The first step towards a DNA barcode reference database for mayflies (Ephemeroptera) of Slovakia

Mayflies (Ephemeroptera) represent a small but diverse order of amphibiotic insects, whose larvae contribute to several essential processes in freshwater habitats, such as bioturbation and bioirrigation, decomposition, nutrient cycling, and also serve as a primary source of nutrients for numerous organisms. Due to their cosmopolitan distribution and high-quality water requirements, they are also important indicators of ecosystem health and an integral part of biomonitoring protocols. Although the Slovak mayfly fauna is well researched, studies on genetic diversity, including DNA barcoding, are still lacking. The absence of the comprehensive DNA barcode reference libraries from various biogeographical regions and the presence of so-called cryptic lineages may prevent further efficient use and application of new approaches to aquatic ecosystem biomonitoring (Biomonitoring 2.0) based on eDNA analyses. Therefore, in the initial stage of our research, we bring the first insight into the genetic diversity of mayflies (based on mtDNA COI-5P barcoding fragment) from 47 localities of Slovakia mostly situated in the biogeographically significant Western Carpathians' territory. A total of 403 sequences of 42 morphologically determined species were added to the BOLD (Barcode of Life Data System) database, representing more than 1/3 of the mayfly fauna of Slovakia and covering 10 of 16 families. Sequences of these species were finally assigned to 62 BINs (Barcode Index Numbers) in BOLD (Fig. 1), whereby sequences of 12 species were divided into more than one BIN, indicating the presence of cryptic lineages. The largest number of BINs was represented by widely distributed species such as Baetis rhodani Pictet, 1843-1845 (6 BINs), Habroleptoides confusa Sartori & Jacob, 1986 (4 BINs) and Ecdyonurus venosus (Fabricius, 1775) (3 BINs). The sequences of the remaining nine species were split into two BINs. Maximum intraspecific variability (calculated by K2P) of some representatives was surprisingly high [e.g., E. venosus – 27.1 %; Baetis muticus (Linneaus, 1758) – 23.6 %; Caenis luctuosa (Burmeister, 1839) – 23.34 %, Baetis rhodani – 18.66 % and B. vernus Curtis, 1834 – 15.25 %] and far exceeded the level of intraspecific variability of the COI fragment based on the BOLD standards. The sequences of 23 individuals determined as Habroleptoides confusa, Baetis rhodani, B. buceratus Eaton, 1870, Caenis beskidensis Sowa, 1973 and Torleya major (Klapálek, 1905) created seven unique BINs, which represent the distant phylogenetic lineages of already existing BINs, that are currently unique to Slovakia. The coexistence of Baetis rhodani individuals of two different BINs was confirmed at five localities. Our study indicates clear importance of more detailed sampling and DNA barcoding due to the presence of unexpected intraspecific genetic diversity of mayflies captured in a relatively small area of the Western Carpathians.

Assessment of the effects of non-aqueous phase liquid (NAPL) pollution on aquatic macroinvertebrates survivorship

<p>Massive production, extensive use and poor disposal practices of organic chemicals from industries and fuel facilities have polluted soil and water resources. Non-aqueous phase liquids (NAPLs), including oils and fuel hydrocarbons are immiscible in water, moving as separate layers based on their density. Due to their high persistence, they are seldom flushed from the system, potentially making it unfit for use later. Along with resource consumption, pollution also poses a threat to dependent aquatic life. NAPLs have the potential to disturb the physiological, behavioral and breeding performance of aquatic organisms, by affecting their oxygen and nutrient uptake from the system. To analyze these effects, this study examined the response of two common European freshwater macroinvertebrates: <em>Gammarus pulex</em> (freshwater shrimp) and <em>Baetis rhodani</em> (mayfly larvae) to controlled exposure of mineral oil (a representative NAPL). The experiments were conducted in the laboratory for a 24-hour period to observe any changes in their locomotion and survivorship at different concentrations of mineral oil. Results indicate that mineral oil has a serious effect on the organism’s movement and survivorship. <em>Gammarus pulex</em> displayed a high tolerance compared to the mayfly (<em>Baetis rhodani</em>). Most mayfly larvae died due to NAPL exposure. The results indicate that both organisms respond rapidly to NAPL contamination, providing the potential for the development of bio-monitoring tools for water quality studies.</p><p><strong>Keywords</strong>: Aquatic macroinvertebrates, NAPLs, pollution, response</p>

