Hydrography and geography of the sampling area

The study was conducted in Lithuania, specifically within the Nemunas Basin, focusing on the sub-basins of the Šventoji and Žeimena Rivers. To ensure accurate data for the comparison, over 200 rivers and streams in Lithuania were screened and evaluated, based on data from the Rivers, Lakes and Ponds Cadastre of the Republic of Lithuania (UETK) data (Lietuvos Respublikos aplinkos ministerija 2024). Chironomidae specimens were collected from two types of streams: dammed (Skerdyksna, Šešuola, Dubinga) and undammed (Plaštaka, Kiauna, Luknelė). The streams were grouped into three pairs and then selected, based on their similarities in terms of geographic location, morphometric and hydrographic characteristics. The sample collection sites are situated in three protected areas: Anyksčiai and Asveja Regional Parks (Plaštaka, Šešuola, Dubinga and Skerdyksna) and Aukštaitija National Park and Labanoras Regional Park (Luknelė and Kiauna) (Fig. 1). All the streams are in the central-eastern part of Lithuania, with altitude ranges from 80 m to 150 m above sea level and traverse various landscapes, including natural or semi-natural forests and meadows, as well as agricultural and urbanised areas. The flow rate varied between the study sites, with a steady increase downstream in the undammed streams. However, the dammed streams varied considerably. For example, the Skerdyksna stream in the upper reaches passes through agricultural fields and is reclaimed, resulting in very low flow velocities. In the middle of the stream, at study sites 6 and 7, the stream is dammed. Upstream of the dam, a pond is formed, leading to stagnant water flow. Downstream of the dam, the flow velocity increases and the stream follows a natural course, characterised by a habitat-rich environment. The Šešuola stream is dammed twice, so all study sites were chosen at the dams. Study sites 23 and 21 are located in the ponds, resulting in stagnant water flow. Study site 24 is situated below the dam and in agricultural fields, where the stream section is reclaimed, with consistently low water levels averaging about 15 cm, leading to very low water velocity. Below the second dam, at study site 22, the river section is natural, which increases the flow rate. However, due to the dam and the varying rainfall, the stream depth fluctuates significantly during the season, ranging from 5 cm to 30 cm. The Dubinga stream, the third dammed stream, is dammed only in its downstream, while the upstream is natural or semi-natural, flowing through several small villages without intensive agriculture. The stream is quite deep in the upper and middle reaches, with study sites 1 and 2 maintaining an average depth of around 40 cm throughout the season. The last two survey sites in this stream were at the dam, with survey site 3 located in a pond, resulting in a stagnant water flow. Behind the dam, the stream's velocity and depth varied due to dam operations, with the depth ranging from 0 cm to 15 cm, causing highly variable flow velocity. The substrate of the studied streams varied not only between different streams, but also within the same stream at different study sites. Sand was the predominant substrate overall, but the composition and distribution of substrates varied between habitats. The substrate composition at the study sites included: silt, clay, mud, sand (the predominant substrate), granules, pebbles, cobbles and boulders. Additionally, some of the pristine sites were rich in detritus, with layers up to 0.5 m thick and contained dead wood trunks, some banks also being covered with abundant aquatic vegetation (Table 1).

Figure 1.  

Map of Lithuania highlighting the studied streams and their locations.

Sampling and identification

The research was conducted in 2021 and 2022, spanning from May to September. Sampling was conducted across six streams with four research sites in each, resulting in sampling from 24 sites in total. Samples were gathered every two weeks using a D-shaped aquatic net mesh size of 1 mm (Fig. 2). At each site, a 1 m² area was randomly selected for sampling, which was conducted using the Kick Sampling method (Letovsky et al. 2012). Samples collected were transferred into 2 litre zip-lock bags filled with 99% propylene glycol. Upon collection, all samples were stored in a refrigerator at 4°C in the Life Sciences Centre of Vilnius University. Subsequently specimens which were identified as belonging to the Chironomidae family underwent meticulous separation and were preserved in containers filled with 97% ethanol.

Figure 2.  

Sample collection using the Kick Sampling method by using D-shaped aquatic net.

