Evaluating Temperature Effects on Bluetongue Virus Serotype 10 and 17 Coinfection in Culicoides sonorensis.

Bluetongue virus (BTV) is a segmented, double-stranded RNA virus transmitted by Culicoides midges that infects ruminants. As global temperatures increase and geographical ranges of midges expand, there is increased potential for BTV outbreaks from incursions of novel serotypes into endemic regions. However, an understanding of the effect of temperature on reassortment is lacking. The objectives of this study were to compare how temperature affected Culicoides survival, virogenesis, and reassortment in Culicoides sonorensis coinfected with two BTV serotypes. Midges were fed blood meals containing BTV-10, BTV-17, or BTV serotype 10 and 17 and maintained at 20 °C, 25 °C, or 30 °C. Midge survival was assessed, and pools of midges were collected every other day to evaluate virogenesis of BTV via qRT-PCR. Additional pools of coinfected midges were collected for BTV plaque isolation. The genotypes of plaques were determined using next-generation sequencing. Warmer temperatures impacted traits related to vector competence in offsetting ways: BTV replicated faster in midges at warmer temperatures, but midges did not survive as long. Overall, plaques with BTV-17 genotype dominated, but BTV-10 was detected in some plaques, suggesting parental strain fitness may play a role in reassortment outcomes. Temperature adds an important dimension to host-pathogen interactions with implications for transmission and evolution.

A sodium-dependent trehalose transporter contributes to anhydrobiosis in insect cell line, Pv11.

Pv11 is the only animal cell line that, when preconditioned with a high concentration of trehalose, can be preserved in the dry state at room temperature for more than one year while retaining the ability to resume proliferation. This extreme desiccation tolerance is referred to as anhydrobiosis. Here, we identified a transporter that contributes to the recovery of Pv11 cells from anhydrobiosis. In general, the solute carrier 5 (SLC5)-type secondary active transporters cotransport Na+ and carbohydrates including glucose. The heterologous expression systems showed that the transporter belonging to the SLC5 family, whose expression increases upon rehydration, exhibits Na+-dependent trehalose transport activity. Therefore, we named it STRT1 (sodium-ion trehalose transporter 1). We report an SLC5 family member that transports a naturally occurring disaccharide, such as trehalose. Knockout of the Strt1 gene significantly reduced the viability of Pv11 cells upon rehydration after desiccation. During rehydration, when intracellular trehalose is no longer needed, Strt1-knockout cells released the disaccharide more slowly than the parental cell line. During rehydration, Pv11 cells became roughly spherical due to osmotic pressure changes, but then returned to their original spindle shape after about 30 min. Strt1-knockout cells, however, required about 50 min to adopt their normal morphology. STRT1 probably regulates intracellular osmolality by releasing unwanted intracellular trehalose with Na+, thereby facilitating the recovery of normal cell morphology during rehydration. STRT1 likely improves the viability of dried Pv11 cells by rapidly alleviating the significant physical stresses that arise during rehydration.

Laboratory and In situ Selenium Bioaccumulation Assessment in the Benthic Macroinvertebrates Hyalella azteca and Chironomus dilutus.

Selenium (Se) bioaccumulation and toxicity in aquatic vertebrates have been thoroughly investigated. Limited information is available on Se bioaccumulation at the base of aquatic food webs. In this study, we evaluated Se bioaccumulation in two benthic macroinvertebrates (BMI), Hyalella azteca and Chironomus dilutus raised in the laboratory and caged in-situ to a Canadian boreal lake e (i.e., McClean Lake) that receives continuous low-level inputs of Se (< 1 µg/L) from a uranium mill. Additional Se bioaccumulation assays were conducted in the laboratory with these BMI to (i) confirm field results, (ii) compare Se bioaccumulation in lab-read and native H. azteca populations and (iii) identify the major Se exposure pathway (surface water, top 1 cm and top 2-3 cm sediment layers) leading to Se bioaccumulation in H. azteca. Field and laboratory studies indicated overall comparable Se bioaccumulation and trophic transfer factors (TTFs) in co-exposed H. azteca (whole-body Se 0.9-3.1 µg/g d.w; TTFs 0.6-6.3) and C. dilutus (whole-body Se at 0.7-3.2 µg Se/g d.w.; TTFs 0.7-3.4). Native and lab-reared H. azteca populations exposed to sediment and periphyton from McClean Lake exhibited similar Se uptake and bioaccumulation (NLR, p = 0.003; 4.1 ± 0.8 µg Se/g d.w), demonstrating that lab-reared organisms are good surrogates to assess on-site Se bioaccumulation potential. The greater Se concentrations in H. azteca exposed to the top 1-3 cm sediment layer relative to waterborne exposure, corroborates the importance of the sediment-detrital pathway leading to greater Se bioaccumulation potential to higher trophic levels via BMI.

