Understanding recombination rates is crucial in evolutionary biology, as recombination shapes genetic diversity, natural selection, and adaptation. We investigated recombination rate variation in Chironomus riparius across different latitudes, seasons, and experimental treatments using Pool-seq data from five studies and the ReLERNN neural network-based method. We examined its relationship with genetic diversity, GC content, and FST, assessing causality through structural equation modeling. In natural populations, recombination rates showed no clear latitudinal pattern, likely due to interactions between climate-driven selection, demographic history and regional environmental heterogeneity. However, seasonal variation was evident, with higher recombination rates in autumn than winter, possibly due to temperature-induced plasticity or seasonal bottlenecks. A cold snap in March 2018 triggered a sharp recombination increase, potentially suggesting a stress-induced adaptive response. Across datasets, recombination rates were correlated with genetic diversity and other genomic parameters, with structural equation models (SEMs) indicating that recombination and selection jointly shape patterns of π and differentiation, while relationships with GC content and TEs counts varied among environmental and experimental contexts. In experimental populations, thermal regimes alone had little effect on recombination; instead, adaptation to laboratory conditions and specific stressors drove recombination changes. Exposure to microplastics led to a genome-wide reduction in recombination, likely due to stress-induced DNA repair prioritizing genome integrity, whereas cadmium exposure generally suppressed recombination. Our results demonstrate that recombination in C. riparius is a highly dynamic trait influenced by environmental conditions, selection, and genomic context. By integrating ecological variation, experimental evolution, and multivariate genomic analyses, this study highlights recombination as a context-dependent process that responds to both natural and anthropogenic stressors and interacts with multiple features of genome architecture.

The modern era is characterized by the vast production of plastics, with aquatic ecosystems acting as major sinks for contaminants, making aquatic species highly susceptible to microplastic (MP) ingestion. Accumulation of MPs in benthic zones enables direct interaction between these particles and benthic organisms. This study utilized the Organisation for Economic Co-operation and Development's chronic exposure test on Chironomus riparius larvae to (a) assess the effects of MP concentrations 10 times higher than environmentally relevant ones on life history traits and (b) monitor particle presence throughout the life cycle. After digestion, MPs were counted in adult C. riparius and exuviae. Results indicated a significant delay in developmental time, a reduction in developmental rate, and a lag in cumulative emergence in the MP-treated group, even though total emergence was not affected. Notably, exuviae retained significantly more MPs than adults, indicating metamorphosis as a potential detoxification route. These findings advance our understanding of life stage-specific MP dynamics in freshwater invertebrates and suggest a novel mechanism for contaminant elimination via exuvial shedding.

Abstract The spontaneous mutation rate (μ) is shaped by two distinct forces: the passage of chronological time, inevitably associated with mutation accumulation, and the speed of development, where rapid replication contributes to error rates. Disentangling these forces has been a major challenge, particularly in ectotherms. Testing the distinct predictions of the classic replication-dependent generation length hypothesis and the time-dependent accumulation model, we experimentally assessed individuals with naturally short (15 days) and long (38 days) generation time (GT), which is mainly determined by differences in developmental speed in the midge Chironomus riparius. To overcome the challenges of swarm-mating, we employed a parent-offspring pedigree reconstruction approach using pooled sequencing of siblings to infer parental allelic composition. We estimated the de novo mutation rates by whole genome sequencing. We found that Long-GT groups accumulated 1.2-fold more total mutations per generation (μ/gen), consistent with time-dependent mutagenic processes. Conversely, Short-GT groups exhibited a nearly two-fold higher mutation rate per day (μ/day) and a trend toward a transition-biased mutational spectrum (Ts/Tv ratio = 1.14 vs. 0.89), a pattern consistent with a replication-dependent errors in DNA. These results suggest that the overall mutation load is the product of these two interacting processes. Integrating our data with previous studies and life-history data, we show that the daily mutation rate followed a non-linear relationship with respect to developmental speed, and that the species’ generation time mode coincides with its minimum. This suggests that the developmental speed is, amongst other factors, selected to optimize the mutational load by balancing between replication accuracy and developmental speed.