Mayfly (Ephemeroptera) assemblages of a Pannonian lowland mountain, with first records of the parasite Symbiocladius rhithrogenae (Zavrel, 1924) (Diptera: Chironomidae)

Despite increasing numbers of studies on aquatic insects in South-East European streams, there are still gaps in the knowledge about their distribution and assemblage composition in many regions. As one of the most abundant and sensitive aquatic insects, mayflies are widely used as bioindicator taxa. With the main goal of improving the knowledge of South-East European mayflies, a study was conducted of 15 streams along a Pannonian lowland mountain, in spring and summer 2017. A total of 18 mayfly taxa were recorded, where Ephemera danica Müller, 1764 and Baetis rhodani (Pictet, 1843) were the most widespread. All sites were characterized with the domination of rhithral elements, with similar shares of grazers/scrapers and detritivores. Nevertheless, sites at lower altitudes generally had a higher share of potamal and littoral elements, and a higher share of detritivores than at higher altitudes. NMDS analysis separated sites mainly based on sampling period. Although Heptageniidae nymphs were recorded in all streams, the parasitic chironomid Symbiocladius rhithrogenae (Zavrel, 1924) was recorded attached to three nymphs of Rhithrogena group semicolorata, each in a different stream. These records represent the first report of this species for Croatia. The current study contributes significantly to our knowledge of mayfly assemblages and species distribution in South-East Europe. Moreover, with a newly recorded species, this study indicates that the knowledge of the Croatian chironomid fauna is still growing.

Development of genomic resources for four potential environmental bioindicator species: Isoperla grammatica, Amphinemura sulcicollis, Oniscus asellus and Baetis rhodani

A low-coverage genome was generated for each of four environmental key-species of macroinvertebrate taxa for the primary purpose of microsatellite marker development. De novo assemblies and microsatellite markers were designed for the freshwater species Isoperla grammatica, Amphinemura sulcicollis, and Baetis rhodani but have not been completed for the common shiny woodlice Oniscus asellus. Here, the data is made available, and the methods and pipeline are described which led to the creation of this resource. As widespread and functionally important organisms, which are often neglected in favour of studies on vertebrates, this data will be a useful resource for further research.

Linkages between reach-scale physical habitat and invertebrate assemblages in upland streams

Determining the influence of physical habitat on biological structure in minimally disturbed settings is important if the effects of alterations to physical habitat are to be understood. This study tested whether reach-scale differences in physical habitat influence macroinvertebrate community composition at 24 sites in the Cairngorm Mountains, Scotland. Stream reaches were classified into channel types based on a geomorphic typology (i.e. step-pool, bedrock, plane-bed and pool-riffle). PERMANOVA indicated an overall significant relationship between the geomorphic typology and macroinvertebrate species-level composition, and among all combinations of channel types (such as step-pool and pool-riffle, step-pool and bedrock). Most channel types were dominated by high abundances of Baetis rhodani, Rhithrogena semicolorata and Leuctra inermis, which are ubiquitous in unpolluted gravel-bedded Scottish streams. However, reflecting significant differences in abundance of commoner taxa between types, indicator value (IndVal) analysis revealed that pool-riffle reaches were characterised by elmids (Limnius sp. and Oulimnius sp.) and Caenis rivulorum, and step-pool reaches by Alainites muticus, B. rhodani, L. inermis and Brachyptera risi. Geomorphic typing of rivers provides a useful basis for the initial assessment of ecological status whereas abundance-based biological data processed at the appropriate taxonomic resolution should be sensitive to physical-habitat modifications.