New records of immature aquatic Diptera from the Foulden Maar Fossil-Lagerstätte, New Zealand, and their biogeographic implications.

Background: The biogeographical and ecological history of true flies (Diptera) in New Zealand is little known due to a scarcity of fossil specimens. Here, we report a fauna of immature aquatic dipterans from freshwater diatomites of the early Miocene Foulden Maar Fossil-Lagerstätte in southern New Zealand. Methods: We document 30 specimens of immature dipterans, mostly pupae, and compare their external morphology to extant aquatic Diptera. Based on the reconstructed paleoenvironment of Foulden Maar, we discuss taxonomic, ecological and taphonomic implications of this early Miocene fauna. Results: Among Chironomidae, one pupal morphotype is attributed to Tanypodinae, one pupal morphotype and one larval morphotype are placed into Chironomus (Chironominae) and a further morphotype into Chironominae incertae sedis. Chaoboridae are represented by a pupal morphotype congeneric or very close to the extant Chaoborus, today globally distributed except for New Zealand. Additional immature specimens are likely larvae and puparia of brachyceran flies but cannot be identified to a narrower range. These finds document an aquatic dipteran fauna in New Zealand in the earliest Miocene and highlight Neogene extinction as a factor in shaping the extant Diptera fauna in New Zealand. Immature aquatic dipterans were a common and likely ecologically important component of the early Miocene Foulden Maar lake. Preservation of larvae and pupae may have been promoted by diatomaceous microbial mats and the light colour of the diatomite likely facilitated spotting of these minute fossils in the field.

Smittia solominae sp. nov. (Diptera: Chironomidae: Orthocladiinae), living on ice of high mountain glaciers of the Elbrus Region (North Caucasus).

Illustrated morphological description with a study of DNA barcoding and biology of adult male, pupa and larva of Smittia solominae sp. nov., living on the ice surface of glaciers at an altitude of about 3000 m above sea level in the Elbrus region of the North Caucasus is given. DNA barcoding provided support that the new species unique within genus Smittia. The average interspecific distances between S. solominae sp. nov. and other Smittia from BOLD above 12% that correspond to species level.

New findings on Nilothauma Kieffer (Diptera: Chironomidae) from Brazil and Peru.

Two new species of Nilothauma from Brazil and Peru are described and illustrated based on adult males collected respectively in the central and western Amazon. In addition, new records of Nilothauma complicatum Mendes & Andersen, 2009 in Brazil are provided.

Parachaetocladius lenferringtoni (Diptera: Chironomidae), a new orthoclad from North America, with keys to adult males and pupae of the Nearctic Parachaetocladius (Wlker, 1959).

In this study, we describe Parachaetocladius lenferringtoni sp. nov., based on all its life stages, and provide keys to adult males and pupae of the Nearctic Parachaetocladius. The new species was originally placed in Parachaetocladius abnobaeus (Wlker 1959) due to the similarity in the hypopygium of these species. However, a detailed examination of adults and associated immature specimens demonstrates that P. lenferringtoni sp. nov. represents a single species that is different from P. abnobaeus. Diagnostic adult and pupal characters were identified to separate this new species from P. abnobaeus and other species of Parachaetocladius. The separation of the larva of P. lenferringtoni sp. nov. and P. abnobaeus is currently tenuous, but some possible diagnostic characters are provided. Material of P. lenferringtoni sp. nov. was obtained for this study from Canada (Manitoba) and the United States of America (Kansas, Minnesota, Nebraska, and Wisconsin), indicating this species is largely limited to central North America. The habitat of this new species matches that of other Parachaetocladius species in that it has been collected from sandy substrates in springs and groundwater-dominated streams.

First Upper Triassic Diptera (Insecta) from Germany.

