analysis showed that the association between fish mass and microbial alpha diversity was strongly negative in the smallest lakes, weakest in intermediate-sized lakes, and strongly positive in the largest lakes. In addition, sex and S. solidus infection were significantly associated with gut microbiota alpha and beta diversity, whereas fibrosis and gravidity showed minimal effects. Differential abundance analysis revealed lake size-dependent associations between body mass and individual taxa. Together, these results demonstrate that both habitat context and host variation interactively shape stickleback gut microbial communities in the wild. Integrating lake-level and individual-level analyses reveals how ecological setting modulates host-microbe associations, offering insights into the role of the gut microbiota in host adaptation and population divergence.

microbiology↗

bioRxiv2025

Temperature modulates PFAS accumulation and energy allocation in sheepshead minnows

Climate warming and chemical pollution shape aquatic ecosystems, yet the physiological mechanisms underlying their combined effects remain unclear. We investigated how projected increases in mean summer surface water temperature alter per- and polyfluoroalkyl substances (PFAS) toxicokinetics and their effects on sheepshead minnows (Cyprinodon variegatus) physiological performance. Adult fish were chronically exposed to an environmentally relevant PFAS mixture (perfluorooctane sulfonate (PFOS) + perfluorooctanoate (PFOA)) under current and projected mean-temperature scenarios. Tissue PFAS concentrations, whole-organism metabolic rates, swimming performance, reproductive parameters, somatic indices were assessed. Temperature modified PFAS tissue concentrations in a compound- and tissue-specific manner, promoting PFOA redistribution to eggs. Metabolic responses were temperature-dependent: at 26 {degrees}C, higher tissue PFAS concentrations were associated with elevated standard and maximum metabolic rates (SMR and MMR), maintaining aerobic scope (AS). At 28.5 {degrees}C, SMR remained stable while MMR and AS declined with rising PFAS, indicating less oxygen for energetically demanding activities. Despite unchanged swimming and reproductive outputs, an increased hepatosomatic index with increasing tissue PFAS concentrations and altered PFAS distribution suggest detoxification costs. These findings indicate that increases in mean water temperature are likely to exacerbate contaminant stress, with consequences for coastal fish population resilience and offspring development. PFAS risk assessment should consider co-stressors under projected warming. SynopsisLimited research addresses how temperature affects PFAS toxicokinetics and toxicity. This study shows that warming reshapes tissue PFAS concentrations and distribution, and influences fish energy-allocation trade-offs.

physiology↗

bioRxiv2025

Rapid genome-wide introgression reveals fitness advantage of immigrant genotypes

Evolutionary biology has long recognized the tendency for populations to be locally adapted to their ancestral habitat, resulting in higher resident fitness. However, immigrants can also introduce beneficial alleles. The resulting adaptive introgression is usually inferred retrospectively, rather than as a contemporary process. Here, we document exceptionally rapid ongoing adaptive introgression in a lake population of threespine stickleback (Gasterosteus aculeatus). In the first generations after a discrete immigration event, all chromosomes exhibited large increases in immigrant ancestry due to linkage disequilibrium. After a decade, the extent of introgression varied across the genome. The fastest-evolving genes included Spi1b, which enables an increased fibrosis defense against a previously common tapeworm, whose prevalence then declined dramatically. This case study highlights the capacity for immigration to supply beneficial alleles that drive rapid genome-wide evolution.

evolutionary biology↗

bioRxiv2025

Changes in gene regulation are associated with the evolution of resistance to a novel parasite

