Aquatic Insects Transfer Pharmaceuticals and Endocrine Disruptors from Aquatic to Terrestrial Ecosystems.

A wide range of pharmaceuticals and endocrine disrupting compounds enter freshwaters globally. As these contaminants are transported through aquatic food webs, understanding their impacts on both aquatic and terrestrial ecosystems remains a major challenge. Here, we provide the first direct evidence of the transfer of pharmaceuticals and endocrine disruptors through the aquatic-terrestrial habitat linkage by emerging aquatic insects. We also show that the type of insect metamorphosis and feeding behavior determine the bioaccumulation patterns of these contaminants. Adult Trichoptera, an important food source for riparian predators, showed an increased body burden of pharmaceuticals and endocrine disruptors. This implies that terrestrial predators, such as spiders, birds, and bats, are exposed to mixtures of pharmaceuticals and endocrine disruptors of aquatic origin, which may impact their physiology and population dynamics. Overall, our study provides valuable insights into the bioaccumulation patterns and trophic cross-ecosystem transfer of these contaminants, from aquatic primary producers to terrestrial predators.

Integrative taxonomy by molecular species delimitation: multi-locus data corroborate a new species of Balkan Drusinae micro-endemics.

BACKGROUND: Taxonomy offers precise species identification and delimitation and thus provides basic information for biological research, e.g. through assessment of species richness. The importance of molecular taxonomy, i.e., the identification and delimitation of taxa based on molecular markers, has increased in the past decade. Recently developed exploratory tools now allow estimating species-level diversity in multi-locus molecular datasets. RESULTS: Here we use molecular species delimitation tools that either quantify differences in intra- and interspecific variability of loci, or divergence times within and between species, or perform coalescent species tree inference to estimate species-level entities in molecular genetic datasets. We benchmark results from these methods against 14 morphologically readily differentiable species of a well-defined subgroup of the diverse Drusinae subfamily (Trichoptera, Limnephilidae). Using a 3798 bp (6 loci) molecular data set we aim to corroborate a geographically isolated new species by integrating comparative morphological studies and molecular taxonomy. CONCLUSIONS: Our results indicate that only multi-locus species delimitation provides taxonomically relevant information. The data further corroborate the new species Drusus zivici sp. nov. We provide differential diagnostic characters and describe the male, female and larva of this new species and discuss diversity patterns of Drusinae in the Balkans. We further discuss potential and significance of molecular species delimitation. Finally we argue that enhancing collaborative integrative taxonomy will accelerate assessment of global diversity and completion of reference libraries for applied fields, e.g., conservation and biomonitoring.

Cryptic diversity in the Western Balkan endemic copepod: Four species in one?

We use mitochondrial (mtCOI) and nuclear (nH3) sequence data to investigate differentiation of Eudiaptomus hadzici, a freshwater copepod endemic to the Western Balkans. E. hadzici has a disjunct distribution and morphological differences were observed at regional scale. In the current study 6 out of 7 known populations are included. We applied several species delimiting approaches, distance based methods (K2P p-distance and Automatic Barcode Gap Discovery, ABGD) using the mtCOI, Bayesian phylogeny and the Bayesian method implemented in bPTP and BPP programs using the concatenated sequences of both genes. Phylogenetic and species delimitation analyses all suggest that the nominal species E. hadzici consists of four isolated, cryptic evolutionary lineages in the Western Balkans. Each of the four lineages inhabits a single lake or a group of lakes in close proximity. They exhibit major differences in secondary sexual characters, e.g. right antennule in males. Denticulation of spine on 13th segment is substantially distinct among the four lineages, having different number and shape of tooth-like protrusions. Gene flow and dispersal are restricted to very small spatial scale, but with local differences, implying that diverse historical and contemporary processes are operating at small spatial scales in E. hadzici. In order to further examine spatial and temporal diversification patterns, we constructed a dated species tree analysis using (*)BEAST. Due to lack of reliable calibration points and taxa specific evolutionary rates, two evolutionary rates were applied and the faster one (2.6% myr) seems more plausible considering the geological history of the region. The divergence of E. hadzici lineages is dated from Early Miocene onwards with geographically close lineages diverging more recently, Late Miocene to Pleistocene and Pleistocene, respectively. Overall, our findings shed light on cryptic genetic complexity of endemics in one of European biodiversity hotspots. Moreover, this study represents one further example of integrative taxonomy, linking DNA methodology and classical taxonomy based on morphology. Therefore, it lays groundwork for future taxonomy and biogeography of freshwater microcrustaceans in the region.