Histological techniques are valuable tools in various biological and medical disciplines. They have proven particularly valuable in ecotoxicological studies, providing critical insight into the sublethal effects of pollutants on aquatic organisms. However, limited reference data on invertebrate histology make these analyses time-consuming, particularly for previously undescribed species. In this study, a deep learning model, based on the Convolutional Neural Network (CNN) was developed to automatically identify 11 tissue types of Chironomus riparius. The model achieved an overall accuracy of 94.21%, with five of the eleven tissues correctly identified in all cases, while the highest misclassification rate (22.72%) occurred in the case of recognizing parietal fat body. This represents the first AI-assisted approach for histological tissue classification in a standard invertebrate Organisation for Economic Co-operation and Development model organism, achieving high accuracy and providing a foundation for integrating deep learning into future histopathological workflows in environmental and ecotoxicological research.

Riparian ecosystems are vital interfaces between aquatic and terrestrial environments but are increasingly impacted by anthropogenic pollution. In these systems, merolimnic insects serve as crucial ecological links, occupying aquatic habitats as larvae and terrestrial environments as adults, thus being an essential food source in both. Consequently, pollutant exposure during the aquatic larval stage can have cascading effects across ecosystem boundaries. While the ecological consequences of such exposure are well documented, the evolutionary potential of merolimnic insects to adapt to chronic pollution remains poorly understood. To address this, we previously conducted a selection experiment exposing populations of the non-biting midge Chironomus riparius to the mosquito larvicide Bacillus thuringiensis israelensis (Bti) or heavy metal copper over approximately eight generations, which revealed only limited evidence of consistent phenotypic adaptation. Here we use whole-genome sequencing of these populations to assess their genomic responses to chronic pollutant exposure. Despite similar phenotypic sensitivity in pre-exposed and naïve populations, we detected distinct stressor-specific genomic responses. Copper exposure induced a significant genome-wide reduction in nucleotide diversity and evidence of selection-driven allele frequency changes, while Bti exposure was associated with heterogeneous, replicate-specific shifts, potentially reflecting drift or selection on multiple redundant pathways. Functional enrichment analyses indicated early-stage adaptation: immune- and apoptosis-related pathways were enriched under Bti, while metal detoxification and DNA repair pathways were enriched under copper, highlighting distinct adaptive mechanisms despite weak genome-wide signals of selection. Our findings demonstrate that Evolve and Resequencing approaches enable the detection of early genomic signals of adaptation even when phenotypic change is subtle or absent, offering a powerful framework for studying evolutionary responses to environmental pollution.

As pesticides negatively affect non-target organisms, their excessive and indiscriminate use poses a threat to the environment and aquatic ecosystems. In this study, it was aimed to examine the lethal and sublethal effects of tau-fluvalinate, a pyrethroid pesticide, on the non-target organism Chironomus riparius. For this aim, acute toxicity of tau-fluvalinate was evaluated through a 96-h mortality test, and changes in the locomotor activity of C. riparius were examined after 72- and 144-h exposures to different sublethal concentrations. Mortality tests indicated that both concentration (F9, 240 = 133,80; P

Anthropogenic stressors, such as pollution and climate change, are altering selective pressures on natural populations, but the evolutionary consequences of chronic exposure to complex mixtures of contaminants remain poorly understood. Addressing this knowledge gap is critical to the emerging field of evolutionary ecotoxicology, which aims to understand how long-term exposure to environmental contaminants shapes adaptive evolution and genome-wide variation. In this study, we used urban runoff sediment as complex and environmentally realistic model stressor to investigate how multigenerational exposure affects fitness and potentially drives genomic adaptation in the freshwater midge Chironomus riparius. We combined an evolutionary life-cycle test with the evolve and resequence approach, exposing replicate populations over seven generations to three treatments: a control and two concentrations of urban runoff sediment (0.5% and 10%). Key fitness traits, including mortality, mean emergence time, fertility, and population growth rate, were measured, and allele frequency changes were tracked to identify genomic signatures of selection. The results revealed distinct and nonlinear fitness responses across treatments, including transgenerational effects, recovery of performance, and evidence of life-history trade-offs. Candidate haplotypes were enriched for genes involved in membrane transport, metabolism, and gene regulation, suggesting selection on general stress-response pathways consistent with polygenic adaptation. Signals of selection were also detected in control populations, underscoring the evolutionary influence of laboratory conditions. Overall, our findings demonstrate how evolutionary ecotoxicology can reveal both the potential and the constraints of rapid adaptation to realistic environmental stressors and highlight the importance of integrating evolutionary perspectives into ecological risk assessment.