Host-parasite interactions are ubiquitous and are important drivers of diversification and evolution. Host immune systems in particular are frequent targets of parasite-driven selection. The resulting rapid evolution of immune genes is usually framed as an ongoing arms race between a co-evolving pair of host and parasite species. But, often immune evolution may be driven by the acquisition of a new and unfamiliar parasite. For instance, when marine populations of threespine stickleback (Gasterosteus aculeatus) colonized freshwater lakes [~]12,000 years ago, they encountered and evolved resistance to a freshwater-restricted cestode Schistocephalus solidus. We compared the transcriptomic responses of lab-reared stickleback from three populations with varying cestode susceptibility (naive marine, susceptible lake, and resistant lake), when exposed to several immune stimuli. The resulting changes in expression reveal strong evidence for shared and population-specific responses during evolution of defense against a new parasite. Our investigation highlights the roles of several key immunological processes in underlying a general physiological response to tissue damage (fibrosis), and the importance of regulation of this fibrosis as a necessary step for its co-option into parasite defense. Furthermore, we highlight changes in expression of fibrosis-associated genes which facilitate faster and more targeted deployment of this defense against parasites; fish from resistant populations have higher and more consistent expression of fibrosis genes, which experience strong negative feedback in response to cestode stimuli only. Combined, our results provide strong evidence that changes in gene regulation and increased negative feedback to mitigate immunopathology are essential steps in the evolution of novel parasite defense.

immunology↗

bioRxiv2025

Local adaptation shapes community composition without affecting performance

Eco-evolutionary theory predicts that local adaptation should influence community assembly and performance, yet empirical tests of these predictions remain limited, particularly at microgeographic scales where gene flow is expected to constrain adaptation. Using yeast communities in apple orchards as a model system, we tested whether local adaptation of a dominant species affects community composition and performance through reciprocal transplant experiments across eight Connecticut orchards and three apple varieties. The dominant yeast Hanseniaspora uvarum varied dramatically in abundance (4.3-97.5%) and exhibited significant local adaptation and maladaptation patterns across orchard-variety combinations. Critically, H. uvarum local adaptation strongly predicted community composition: locally adapted populations achieved higher abundance while suppressing competitors. However, local adaptation had no effect on community-level performance in apple decomposition. Communities dominated by locally adapted H. uvarum performed equivalently to diverse assemblages, indicating functional redundancy among species. These results demonstrate that rapid evolutionary change can decouple community composition from performance, suggesting that functional redundancy can maintain community performance despite dramatic compositional shifts driven by local adaptation.

ecology↗

bioRxiv2025

Hi-reComb: constructing recombination maps from bulk gamete Hi-C sequencing

Recombination is central to genetics and to evolution of sexually reproducing organisms. However, obtaining accurate estimates of recombination rates, and of how they vary along chromosomes, continues to be challenging. To advance our ability to estimate recombination rates, we present Hi-reComb, a new method and software for estimation of recombination maps from bulk gamete chromosome conformation capture sequencing (Hi-C). Simulations show that Hi-reComb produces robust, accurate recombination landscapes. With empirical data from sperm of five fish species we show the advantages of this approach, including joint assessment of recombination maps and large structural variants, map comparisons using bootstrap, and workflows with trio phasing vs. Hi-C phasing. With off-the-shelf library construction and a straightforward rapid workflow, our approach will facilitate routine recombination landscape estimation for a broad range of studies and model organisms in genetics and evolutionary biology. Hi-reComb is open-source and freely available at https://github.com/millanek/Hi-reComb.

genetics↗

bioRxiv2025

Dynamics of infection and immunity over 50 years as marine stickleback adapt to freshwater