A hairy case: The evolution of filtering carnivorous Drusinae (Limnephilidae, Trichoptera).

The caddisfly subfamily Drusinae BANKS comprises roughly 100 species inhabiting mountain ranges in Europe, Asia Minor and the Caucasus. A 3-gene phylogeny of the subfamily previously identified three major clades that were corroborated by larval morphology and feeding ecologies: scraping grazers, omnivorous shredders and filtering carnivores. Larvae of filtering carnivores exhibit unique head capsule complexities, unknown from other caddisfly larvae. Here we assess the species-level relationships within filtering carnivores, hypothesizing that head capsule complexity is derived from simple shapes observed in the other feeding groups. We summarize the current systematics and taxonomy of the group, clarify the systematic position of Cryptothrix nebulicola, and present a larval key to filtering carnivorous Drusinae. We infer relationships of all known filtering carnivorous Drusinae and 34 additional Drusinae species using Bayesian species tree analysis and concatenated Bayesian phylogenetic analysis of 3805bp of sequence data from six gene regions (mtCOI5-P, mtCOI3-P, 16S mrDNA, CADH, WG, 28S nrDNA), morphological cladistics from 308 characters, and a total evidence analysis. All analyses support monophyly of the three feeding ecology groups but fail to fully resolve internal relationships. Within filtering carnivores, variation in head setation and frontoclypeus structure may be associated with progressive niche adaptation, with less complex species recovered at a basal position. We propose that diversification of complex setation and frontoclypeus shape represents a recent evolutionary development, hypothetically enforcing speciation and niche specificity within filtering carnivorous Drusinae.

Larval morphology and phylogenetic position of Drusus balcanicus, Drusus botosaneanui, Drusus serbicus and Drusus tenellus (Trichoptera: Limnephilidae: Drusinae).

In a recent 3-gene phylogeny of the Trichoptera subfamily Drusinae Banks, 1916 molecular data clearly correlated with the morphology and feeding ecology of larvae. The largest of three main groups, the Drusinae grazer clade, exhibits an unusual larval feeding ecology for Limnephilidae, and is the most diverse group. In this paper we describe four previously unknown Drusinae larvae from this clade: Drusus balcanicus Kumanski, 1973 (micro-endemic to Eastern Balkans); Drusus botosaneanui Kumanski, 1968 (Dinaric Western Balkans, Hellenic and Eastern Balkan, Asia Minor), Drusus serbicus Marinkovic-Gospodnetic, 1971a (micro-endemic to Dinaric Western Balkans); and Drusus tenellus (Klapálek, 1898) (Carpathians, Dinaric Eastern Balkans). Characteristically, the larvae of these species develop toothless mandibles typical for the Drusinae grazer clade. Larvae and adults were unambiguously associated by a phylogenetic approach based on two mitochondrial (mtCOI, mtLSU= 16S rDNA) and two nuclear genes (nuWG, nuCAD). In addition, information on the morphology of the larvae is given and the diagnostic features necessary for identification are illustrated.

Pharmaceuticals and endocrine disrupting compounds modulate adverse effects of climate change on resource quality in freshwater food webs.

Freshwater biodiversity, ecosystem functions and services are changing at an unprecedented rate due to the impacts of vast number of stressors overlapping in time and space. Our study aimed at characterizing individual and combined impacts of pollution with pharmaceuticals (PhACs) and endocrine disrupting compounds (EDCs) and increased water temperature (as a proxy for climate change) on primary producers and first level consumers in freshwaters. We conducted a microcosm experiment with a simplified freshwater food web containing moss (Bryophyta) and shredding caddisfly larvae of Micropterna nycterobia (Trichoptera). The experiment was conducted with four treatments; control (C), increased water temperature + 4 °C (T2), emerging contaminants' mix (EC = 15 PhACs & 5 EDCs), and multiple stressor treatment (MS = EC + T2). Moss exhibited an overall mild response to selected stressors and their combination. Higher water temperature negatively affected development of M. nycterobia through causing earlier emergence of adults and changes in their lipidome profiles. Pollution with PhACs and EDCs had higher impact on metabolism of all life stages of M. nycterobia than warming. Multiple stressor effect was recorded in M. nycterobia adults in metabolic response, lipidome profiles and as a decrease in total lipid content. Sex specific response to stressor effects was observed in adults, with impacts on metabolome generally more pronounced in females, and on lipidome in males. Thus, our study highlights the variability of both single and multiple stressor impacts on different traits, different life stages and sexes of a single insect species. Furthermore, our research suggests that the combined impacts of warming, linked to climate change, and contamination with PhACs and EDCs could have adverse consequences on the population dynamics of aquatic insects. Additionally, these findings point to a potential decrease in the quality of resources available for both aquatic and potentially terrestrial food webs.