Chironomidae larvae were identified by using taxonomic keys, based on morphology (morpho) available from Epler (2001), Orendt et al. (2011) and Andersen et al. 2013. The systematics and nomenclature of taxa follow Andersen et al. (2013). For molecular analysis, larvae from 200 specimens were selected. Total genomic DNA was extracted from each larva using the DNeasy Blood and Tissue kit (Qiagen) according to the manufacturer's protocol. Partial sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene were amplified using primers LCO-1490 and HCO-2198 (Folmer et al. 1994). PCR amplification was performed in a thermal cycler (Eppendorf) in 30 μl reaction volumes containing 3 μl genomic DNA, 1.5 μl of each primer (0.5 μM), 15 μl of DreamTaq PCR Master Mix (Thermo Scientific) and 9 μl of nuclease free water (Thermo Scientific). The cycling parameters were as follows: initial denaturation at 95°C for 3 minutes (1 cycle), denaturising at 95°C for 30 seconds, annealing at 49°C for 30 seconds, extension at 72°C for 60 seconds (35 cycles in total) and final extension at 72°C for 10 minutes (1 cycle). PCR products were purified using the GeneJet PCR purification kit (Thermo Scientific) and sequenced at Macrogen Europe BV (Amsterdam, the Netherlands). The amplification primers were also used as sequencing primers. DNA sequences for each specimen were aligned in the BioEdit Sequence Alignment Editor (Hall 1999) and compared by BLAST (National Library of Medicine 2024). GenBank accession numbers for each individual are given in the Check List.

Sample-based rarefaction curves were produced to visually compare the genera richness of non-biting midges amongst the researched streams and to assess the sampling effort (Gotelli and Colwell 2001). The similarity of chironomid assemblages collected over two study years from dammed and undammed streams was visualised using non-metric multidimensional scaling (NMDS) ordination, based on the Bray–Curtis similarity index. The analyses were conducted using PAST 4.07b software (Hammer et al. 2001).

A grand total of 11,296 non-biting midge specimens, comprising 89 species representing 65 genera and five subfamilies, were gathered from the sampling sites from six streams. Most specimens were identified to the subfamily and genus level, based on both morphological characteristics and analysis of partial COI sequences.

The highest richness of chironomids was observed in the subfamily Chironominae, which included 28 genera and 45 species. In contrast, the lowest richness was found in Diamesinae, with two genera and two species (Fig. 3). The Orthocladiinae exhibited a richness of 21 genera and 21 species, while Prodiamesinae showed a richness of 13 genera and 19 species.

Figure 3.  

The amount of individuals (blue bars) and the distribution across genera (orange bars) of non-biting midges’ (Chironomidae) subfamilies collected during the study.

In terms of abundance, the subfamilies Chironominae (6816 specimens) and Tanypodinae (2476 specimens) were the most prevalent, together accounting for 82.46% of collected specimens. Conversely, the subfamilies Prodiamesinae and Diamesinae subfamilies had the fewest individuals, with specimens 176 and six specimens, respectively.

Amongst the genera, the most abundant were Ablabesmyia Johannsen, 1905 (944 specimens), Microtendipes Kieffer, 1915 (661 specimens), Procladius Skuse, 1889 (474 specimens), Tanytarsus van der Wulp, 1874 (349 specimens), Polypedilum Kieffer, 1912 (290 specimens), Chironomus Meigen, 1803 (254 specimens), Conchapelopia Fittkau, 1957 (168 specimens), Prodiamesa Kieffer, 1906 (156 specimens), Micropsectra Kieffer, 1908 (151 specimens), Paratendipes Kieffer, 1911 (104 specimens) and Psectrocladius Kieffer, 1906 (103 specimens) (Fig. 4).

Figure 4.  

The number of specimens of non-biting midges (Chironomidae) representing each genus after excluding those with less than 100 collected specimens.

The Table below lists the subfamilies, genera and the abundance of specimens in each of the studied streams: Dubinga, Kiauna, Luknelė, Plaštaka, Skerdyksna and Šešuola in 2021 and 2022, spanning from May to September (Table 2).

The genera richness of non-biting midges reached an asymptote in all the streams studied, except for the Kiauna stream, suggesting that additional genera may still be discovered (Fig. 5).

Figure 5.  

Sample-based genera accumulation (rarefaction) curves, with 95% confidence intervals, of the Chironomidae genera’ richness collected in six streams.

Amongst the undammed streams, the Luknelė had the highest abundance of individuals and the greatest number of genera identified. It also emerged as a stream with the highest abundance and diversity of chironomids in terms of genera across all the streams studied. In contrast, the Šešuola stream had the lowest number of individuals, the least diversity of genera across all the studied streams and was the only stream that was dammed twice (Fig. 6). Since three of the streams studied were dammed and three were undammed, NMDS analysis was conducted to compare them based on chironomid assemblages. The visual analysis showed that the genera composition of chironomids in dammed and undammed streams partially overlapped (Fig. 7).

Figure 6.  

Number of genera (orange bars) and specimens (blue bars) of non-biting midges (Chironomidae) in all streams researched.

Figure 7.  

Non-metric multidimensional scaling (NMDS) ordination (stress = 0.167) representing Chironomidae assemblages in dammed (purple) and undammed (blue) streams.