First Upper Triassic dipterans (Nematocera) are described based on isolated wings from Fuchsberg near Seinstedt (Sevatian, uppermost Norian of Lower Saxony, Germany): Crivoptychoptera nebrias gen. et sp. nov. (Psychodomorpha, Ptychopteridae), Ptychopteridae incertae sedis, Dohloboyia triassica gen. et sp. nov. (Bibionomorpha, Boholdoyidae) and Chironomidae incertae sedis (Culicomorpha). All taxa under discussion present the oldest occurrences of their families. Geological background and composition of the Upper Triassic insect fauna of Seinstedt are briefly discussed.

Biofragmentation of Polystyrene Microplastics: A Silent Process Performed by Chironomus sancticaroli Larvae.

Polystyrene (PS) is one of the main synthetic polymers produced around the world, and it is present in the composition of a wide variety of single-use objects. When released into the environment, these materials are degraded by environmental factors, resulting in microplastics. We investigated the ability of Chironomus sancticaroli (Diptera, Chironomidae) to promote the fragmentation of PS microspheres (24.5 ± 2.9 µm) and the toxic effects associated with exposure to this polymer. C. sancticaroli larvae were exposed to 3 different concentrations of PS (67.5, 135, and 270 particles g-1 of dry sediment) for 144 h. Significant lethality was observed only at the highest concentration. A significant reduction in PS particle size as well as evidence of deterioration on the surface of the spheres, such as grooves and cracks, was observed. In addition, changes in oxidative stress biomarkers (SOD, CAT, MDA, and GST) were also observed. This is the first study to report the ability of Chironomus sp. to promote the biofragmentation of microplastics. The information obtained demonstrates that the macroinvertebrate community can play a key role in the degradation of plastic particles present in the sediment of freshwater environments and can also be threatened by such particle pollution.

Beneath the surface: Decoding the impact of Chironomus riparius bioturbation on microplastic dispersion in sedimentary matrix.

A detailed understanding of microplastics (MPs) behaviour in freshwater ecosystems is crucial for a proper ecological assessment. This includes the identification of significant transport pathways and net accumulation zones, considering their inherent, and already proven influence on aquatic ecosystems. Bioavailability of toxic agents is significantly influenced by macroinvertebrates' behaviour, such as bioturbation and burrowing, and their prior exposure history. This study investigates the effect of bioturbation activity of Chironomus riparius Meigen, 1804 on the vertical transfer of polyethylene MPs ex-situ. The experimental setup exposes larvae to a scenario of 10× the environmentally relevant high concentration of MPs (80 g m-2). Bioturbation activity was estimated using sediment profile imaging with luminophore tracers. This study demonstrated that spherical MPs are vertically transferred in the sediment due to the bioturbation activity of C. riparius larvae and that their presence influences the intensity of the bioturbation activity over time. The present findings provide a noteworthy contribution to the understanding of the relationship between ecosystem engineers and the dispersion and accumulation of MPs within freshwater ecosystems.

Polyethylene microplastics induced lipidomic responses in Chironomus tepperi: A two-generational exploration.

Microplastics (MPs) accumulating in freshwater sediment have raised concerns about potential risks to benthic dwelling organisms, yet few studies have examined the long-term impacts caused by MP exposure. This study investigated alterations to lipid profiles in an Australian freshwater invertebrate, Chironomus tepperi, induced by polyethylene MP fragments (1-45 µm) at environmentally relevant concentrations (125, 250, 500 and 1000 MPs/kg sediment), using a two-generational experimental design. In the parental generation, the relative abundance of triacylglycerols, total fatty acids and unsaturated fatty acids exhibited apparent hormetic patterns, with low-concentration stimulation and high-concentration inhibition observed. The overall trend in these lipid classes is consistent with previously observed changes to polar metabolite profiles, indicating that ingestion of MPs could inhibit nutrient assimilation from food leading to disruption of energy availability. In the first filial generation continuously exposed to MPs, however, abundance of cholesterol and total fatty acids increased with increasing exposure concentrations, suggesting different effects on energy metabolism between the parental generation and offspring. No differences in the lipidome were observed in first filial larvae that were not exposed, implying that MPs pose negligible carry-over effects. Overall, the combined results of this study together with a preceding metabolomics study provide evidence of a physical effect of MPs with subsequent impacts to bioenergetics. Nevertheless, future research is required to explore the potential long-term impacts caused by MPs, and to unravel the impacts of the surfactant control as a potential contributor to the observed hormetic response, particularly for studies exploring sub-lethal effects of MP exposure using sensitive omics techniques.