The larvae of Drusus franzressli Malicky 1974 and Drusus spelaeus (Ulmer 1920) (Trichoptera: Limnephilidae: Drusinae) with notes on ecology and zoogeography.

Water quality monitoring is greatly dependent on identification tools for aquatic and semi-aquatic insects. Species-level identification improves resolution and precision of water quality assessment and requires comprehensive keys. With the aim of increasing the suitability of Drusinae for such applications, this paper gives a description of the hitherto unknown larvae of Drusus franzressli Malicky 1974 and Drusus spelaeus (Ulmer 1920). Information on the morphology of the larvae is given and the most important diagnostic features are illustrated. In the context of already available keys, the larvae of D. franzressli and D. spelaeus key together with Metanoea flavipennis (Pictet 1834), M. rhaetica Schmid 1956, D. improvisus McLachlan 1884, D. nigrescens Meyer-Dür 1875 and Ecclisopteryx malickyi Moretti 1991. These species are easily separated by differences in larval morphology (dorsal outline and sculpturing of pronotum, presence/absence of lateral gills at 2nd and 3rd abdominal segments, start of lateral fringe) and their distribution ranges. Drusus franzressli is endemic to the Hellenic western Balkans whereas D. spelaeus is endemic to the western Alps (Grenoble area). In addition, ecological characteristics are briefly discussed.

Fate and effects of microplastics in combination with pharmaceuticals and endocrine disruptors in freshwaters: Insights from a microcosm experiment.

Microplastic contamination of freshwater ecosystems has become an increasing environmental concern. To advance the hazard assessment of microplastics, we conducted a microcosm experiment in which we exposed a simplified aquatic ecosystem consisting of moss and caddisflies to microplastics (polyethylene, polystyrene and polypropylene) and pharmaceuticals and personal care products (1H-benzotriazole, bisphenol A, caffeine, gemfibrozil, ketoprofen, methylparaben, estriol, diphenhydramine, tris (1-chloro-2-propyl) phosphate) over the course of 60 days. We monitored the flux of microplastics within the microcosm, as well as the metabolic and total protein variation of organisms. This study offers evidence highlighting the capacity of moss to act as a sink for free-floating microplastics in freshwater environments. Moss is also shown to serve as a source and pathway for microplastic particles to enter aquatic food webs via caddisflies feeding off of the moss. Although most ingested microparticles were eliminated between caddisflies life stages, a small fraction of microplastics was transferred from aquatic to terrestrial ecosystem by emergence. While moss exhibited a mild response to microplastic stress, caddisflies ingesting microplastics showed stress comparable to that caused by exposure to pharmaceuticals. The molecular responses that the stressors triggered were tentatively identified and related to phenotypic responses, such as the delayed development manifested through the delayed emergence of caddisflies exposed to stress. Overall, our study provides valuable insights into the adverse effects of microplastics on aquatic species, compares the impacts of microplastics on freshwater biota to those of pharmaceuticals and endocrine disrupting compounds, and demonstrates the role aquatic organisms have in redistributing microplastics between aquatic and terrestrial ecosystems.

The curious case of methylparaben: Anthropogenic contaminant or natural origin?

The widespread use of methylparaben as a preservative has caused increased exposure to natural aquatic systems in recent decades. However, current studies have suggested that exposure to this compound can result in endocrine disrupting effects, raising much concern regarding its environmental impact. In contast, methylparaben has also been found to be part of the metabolome of some organisms, prompting the question as to whether this compound may be more natural than previously assumed. Through a combination of field studies investigating the natural presence of methylparaben across different taxa, and a 54-day microcosm experiment examining the bioaccumulation and movement of methylparaben across different life stages of aquatic insects (order Trichoptera), our results offer evidence suggesting the natural origin of methylparaben in aquatic and terrestrial biota. This study improves our understanding of the role and impact this compound has on biota and challenges the current paradigm that methylparaben is exclusively a harmful anthropogenic contaminant. Our findings highlight the need for further research on this topic to fully understand the origin and role of parabens in the environment which will allow for a comprehensive understanding of the extent of environmental contamination and result in a representative assessment of the environmental risk that may pose.