For the analysis of micropollutants in biota samples such as insects with aquatic life stages with inherently low body weight, miniaturization of common extraction methods is necessary. For QuEChERS extractions, miniaturization down to a few individual organisms was reported for invertebrates. For the investigation of interindividual differences in insects, even further miniaturization is necessary. Our salt-free SWIEET extraction method recently developed as an alternative to QuEChERS was originally optimized for a total volume of 5 mL. To enable the determination of carbendazim in midges, that weigh between 0.3 and 0.5 mg, the method required miniaturization. Using a model analyte mix, we investigated how reducing the total extraction volume affected analyte recoveries and investigated matrix effects using Chironomus riparius as model organisms. The miniaturized SWIEET extraction was then applied to the analysis of the pesticide carbendazim in adult midges of Chironomus riparius from an exposure experiment at the larval stage. Since matrix effects played a significant role in the extraction of carbendazim, particularly for samples from lower exposure concentrations, sample preparation and calibration modes had to be further optimized. With the final protocol, we were able to successfully extract carbendazim from single midges. The effects of pooling midges were investigated to better understand matrix effects and effects on robustness. With the optimized and miniaturized method, we achieved repeatable results, making it suitable for determining carbendazim loads in midges and correlating them to the exposure concentrations.

Chironomus riparius midge larvae are ubiquitous in freshwater ecosystems throughout the Northern Hemisphere. Recently, freshwater in temperate regions has experienced a threat from salinization due to the use of road de-icers. As ambient temperatures fluctuate, snow and ice melt carry the de-icers into nearby freshwater and significantly raise salt levels. Recently, organic de-icers including brine beet juice de-icer (BBJD) have been implemented as an "ecofriendly" alternative to traditional road salt. Not much is known about the effects of BBJD on freshwater invertebrates. Aquatic insects respond to salinity by adjusting the ion transport functions of osmoregulatory organs. A key component in insect osmoregulation is the presence of water channel proteins known as aquaporins (AQPs) that allow movement of water along an osmotic gradient. To date, there is limited knowledge on the effects of salinity on AQP function in aquatic insects. In this study, we characterized a water-specific AQP known as CrAQP2 (a PRIP homolog) in the osmoregulatory organs of C. riparius larvae. CrAQP2 was immunolocalized in osmoregulatory organs with greatest transcript abundance in the Malpighian tubules. NaCl caused differential Craqp2 transcript expression in some of the organs, whereas BBJD had little effect on Craqp2 transcript levels. Craqp2 knockdown decreased total body water regardless of treatment and reduced survival of larvae in BBJD and NaCl. Therefore, CrAQP2 appears to be important in maintaining total body water levels stable and likely plays a role in the ability of midge larvae to respond to salinity.

Chironomus riparius (Insecta: Diptera) is a globally distributed non-biting midge species of considerable ecological and economic importance. In the present study, the genetic diversity, population structure, and demographic history of this species were assessed using the mitochondrial cytochrome c oxidase subunit I (COI) gene sequences. A total of 70 C. riparius larvae representing 11 populations across Türkiye were collected, sequenced, and analysed. Nineteen haplotypes were identified, 14 of which were unique (73.7%) and five shared (26.3%). Analyses of C. riparius populations revealed high haplotype diversity (h = 0.936) coupled with low nucleotide diversity (π = 0.007), significant genetic structuring among populations in Türkiye at several levels (p<0.001), and multiple indicators of a historical population expansion event. These findings suggest that C. riparius populations in Türkiye have recently expanded demographically following a period of isolation, likely driven by climatic or hydrological fluctuations. Limited gene flow among regional populations reflects environmental heterogeneity and habitat fragmentation. This study provides significant genetic evidence on the influence of historical and ecological processes on the population dynamics of C. riparius in Türkiye.

ABSTRACTOxidative stress, caused by reactive oxygen species (ROS), poses a major challenge for organisms facing temperature fluctuations. This study provides the first direct in vivo measurements of ROS production together with transcriptome analysis of oxidative stress genes across a broad range of ecologically relevant temperatures in insect larvae of the dipteran midge Chironomus riparius. We observed a U‐shaped pattern of oxidative stress, with minimal ROS levels within an optimal thermal window (12°C–18°C) and significantly elevated stress at both cold and warm extremes. Crucially, our findings reveal distinct underlying molecular mechanisms for ROS generation at these extremes: at low temperatures, predominantly ROS produced is of the superoxide group, linked to hypoxia‐induced hemoglobin autoxidation. Conversely, at high temperatures, the hydrogen peroxide group dominates, associated with increased metabolic rate and heat stress signaling pathways. Transcriptomic analysis shows that C. riparius's antioxidant defense system adapts accordingly, selectively upregulating mechanisms to counteract the specific dominant ROS type at different temperatures. This mechanistically differentiated oxidative stress at different temperatures and the modulated organismic response reflect the ecological niche and evolution of C. riparius.