Photoaging of biodegradable nanoplastics regulates their toxicity to aquatic insects (Chironomus kiinensis) by impairing gut and disrupting intestinal microbiota.

Biodegradable plastic, a widely used ecofriendly alternative to conventional plastic, easily form nanoplastics (NPs) upon environmental weathering. However, the effects and underlying mechanisms governing the toxicity of photoaged biodegradable NPs to aquatic insects are not understood. In this study, we investigated the photoaging of polylactic acid nanoplastics (PLA-NPs, a typical biodegradable plastic) that were placed under xenon arc lamp for 50 days and 100 days and compared the toxicity of virgin and photoaged PLA-NPs to Chironomus kiinensis (a dominant aquatic insect). The results showed that photoaged PLA-NPs significantly decreased the body weight, body length and emergence rate of C. kiinensis. Additionally, photoaged PLA-NPs induced more severe gut oxidative stress, histological damage, and inflammatory responses than virgin PLA-NPs. Furthermore, the alpha diversity of gut microbiota was lower in photoaged PLA-NPs group than virgin PLA-NPs. The relative abundance of key gut bacteria related to intestinal barrier defense, immunity, and nutrient absorption was reduced more significantly in photoaged PLA-NPs group than virgin PLA, indirectly leading to stronger gut damage and growth reduction. A stronger impact of photoaged PLA-NPs on the gut and its microbiota occurred because photoaging reduced the size of NPs from 255.5 nm (virgin PLA) to 217.1 nm (PLA-50) and 182.5 nm (PLA-100), induced surface oxidation and enhancement of oxidative potential, and improved the stability of NPs, thereby exacerbating toxicity on the gut and its microbiota. This study provides insights into the effects of biodegradable NPs on aquatic insects and highlights the importance of considering biodegradable nanoplastic aging in risk assessments.

Fate of Planktothrix-derived toxins in aquatic food webs: A case study in Lake Mindelsee (Germany).

Blooms of the red, filamentous cyanobacterium Planktothrix rubescens occur frequently in pre-alpine lakes in Europe, often with concomitant toxic microcystin (MC) production. Trophic transfer of MCs has been observed in bivalves, fish, and zooplankton species, while uptake of MCs into Diptera species could facilitate distribution of MCs into terrestrial food webs and habitats. In this study, we characterized a Planktothrix bloom in summer 2019 in Lake Mindelsee and tracked possible trophic transfer and/or bioaccumulation of MCs via analysis of phytoplankton, zooplankton (Daphnia) and emergent aquatic insects (Chaoborus, Chironomidae and Trichoptera). Using 16 S rRNA gene amplicon sequencing, we found that five sequence variants of Planktothrix spp. were responsible for bloom formation in September and October of 2019, and these MC-producing variants, provisionally identified as P. isothrix and/or P. serta, occurred exclusively in Lake Mindelsee (Germany), while other variants were also detected in nearby Lake Constance. The remaining cyanobacterial community was dominated by Cyanobiaceae species with high species overlap with Lake Constance, suggesting a well-established exchange of cyanobacteria species between the adjacent lakes. With targeted LC-HRMS/MS we identified two MC-congeners, MC-LR and [Asp3]MC-RR with maximum concentrations of 45 ng [Asp3]MC-RR/L in lake water in September. Both MC congeners displayed different predominance patterns, suggesting that two different MC-producing species occurred in a time-dependent manner, whereby [Asp3]MC-RR was clearly associated with the Planktothrix spp. bloom. We demonstrate an exclusive transfer of MC-LR, but not [Asp3]MC-RR, from phytoplankton into zooplankton reaching a 10-fold bioconcentration, yet complete absence of these MC congeners or their conjugates in aquatic insects. The latter demonstrated a limited trophic transfer of MCs from zooplankton to zooplanktivorous insect larvae (e.g., Chaoborus), or direct transfer into other aquatic insects (e.g. Chironomidae and Trichoptera), whether due to avoidance or limited uptake and/or rapid excretion of MCs by higher trophic emergent aquatic insects.

Once upon a fly: The biogeographical odyssey of Labrundinia (Chironomidae, Tanypodinae), an aquatic non-biting midge towards diversification.