Bioaccumulation and bioamplification of pharmaceuticals and endocrine disruptors in aquatic insects.

Environmental fate of emerging contaminants such as pharmaceuticals and endocrine disrupting compounds at the aquatic terrestrial boundary are largely unexplored. Aquatic insects connect aquatic and terrestrial food webs as their life cycle includes aquatic and terrestrial life stages, thus they represent an important inter-habitat linkage not only for energy and nutrient flow, but also for contaminant transfer to terrestrial environments. We measured the concentrations of pharmaceuticals and endocrine disrupting compounds in the larval and adult tissues (last larval stages and teneral adults) of five Odonata species sampled in a wastewater-impacted river, in order to examine their bioaccumulation and bioamplification at different taxonomic levels. Twenty different compounds were bioaccumulated in insect tissues, with majority having higher concentrations (up to 90% higher) in aquatic larvae compared to terrestrial adults (reaching 88 ng/g for 1H-benzotriazole). However, increased concentration in adults was observed for seven compounds in at least one suborder (41% of the accumulated), confirming contaminants bioamplification across the metamorphosis. Both, bioaccumulation and bioamplification differed at various taxa levels; the order (Odonata), suborder (Anisoptera and Zygoptera) and species level. Highest variability was observed between Anisoptera and Zygoptera, due to the underlying differences in their ecology. Generally, Zygoptera had higher concentrations of contaminants in both larvae and adults. Additionally, we aimed at predicting effects of contaminant properties on bioaccumulation and bioamplification patterns using the commonly used physicochemical and pharmacokinetic descriptors on both order and suborder levels, however, neither of the two processes could be consistently predicted with simple linear models. Our study highlights the importance of taxonomy in studies aiming at advancing the understanding of contaminant exchange between aquatic and terrestrial food webs, as higher taxonomic categories include ecologically diverse groups, whose contribution to "the dark side of subsidies" could substantially differ.

Description of the larval stage of Drusus mixtus (Pictet, 1834) (Trichoptera: Limnephilidae: Drusinae) with notes on its ecology and zoogeography.

In the Swiss Jura adults of Drusus mixtus and unknown Drusinae larvae which could not be identified with existing keys were sampled. Based on ripe pupae, the unknown larvae were identified as D. mixtus. The association was confirmed by specimen rearing in aquaria. Based on morphology, larvae of Drusus mixtus key out together with D. croaticus in existing keys. D. mixtus is separated from the latter species by the shape of the anteromedian metanotal sclerites which are broadly triangular, whereas in D. croaticus the sclerites are almost parallel-sided, resembling a stretched rectangle. In addition, the two species are geographically well separated: D. croaticus is restricted to the confines of Croatia and Slovenia, whereas D. mixtus is only present in Switzerland and eastern France. With this present paper, all Central European Drusinae species except Drusus chapmani McL, 1901 (France, Switzerland) and D. noricus Malicky, 1981, an endemic from the Saualpe (Carinthia, Austria), are known in the larval stage.

Holo- and hemimetabolism of aquatic insects: Implications for a differential cross-ecosystem flux of metals.

Increased metal concentrations in aquatic habitats come as a result of both anthropogenic and natural sources. Emerging aquatic insects that play an indispensable role in these environments, transferring resources and energy to higher trophic levels in both aquatic and terrestrial habitats, may inadvertently also act as biovectors for metals and other contaminants. This study measured levels of 22 different metals detected in biofilm, aquatic and terrestrial life stages of Trichoptera and Odonata, as well as riparian spiders, to examine the uptake and transfer from freshwater to terrestrial ecosystems. We show that emerging insects transfer metals from aquatic to terrestrial ecosystems, however with large losses observed on the boundary of these two environments. Significantly lower concentrations of most metals in adult insects were observed in both hemimetabolous (Odonata) and holometabolous insect orders (Trichoptera). In holometabolous Trichoptera, however, this difference was greater between aquatic life stages (larvae to pupae) compared to that between pupae and adults. Trophic transfer may have also played a role in decreasing metal concentrations, as metal concentrations generally adhered to the following pattern: biofilm > aquatic insects > terrestrial invertebrates. Exceptions to this observation were detected with a handful of essential (Cu, Zn, Se) and non-essential metals (Cd, Ag), which measured higher concentrations in adult aquatic insects compared to their larval counterparts, as well as in aquatic and terrestrial predators compared to their prey. Overall, all metals were found to be bioavailable and biotransferred from contaminated waters to terrestrial invertebrates to some degree, suggesting that risks associated with metal-contaminated freshwaters could extend to terrestrial systems through the emergence of these potential invertebrate biovectors.