Antibiotics and benthic fauna diversity affect nitrogen removal and greenhouse gas emissions in constructed wetlands by changing enzyme activity and microbial community.

Reaction of Midge Larvae Chironomus riparius Meigen (Diptera, Chironomidae) to Exposure to Lanthanum, Copper and Their Mixture

BackgroundBroadening our taxonomic scope beyond model species offers deeper insights into the evolutionary dynamics of genomic processes such as recombination and the proliferation of transposable elements (TEs). TEs can drive substantial genomic rearrangements, yet the interplay between TEs and recombination remains poorly understood.ResultsTo investigate population-specific recombination patterns, we analysed the distribution of the species-specific Cla-element in the non-biting midge Chironomus riparius. This TE is known for its dynamic behaviour, exhibiting high numbers of unique insertions and population-specific distribution patterns. Its distribution showed no consistent association with recombination rates at the chromosome-wide scale. However, the Cla-element was often found outside haplotype blocks, suggesting it may be spatially separated from regions with low recombination.ConclusionsNo strong association was found between the overall recombination landscape in C. riparius and the transposition activity of repetitive elements. Highlighting how the dynamics of transposable elements contribute to the complexity of genome evolution.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-025-12130-7.

Radiocarbon (14C) is a key radionuclide in radioactive waste management due to its long half-life and potential integration into the global carbon cycle. However, its incorporation into freshwater organisms remains poorly understood. This study investigated the transfer of 14C into two benthic invertebrates, Lumbriculus variegatus and Chironomus riparius, in a controlled experimental system. Naturally 14C depleted peat was used as the primary carbon source and substrate for the organisms, enabling clear source tracing without artificial labeling but represented a simplified scenario compared to natural sediments with higher nutritional quality. An isotope mixing model based on the natural 14C abundance partitioned C sources between peat and dietary inputs (fish food and unbleached strips of paper towel). Results showed significantly higher peat-derived C contribution in C. riparius (40 %) than in L. variegatus (<3 %), likely due to more active substrate-feeding of recalcitrant peat during early instars. In contrast, L. variegatus with slower C turnover likely relied on previous dietary C (fish food and unbleached strips of paper towel) or assimilation from fish food in the feeding group rather than feeding on recalcitrant peat with larger particles. Survival rates differed, with lower chironomid survival possibly influenced by a combination of peat characteristics (recalcitrant and acidic), food distribution, and sensitivity of early instars. While these findings provide baseline data for biosphere models of 14C transfer, the use of peat and simplified conditions limits direct extrapolation to natural systems. Further studies with representative sediments and ecological complexity are needed to improve risk assessments.

Copper-based plant protection products (PPPs) are widely used in agriculture to control fungal and bacterial diseases. However, concerns regarding copper accumulation in the environment have led to restrictions on its use. Copper-based nano-formulations have been proposed as an alternative, offering improved efficiency and reduced application rates. However, their environmental risks remain poorly understood, particularly their effects on aquatic ecosystems. This study evaluates the ecotoxicity of conventional and nano-formulated copper pesticides using Chironomus riparius as a holobiont model, considering both host life history traits and microbiota responses. Larvae were exposed to environmentally relevant concentrations (5, 50, and 500 µg Cu/L) of conventional and nano-formulated products under controlled laboratory conditions. Exposure to the highest concentration resulted in 100 % mortality. Following exposure to 50 µg Cu/L, sublethal effects were observed, including alteration of the larval microbiota composition, apparently shifting towards a copper-tolerant profile following exposure to the conventional formulation. Despite the distinct shifts in bacterial taxa between conventional and nano-formulated treatments, similar decreased adult emergences are noticed following exposure to both formulations. Despite comparable toxicity profiles, nano-formulations resulted in higher copper bioaccumulation in emerging adults, raising concerns about trophic transfer and ecosystem impacts. Our findings highlight the need for a holistic approach integrating host-microbiota interactions in ecotoxicological assessments. The comparable toxicity of nano-formulated and conventional copper pesticides, coupled with increased bioaccumulation potential, questions the assumption that nano-formulations represent a safer alternative. These results emphasize the importance of comprehensive risk assessments to ensure sustainable agricultural practices while minimizing environmental harm.