Labrundinia is a highly recognizable lineage in the Pentaneurini tribe (Diptera, Chironomidae). The distinct predatory free-swimming larvae of this genus are typically present in unpolluted aquatic environments, such as small streams, ponds, lakes, and bays. They can be found on the bottom mud, clinging to rocks and wood, and dwelling among aquatic vegetation. Labrundinia has been extensively studied in ecological research and comprises 39 species, all but one of which has been described from regions outside the Palearctic. Earlier phylogenetic studies have suggested that the initial diversification of the genus likely occurred in the Neotropical Region, with its current presence in the Nearctic Region and southern South America being the result of subsequent dispersal events. Through the integration of molecular and morphological data in a calibrated phylogeny, we reveal a complex and nuanced evolutionary history for Labrundinia, providing insights into its biogeographical and diversification patterns. In this comprehensive study, we analyze a dataset containing 46 Labrundinia species, totaling 10,662 characters, consisting of 10,616 nucleotide sites and 46 morphological characters. The molecular data was generated mainly by anchored enrichment hybrid methods. Using this comprehensive dataset, we inferred the phylogeny of the group based on a total evidence matrix. Subsequently, we employed the generated tree for time calibration and further analysis of biogeography and diversification patterns. Our findings reveal multiple dispersal events out of the Neotropics, where the group originated in the late Cretaceous approximately 72 million years ago (69-78 Ma). We further reveal that the genus experienced an early burst of diversification rates during the Paleocene, which gradually decelerated towards the present-day. We also find that the Neotropics have played a pivotal role in the evolution of Labrundinia by serving as both a cradle and a museum. By "cradle," we mean that the region has been a hotspot for the origin and diversification of new Labrundinia lineages, while "museum" refers to the region's ability to preserve ancestral lineages over extended periods. In summary, our findings indicate that the Neotropics have been a key source of genetic diversity for Labrundinia, resulting in the development of distinctive adaptations and characteristics within the genus. This evidence highlights the crucial role that these regions have played in shaping the evolutionary trajectory of Labrundinia.

Molecular and morphological characterisation of larvae of the genus Diamesa Meigen, 1835 (Diptera: Chironomidae) in Alpine streams (Ötztal Alps, Austria).

Diamesa species (Diptera, Chironomidae) are widely distributed in freshwater ecosystems, and their life cycles are closely linked to environmental variables such as temperature, water quality, and sediment composition. Their sensitivity to environmental changes, particularly in response to pollution and habitat alterations, makes them valuable indicators of ecosystem health. The challenges associated with the morphological identification of larvae invoke the use of DNA barcoding for species determination. The mitochondrial cytochrome oxidase subunit I (COI) gene is regularly used for species identification but faces limitations, such as similar sequences in closely related species. To overcome this, we explored the use of the internal transcribed spacers (ITS) region in addition to COI for Diamesa larvae identification. Therefore, this study employs a combination of molecular markers alongside traditional morphological identification to enhance species discrimination. In total, 129 specimens were analysed, of which 101 were sampled from a glacier-fed stream in Rotmoostal, and the remaining 28 from spring-fed streams in the neighbouring valleys of Königstal and Timmelstal. This study reveals the inadequacy of utilizing single COI or ITS genes for comprehensive species differentiation within the genus Diamesa. However, the combined application of COI and ITS markers significantly enhances species identification resolution, surpassing the limitations faced by traditional taxonomists. Notably, this is evident in cases involving morphologically indistinguishable species, such as Diamesa latitarsis and Diamesa modesta. It highlights the potential of employing a multi-marker approach for more accurate and reliable Diamesa species identification. This method can be a powerful tool for identifying Diamesa species, shedding light on their remarkable adaptations to extreme environments and the impacts of environmental changes on their populations.

Two chromosome-level genomes of Smittia aterrima and Smittia pratorum (Diptera, Chironomidae).

Chironomids are one of the most abundant aquatic insects and are widely distributed in various biological communities. However, the lack of high-quality genomes has hindered our ability to study the evolution and ecology of this group. Here, we used Nanopore long reads and Hi-C data to produce two chromosome-level genomes from mixed genomic data. The genomes of Smittia aterrima (SateA) and Smittia pratorum (SateB) were assembled into three chromosomes, with sizes of 78.45 Mb and 71.56 Mb, scaffold N50 lengths of 25.73 and 23.53 Mb, and BUSCO completeness of 98.5% and 97.8% (n = 1,367), 5.68 Mb (7.24%) and 1.94 Mb (2.72%) of repetitive elements, and predicted 12,330 (97.70% BUSCO completeness) and 11,250 (97.40%) protein-coding genes, respectively. These high-quality genomes will serve as valuable resources for comprehending the evolution and environmental adaptation of chironomids.