Peeling the Layers of Caddisfly Diversity on a Longitudinal Gradient in Karst Freshwater Habitats Reveals Community Dynamics and Stability.

Freshwater biodiversity is facing a severe crisis due to many human impacts, yet the diversity dynamics of freshwater communities and possibilities of assessing these are vastly unexplored. We aimed at emphasizing different aspects of portraying diversity of a species-rich, aquatic insect group (caddisflies; Trichoptera) across four different habitats in an anthropogenically unimpacted, connected karst barrage lake/riverine system. To define diversity, we used common indices with pre-set sensitivity to species abundance/dominance; i.e., sensitivity parameter (species richness, Shannon, Simpson, Berger-Parker) and diversity profiles based on continuous gradients of this sensitivity parameter: the naïve and non-naïve diversity profiles developed by Leinster and Cobbold. The non-naïve diversity profiles show diversity profiles with regard to the similarity among species in terms of ecological traits and preferences, whereas the naïve diversity profile is called mathematically "naïve" as it assumes absolute dissimilarity between species that is almost never true. The commonly used indices and the naïve diversity profile both ranked the springs as least diverse and tufa barriers as most diverse. The non-naïve diversity profiles based on similarity matrices (using feeding behavior and stream zonation preferences of species), showed even greater differences between these habitats, while ranking stream habitats close together, regardless of their longitudinal position. We constructed the Climate Score index (CSI) in order to assess how diversity and species' vulnerability project the community's resistance and/or resilience to climate change. The CSI ranked the springs as most vulnerable, followed by all habitats longitudinally placed below them. We highlight the importance of integrating ecological information into biodiversity and vulnerability assessment of freshwater communities.

Aquatic macroinvertebrates under stress: Bioaccumulation of emerging contaminants and metabolomics implications.

The current knowledge on bioaccumulation of emerging contaminants (ECs) in aquatic invertebrates exposed to the realistic environmental concentrations is limited. Even less is known about the effects of chemical pollution exposure on the metabolome of aquatic invertebrates. We conducted an in situ translocation experiment with passive filter-feeding caddisfly larvae (Hydropsyche sp.) in an effluent-influenced river in order to i) unravel the bioaccumulation (and recovery) dynamics of ECs in aquatic invertebrates, and ii) test whether exposure to environmentally realistic concentrations of ECs will translate into metabolic profile changes in the insects. The experiment was carried out at two sites, upstream and downstream of the discharge of an urban wastewater treatment plant effluent. The translocated animals were collected at 2-week intervals for 46 days. Both pharmaceuticals and endocrine disrupting compounds (EDCs) were detected in water (62 and 7 compounds, respectively), whereas in Hydropsyche tissues 5 EDCs accumulated. Overall, specimens from the upstream site translocated to the impacted site reached higher ECs concentrations in their tissues, as a reflection of the contaminants' water concentrations. However, bioaccumulation was a temporary process susceptible to change under lower contaminant concentrations. Non-targeted metabolite profiling detected fine metabolic changes in translocated Hydropsyche larvae. Both translocations equally induced stress, but it was higher in animals translocated to the impacted site.

Pleistocene divergence of Dinaric Drusus endemics (Trichoptera, Limnephilidae) in multiple microrefugia within the Balkan Peninsula.

The Balkan Peninsula is one of three major European refugial areas. It has high biodiversity and endemism, but data on the age and origin of its fauna, especially endemics, are limited. Mitochondrial sequence data (COI and 16S genes) were used to study the population structure and phylogeography of the caddisfly Drusus croaticus and the phylogeny and divergence of seven other Drusus species, mostly range-restricted endemics of the Dinaric region of the Balkan Peninsula. The divergence of D. croaticus populations in Croatia and allopatric Drusus species in Bosnia dated to the Pleistocene, showing the importance of this time period for the origin and diversification of Balkan endemic taxa. The divergence of more distantly related species dated to the Late Miocene/Early Pliocene. Population genetic and phylogeographic analysis of 115 individuals from 11 populations of D. croaticus revealed a high level of genetic differentiation and absence of gene flow between populations separated by more than 10 km. The existence of allopatrically fragmented lineages in D. croaticus and the endemic Bosnian species is most likely the result of long-term isolation in multiple microrefugia, probably due to the specific habitat requirements and life-history traits of Drusinae coupled with the topographic complexity and historical changes in geomorphology of the region. Overall, these findings shed light on the processes generating the high genetic complexity of this refugial region that parallels the 'refugia within refugia' pattern widely reported from the Iberian refugium.