The aquatic midge Chironomus riparius is an established indicator taxon for the assessment of water quality as of the European Water Framework Directive. Here, we present a novel long-read genome assembly generated with PacBio HiFi and Hi-C sequencing, which achieves chromosome-scale resolution with an assembly size of 192 Mb, an N50 of 59 Mb, and a BUSCO completeness of 99.0%. Four chromosomes with their predicted centromeric regions and 10 unplaced scaffolds were assembled containing 15,439 protein-coding genes. Chromosome-level resolution in nonmodel species is often limited, posing challenges for population genomic studies that depend on high-quality reference genomes. Reanalyzing genomic data of natural C. riparius populations, we demonstrate the improved accuracy of population genomic estimators based on the high-quality reference genome. The high contiguity and completeness of the assembly enhanced demographic inference with Sequential Markovian Coalescent (MSMC2) modeling. Our results suggest that population divergence began in an ancestral lineage during the late Pleistocene to early Holocene, consistent with paleoclimate records from Central Europe.

Silver (Ag) is widely released into aquatic environments through industrial and municipal discharges, with concentrations often reaching toxic levels for aquatic organisms. Its further extensive use in antimicrobials, especially during the COVID-19 pandemic, has increased environmental inputs. As Ag+ is the most toxic form of Ag, understanding its ecological risks remains critical for environmental regulation and ecosystem protection. Thus, we investigated multigenerational and transgenerational toxicity of Ag+ as AgNO3 on the ecologically important species midge Chironomus riparius using two complementary long-term life-cycle experiments. Experiment 1 simulated exposures with pulsed high environmentally relevant concentrations and recovery phases (nominal 3 µg/L), while Experiment 2 assessed continuous low environmentally relevant concentrations (nominal 0.01, 0.1, 1 and 3 µg/L) across four exposed generations of C. riparius followed by three recovery generations. Endpoints included survival, development, reproduction, growth as well as the population growth rate (PGR). Continuous Ag+ exposure produced cumulative increases in mortality and declines in emergence, reduced fertility and eggs per rope, delayed development (especially in females), and progressive reductions in PGR. Notably, adverse effects emerged or intensified over generations and were detectable at very low concentrations: some reproductive and survival endpoints showed significant impairment at the European Union’s environmental quality standard (EU-EQS) level (0.01 µg/L) by the fourth generation, while transgenerational effects persisted at ≥0.1 µg/L. Partial recovery occurred after removal of contamination at the lowest concentrations but not after higher exposures. The present study not only indicates that chronic, low-level Ag+ contamination can produce persistent, population-level adverse impacts on C. riparius, but also underscores the necessity for long-term ecological assessments to establish more protective standards and maintain ecosystem stability.

The expanding demand for platinum group elements (PGEs) in industrial and medical applications has led to their increasing accumulation in aquatic sediments. However, their ecological impacts remain poorly understood, particularly for sediment-dwelling invertebrates. This study assessed the toxicity of platinum (Pt) and palladium (Pd) across a concentration range of 29-1214 μg·g-1 dw in two freshwater benthic species, Chironomus riparius (C) and Hyalella azteca (H), under both acute and chronic exposures. Pt was more toxic (LC50 in μg·g-1 dw; H: 289 ± 28; C: 84 ± 7) than Pd (H: 1192 ± 356; C: 209 ± 44) for acute survival, whereas Pd caused more pronounced sublethal effects on growth. Bioaccumulation patterns showed that H. azteca accumulated more Pt, whereas C. riparius retained more Pd. In C. riparius, chronic exposure to Pd impacted survival, emergence, and female adult weight, indicating developmental disruption. Compared with other sediment-associated metals, Pd and Pt showed moderate to high toxicity: more toxic than uranium, nickel, arsenic, and molybdenum; comparable with copper; and less toxic than cadmium and lead. Although biological responses varied across metals, species, and endpoints, a consistent pattern of toxicity emerged. This study addresses a significant knowledge gap and reinforces the need to include PGEs in sediment quality guidelines. Although current environmental concentrations remain below toxicity thresholds, growing industrial use raises concerns for future ecological risk. Our findings support regulatory efforts by providing essential toxicity benchmarks and call for further research on mixture toxicity and mechanisms of action.

Exploring aquatic toxicity of diverse pesticides against Chironomus riparius (harlequin fly) and Lemna gibba (swollen duckweed): Applications of QSTR, i-QSTTR, and novel q-RA approaches.