Impacts of climate warming and atmospheric deposition on recent shifts in chironomid communities in two alpine lakes, eastern China.

Climate warming and atmospheric deposition are altering alpine lake ecosystems at unprecedented rates, whereas their direct and indirect effects on primary consumer communities are unclear. This study presents sedimentary multi-proxy records including chironomids, diatoms, elements and stable isotopes of carbon and nitrogen in 210Pb-dated cores from two alpine lakes located above the timberline in the Taibai Mountain, eastern China. Before ∼2000 CE, chironomid communities were co-dominated by Heterotrissocladius marcidus-type and Micropsectra atrofasciata-type in the two lakes. Thereafter, Tanytarsus glabrescens-type increased rapidly to be a dominant species. Redundancy analyses (RDAs) revealed that chironomid fauna shifts were significantly correlated with rising diatom concentrations in both lakes, declining Ti content in the upstream lake and δ13C depletion in the downstream lake. Although temperature, precipitation and δ15N were not significant explanatory variables in RDAs, climate warming and atmospheric deposition likely promoted terrestrial and aquatic primary production, indicated by synchronous increases in organic matter contents and diatom concentrations in the two sediment cores. Since diatoms contain essential polyunsaturated fatty acids that are essential for chironomids, rising diatom concentrations can promote food quantity and quality. In addition, increased primary production would create organic substrates for chironomid larvae. Recent shifts in chironomid fauna driven by indirect effects of global warming and atmospheric deposition might be a widespread phenomenon in alpine lakes, probably triggering regime shifts in headwater lake ecosystems.

New species and record of Diplosmittia Sther, 1981 (Diptera: Chironomidae, Orthocladiinae) from Brazil and Colombia.

Chironomidae is a diverse Diptera family found worldwide. The genus Diplosmittia Sther, 1981, currently with 11 valid species, has been found exclusively in the New World, mainly in the Caribbean and in the northern parts of South America; only three species are recorded from Brazil and none from Colombia so far. In the present study a new species of Diplosmittia is described and illustrated based on material collected in northeastern Brazil. It appears to be most closely related to Diplosmittia beluina Andersen, 1996, but can be distinguished by morphological differences in the virga and gonostylus. Diplosmittia harrisoni Sther, 1981 is documented for the first time from Colombia, and D. carinata Sther, 1985 is formally placed as a junior synonym of D. harrisoni. In addition, an update identification key to all known species of the genus has been provide.

How non-target chironomid communities respond to mosquito control: Integrating DNA metabarcoding and joint species distribution modelling.

The conservation and management of riparian ecosystems rely on understanding the ecological consequences of anthropogenic stressors that impact natural communities. In this context, studies investigating the effects of anthropogenic stressors require reliable methods capable of mapping the relationships between taxa occurrence or abundance and environmental predictors within a spatio-temporal framework. Here, we present an integrative approach using DNA metabarcoding and Hierarchical Modelling of Species Communities (HMSC) to unravel the intricate dynamics and resilience of chironomid communities exposed to Bacillus thuringiensis var. israelensis (Bti). Chironomid emergence was sampled from a total of 12 floodplain pond mesocosms, half of which received Bti treatment, during a 16-week period spanning spring and summer of 2020. Subsequently, we determined the community compositions of chironomids and examined their genus-specific responses to the Bti treatment, considering their phylogenetic affiliations and ecological traits of the larvae. Additionally, we investigated the impact of the Bti treatment on the body size distribution of emerging chironomids. Our study revealed consistent responses to Bti among different chironomid genera, indicating that neither phylogenetic affiliations nor larval feeding strategies significantly contributed to the observed patterns. Both taxonomic and genetic diversity were positively correlated with the number of emerged individuals. Furthermore, our findings demonstrated Bti-related effects on chironomid body size distribution, which could have relevant implications for size-selective terrestrial predators. Hence, our study highlights the value of employing a combination of DNA metabarcoding and HMSC to unravel the complex dynamics of Bti-related non-target effects on chironomid communities. The insights gained from this integrated framework contribute to our understanding of the ecological consequences of anthropogenic stressors and provide a foundation for informed decision-making regarding the conservation and management of riparian ecosystems.