The larva and life cycle of Annitella apfelbecki Klapálek, 1899, including a redescription of Melampophylax nepos McLachlan, 1880 (Trichoptera: Limnephilidae).

The hitherto unknown larva of Annitella apfelbecki (Klapálek, 1899) is described and discussed in the context of contemporary Limnephilidae keys. In addition, its life cycle is discussed, and zoogeographical and ecological notes are included. Finally, we provide some additional information on the morphologically very similar larva of Melampophylax nepos (McLachlan, 1880).

The Larva, ecology and distribution of Tinodes braueri McLachlan, 1878 (Trichoptera: Psychomyiidae).

The hitherto unknown larva of Tinodes braueri McLachlan, 1878, is described and discussed in the context of contemporary Psychomyiidae keys. In addition, zoogeographical and ecological notes are included.

Morphological features of larvae of Drusus plicatus Radovanovic (Insecta, Trichoptera) from the Republic of Macedonia with molecular, ecological, ethological, and distributional notes.

A description of the larva of Drusus plicatus Radovanovic is given for the first time. The most important diagnostic characters enabling separation from larvae of the other Drusinae from the southeast Europe are listed. Molecular, ecological, and ethological features and distribution patterns of the species are given. Additionally, information on the sympatric caddisfly species of the three springs where larvae and adults of Drusus plicatus were found and presented.

Drusus sharrensis sp. n. (Trichoptera, Limnephilidae), a new species from Sharr National Park in Kosovo, with molecular and ecological notes.

In this paper we describe Drusus sharrensis sp. n., from the Sharr Mountains in Kosovo. Males of the new species are morphologically most similar to Drusus krusniki Malicky, 1981, Drusus kerek Oláh, 2011 and Drusus juliae Oláh, 2011 but differ mainly in exhibiting (1) a differently shaped spinose area on tergite VIII; (2) intermediate appendages anteriorly curved in lateral view with broad tips in dorsal view; (3) inferior appendages with a distinct dorsal protrusion in the proximal half. Females of the new species are morphologically most similar to Drusus krusniki, Drusus kerek, Drusus juliae, and Drusus plicatus Radovanovic, 1942 but mainly differ in (1) segment X that is longer than the supragenital plate with distinctly pointed tips; (2) supragenital plate quadrangular with a distinct round dorsal protrusion; (3) a vulvar scale with a small median lobe. Results of phylogenetic species delimitation support monophyly of Drusus sharrensis sp. n. and recover it as sister to a clade comprising (Drusus pelasgus Oláh, 2010 + Drusus juliae + Drusus arbanios Oláh, 2010 + Drusus plicatus + (Drusus dacothracus Oláh, 2010 + Drusus illyricus Oláh, 2010)). The new species is a micro-endemic of the Sharr Mountains, a main biodiversity hotspot in the Balkan Peninsula. Main threats to the aquatic ecosystems of this part of the Balkan Peninsula are discussed.

Larval morphology of the Western Balkans endemic caddisflies Drusus krusniki Malicky 1981, D. vernonensis Malicky 1989, and D. vespertinus Marinkovic 1976 (Trichoptera, Limnephilidae, Drusinae).

Drusinae (Trichoptera, Limnephilidae) are highland caddisflies inhabiting high-gradient, turbulent running water and spring habitats. They are disjunctly distributed over the Eurasian mountain ranges, and the majority of species is endemic to particular mountain areas. The most diverse of three main groups of the Drusinae, the grazer clade, consists of species in which larvae feed on epiltihic biofilm and algae. In this paper we describe three previously unknown grazer-clade Drusinae larvae: Drusus krusniki Malicky 1981 (endemic to the Dinaric western Balkans), D. vernonensis Malicky 1989 (endemic to the Hellenic western Balkans), and D. vespertinus Marinkovic 1976 (endemic to the Dinaric western Balkans). The larvae of these species have toothless mandibles typical of the Drusinae grazer clade. Larvae and adults were unambiguously associated using molecular genetic data, i.e., the mitochondrial cytochrome oxidase I gene fragment (mtCOI3-P). Morphological characteristics of the larvae are described and the diagnostic features enabling species-level identification are illustrated. We further discuss the ecology and distribution of three Western Balkan endemic species.