Our study represents the first comprehensive investigation into the Chironomidae family in Lithuania. Despite studying only six streams with similar characteristics within one region of Lithuania, the gathered chironomids material encompasses over 50% of all known non-biting midges species in the country. However, given the limited scope of our research and its outcomes, it is apparent that the findings may not entirely reflect the actual state of Chironomidae in Lithuania.

The prevalence of subfamilies in terms of genera and number of individuals reveals notable trends, aligning closely with observations made in Croatia by Čerba et al. (2020), who investigated chironomid fauna across diverse freshwater habitats. Their study, like ours, underscores the dominance of the Chironominae subfamily, while also noting a relatively lower richness within the Prodiamesinae subfamily.

According to the latest data on Lithuanian non-biting midge species (Pakalniškis et al. 2006, Ruginis 2007, Móra and Kovács 2009), our research contributes significantly to the understanding of the biodiversity within the Chironomidae family in Lithuania. The discovery of several species and genera new to Lithuania underscores the richness of the region's chironomid fauna and highlights the potential for further entomological exploration. By identifying a substantial proportion of the known Orthocladiinae genera and species, our study sheds light on the ecological complexity and diversity of this subfamily. The identification of new species and genera within the Chironominae and Tanypodinae subfamilies further enhances the existing taxonomic knowledge and provides a valuable foundation for future ecological and environmental studies. These findings not only expand the taxonomic records, but also offer insights into the distribution and ecological roles of these subfamilies in Lithuanian freshwater ecosystems.

Considering the research on Chironomidae conducted in neighbouring countries, it becomes evident that the faunistic knowledge of non-biting midges in Lithuania is relatively limited. In Europe, there are over 190 genera and more than 1260 species of Chironomidae (Paasivirta 2014, Serra et al. 2016, Serra et al. 2017). Germany, in particular, is known for extensive Chironomidae research, with significant efforts concentrated in the Land of Brandenburg (Orendt 2018). While major studies have been conducted in Brandenburg, research has also been carried out in other regions of Germany (Orendt 2000, Brunke 2004, Orendt et al. 2014). Currently, over 165 genera and 780 species of Chironomidae are known in Germany, although there is still a need for further investigation (Chimeno et al. 2022, Chimeno et al. 2023).

Poland has also made significant contributions to Chironomidae research, with studies covering diversity, ecology, biology and other related areas (Płóciennik 2009, Płóciennik et al. 2015, Płóciennik et al. 2018, Leszczyńska et al. 2019, Pleskot et al. 2019, Głowacki et al. 2023). More than 420 species of Chironomidae are known in Poland (Płóciennik 2009). Ukraine, another country in the region where research on Chironomidae has been conducted since the early 20^th century, still faces a significant lack of comprehensive study (Baranov 2011). Despite numerous surveys and the documentation of over 300 Chironomidae species, recent investigations have identified 40 additional species and one new genus, suggesting that the research remains incomplete (Baranov 2011, Bitušík et al. 2024). In the Baltic States of Latvia and Estonia, the level of faunistic knowledge of Chironomidae is similar to Lithuania, with a relatively low number of known species (Spuņģis and Kalniņš 2003). This indicates a general trend of limited research in the region regarding non-biting midges.

According to data from Finland, their national Chironomidae assemblage is extensively researched, with over 780 species of non-biting midges currently documented. However, the dynamic nature of changing climate is impacting the diversity, leading to fluctuations in species composition, with several chironomids already listed on the Red List of Finnish Species (Paasivirta 2014, Engels et al. 2019, Hyvärinen et al. 2019). In Sweden, approximately 900 species of Chironomidae are known, which accounts for more than 70% of the total number of Chironomidae species documented in Europe and the country is one of the regional leaders in the research of this group (SLU Swedish Species Information Centre 2024). Norway is another country in the region actively involved in Chironomidae research, with ongoing studies conducted even in challenging environments such as Svalbard and Jan Mayen. More than 70 species of chironomids are currently known in these areas, contributing to a total of over 650 species of Chironomidae documented in Norway (Elven and Søli 2016, Stur and Ekrem 2020).

Based on the rarefaction results analysis, we can conclude that the detection of non-biting midges in the six streams was effective. Although the NMDS analysis revealed significant overlap in genus composition between dammed and non-dammed streams, this does not imply that dams have no impact on Chironomidae diversity and ecology. In conclusion, the understanding of Chironomidae diversity in Lithuania is still evolving. By leveraging the insights gleaned from neighbouring countries, there is the urgent need for Lithuania to continue its research efforts. This should extend beyond the borders of our country, encompassing regional and global initiatives aimed at conserving biodiversity and grappling with the challenges posed by environmental shifts on non-biting midge communities and populations. It would not only enrich the scientific landscape of Lithuania, but also contribute meaningfully to the collective endeavour of safeguarding our natural heritage for future generations.