When a species colonizes a new environment, it may encounter new parasites to which its immune system is poorly adapted. After an initial spike in infection rates in the naive founder population, the host may subsequently evolve increased immunity thereby reducing infection rates. Here, we present an example of this eco-evolutionary process, in a population of threespine stickleback (Gasterosteus aculeatus) that was founded in Heisholt Quarry, a man-made quarry pond, in 1967. Marine stickleback rarely encounter Schistocephalus solidus tapeworms (which require freshwater to hatch), and so remain highly susceptible to infection. Initially, introduced marine fish were heavily infected by S.solidus. They exhibited low levels of fibrosis, a heritable immune trait which some genotypes activate in response to infection, thereby suppressing tapeworm growth and viability. By the 1990s, the Heisholt Quarry population exhibited high rates of fibrosis, which partly suppressed S.solidus infection. This increased immune response led to reduced infection rates and the tapeworm was apparently extirpated by 2021. Because fibrosis has a strong genetic basis in other stickleback populations, we infer that the newly founded stickleback-parasite interaction exhibit an eco-evolutionary process of increased immunity that e_ectively reduced infection. The infection and immune dynamics documented here closely match those expected from a simple eco-evo dynamic model presented here. IMPACT SUMMARYParasite-host relationships are a great framework within which to study the mechanisms driving evolutionary change, both in terms of ecological dynamics, selection pressures, and the phenotypic basis of adaptation to environmental change. One classic example of adaptation is the loss of armor plating as marine stickleback fish colonize freshwater habitats. However, the parasitological and immunological adaptations in new freshwater populations are less clear. When marine fish colonize freshwater they encounter an unfamiliar tapeworm parasite, Schistocephalus solidus, to which they are poorly adapted. Many (but not all) freshwater populations evolve an inducible fibrosis response to S.solidus, a build-up of scar tissue that limits parasite growth and survival. However, we did not know the time frame over which marine fish, invading freshwater, will evolve this immune trait. To resolve this, we tracked the eco-evolutionary dynamics of Schistocephalus solidus tapeworm infection and the fibrosis immune response over 50 years, in an artificial quarry pond populated by susceptible marine fish in 1967. We show that infection rates were initially high, as the marine fish encountered a freshwater parasite to which they were not adapted. Fibrosis rates subsequently increased, reducing the infection rates and by 2021 S.solidus was apparently eliminated. This process matches a simple eco-evolutionary model of an inducible immune response, and provides a clear example of how hosts can adapt to and suppress novel parasites. Although fibrosis is typically viewed as an immune pathology contributing to disease in humans, we show that in stickleback it has a key adaptive value in protection against helminth infection.

evolutionary biology↗

bioRxiv2025

Needle in a haystack: A droplet digital polymerase chain reaction assay to detect rare helminth parasites infecting natural host populations

Helminths infect humans, livestock, and wildlife, yet remain understudied despite their significant impact on public health and agriculture. Because many of the most prevalent helminth-borne diseases are zoonotic, the health of diverse host species are closely interconnected. Therefore, understanding helminth transmission among wildlife could improve predictions and management of infection risks across species. A key challenge to understanding helminth transmission dynamics in wildlife is accurately and quantitatively tracking infection levels across hosts and environments. Traditional methods, such as visual parasite identification from environmental samples or infected hosts, are time-consuming, while standard molecular techniques (e.g., PCR and qPCR) often lack the sensitivity to reliably detect lower parasit burdens. These limitations often underestimate the prevalence and severity of infection, hindering efforts to manage infectious diseases. Here, we developed a multiplexed droplet digital PCR (ddPCR) assay to quantify helminth levels in aquatic habitats using 18S rRNA target genes. Using Schistocephalus solidus and their copepod hosts as a case study, we demonstrate ddPCRs sensitivity and precision. By establishing a 1:1 infection standard in the lab, we contextualize ddPCR gene concentration data to quantify both host and parasite numbers in field samples. The assay is highly reproducible, reliably detecting target genes at concentrations as low as 1 picogram of DNA in lab standards and field samples (multi-species and eDNA). Thus, we provide a toolkit for quantifying infection loads in intermediate hosts and monitoring infection dynamics across spatio-temporal scales in multiple helminth systems of concern for public health, agriculture, and conservation biology. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/634533v1_ufig1.gif" ALT="Figure 1"> View larger version (66K): org.highwire.dtl.DTLVardef@1bc2e40org.highwire.dtl.DTLVardef@180822eorg.highwire.dtl.DTLVardef@1e2c2c8org.highwire.dtl.DTLVardef@621909_HPS_FORMAT_FIGEXP M_FIG Applications of ddPCR probe-primer design to parallel systems. Cyclopoid copepods serve as initial hosts for diverse helminthic diseases distributed globally. The primers designed in this assay are suitable for other systems, with minimal work required for probe design specific to each helminth species. C_FIG

molecular biology↗