Holocene ecosystem and temperature development inferred from invertebrate remains in Zminje Jezero (Dinaric Alps, Montenegro).

Lake Zminje Jezero (1535 m a.s.l.) in Montenegro was studied for chironomid and other aquatic invertebrate remains in a sediment sequence dating back to 12,000 calibrated 14C years before present (cal yr BP), providing, to our knowledge, the first lake-sediment record studied for chironomids and other associated chitinous aquatic invertebrate remains in the Dinaric Alps. Changes in chironomid and other invertebrate remains along the record make it possible to constrain changes of relevant environmental variables for aquatic invertebrates in the lake including temperature, oxygen availability, trophic status and water depth. The results suggest moderate changes in trophic conditions with chironomid assemblages indicating meso- to eutrophic conditions during the analysed interval. Invertebrate assemblages were typical for lakes with relatively high oxygen availability with a minor trend during the middle to late Holocene to conditions observed in lakes that are more hypoxic. A change in temperature is suggested in the earlier part of the record when the share of chironomid taxa adapted to warmer conditions increased, at the Younger Dryas to Holocene transition, whereas later no particularly pronounced shifts in temperature-sensitive taxa were observed. July air temperatures were estimated based on chironomid assemblages using a transfer function based on calibration data consisting of 117 lakes sampled in the Swiss Alps and northern Switzerland. The results suggest that temperatures rapidly increased by 5 °C at the onset of the Holocene (11,500 cal yr BP) leading to a relatively warm early to mid-Holocene and thereafter slightly decreased during the late Holocene. Reconstructed temperatures are discussed together with a previously published pollen record for our study site and are broadly consistent with other chironomid-based temperature reconstructions from Central, Eastern and Southern Europe. From ca. 3000 cal yr BP sedimentation rates increased and from ca. 500 cal yr BP onwards pollen data suggest that the vegetation and lake catchment were affected by human activities, possibly influencing chironomid and other invertebrate assemblages in the lake and thereby also reconstructed temperatures. Overall, our results show that combining analyses of chironomid and other invertebrate assemblages can provide valuable insights into long-term environmental changes and can provide temperature reconstructions for small mountain lakes in the Balkans, such as Zminje Jezero. Supplementary Information: The online version contains supplementary material available at 10.1007/s10933-024-00334-y.

Climate change drove the route shift of the ancient Silk Road in two distinct ways.

Although climate change has convincingly been linked to the evolution of human civilization on different temporal scales, its role in influencing the spatial patterns of ancient civilizations has rarely been investigated. The northward shift of the ancient Silk Road (SR) route from the Tarim Basin (TB) to the Junggar Basin during ∼420-850 CE provides the opportunity to investigate the relationship between climate change and the spatial evolution of human societies. Here, we use a new high-resolution chironomid-based temperature reconstruction from arid China, combined with hydroclimatic and historical datasets, to assess the possible effects of climate fluctuations on the shift of the ancient SR route. We found that a cooling/drying climate in the TB triggered the SR route shift during ∼420-600 CE. However, a warming/wetting climate during ∼600-850 CE did not inhibit this shift, but instead promoted it, because of the favorable climate-induced geopolitical conflicts between the Tubo Kingdom and the Tang Dynasty in the TB. Our findings reveal two distinct ways in which climate change drove the spatial evolution of human civilization, and they demonstrate the flexibility of societal responses to climate change.

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.

Asynchronous multitrophic level regime shifts show resilience to lake browning.

Lake browning is widespread due to increased supply of dissolved organic carbon under climate warming and nitrogen deposition. However, multitrophic level responses to lake browning are poorly understood. Our study aims to explore such responses across multitrophic levels based on sedimentary records of diatoms, chrysophyte stomatocysts and chironomids in a remote headwater lake in the Three Gorges Reservoir region, central China. Although all biotic proxies were analysed in the same core, the timing of shifts in chironomids (1886 ± 18 CE) preceded that in chrysophyte stomatocysts (∼1914 ± 10 CE) and diatoms (∼1941 ± 6 CE). Shifts in biotic communities were closely linked to rising temperature, δ15N depletion (a proxy for nitrogen deposition), δ13C enrichment (a proxy for littoral moss expansion), as well as biotic interactions, whereas the relative importance of the driving forces varied among the three biotic groups. Our results suggest that the zoobenthos grazing effect might be more important than bottom-up pathways in humic environments. Additionally, the coexistence of benthic, littoral and pelagic algae after the 1950s suggested that mixotrophic chrysophytes could reduce lake browning through heterotrophic processes and sustain the ecological equilibrium between littoral, pelagic and benthic productivity. Therefore, lake browning ecosystem regime shifts require analyses of multiple trophic levels. Our results suggest that heterotrophy may become more important in lake ecosystem carbon cycling with water brownification in Mulong Lake, as well as similar montane lakes.

Environmentally Relevant Concentrations of the Insecticide Fipronil Modulated Molecular Response in Chironomus riparius.

Pesticides employed worldwide for crop protection easily reach aquatic systems, which act as the main reservoirs, and become a risk factor for aquatic fauna. Fipronil is a broad-spectrum insecticide acting on the insect nervous system; however, other effects and systems unrelated to this mechanism could be affected in non-target organisms. Thus, the present study aimed to assess the impact of fipronil on the suborganismal response (gene expression and enzymatic activity) of Chironomus riparius larvae as a model organism in ecotoxicology. To this end, short-term toxicity tests were carried out with fourth-instar larvae exposed to 0.001, 0.01, and 0.1 µg L-1 of fipronil for 24 and 96 h. Messenger RNA levels of 42 genes related to diverse metabolic pathways were analyzed by real-time polymerase chain reaction, complemented with catalase (CAT), glutathione S-transferase (GST), and acetylcholinesterase (AChE) activities. Few effects were observed at 24 h; however, after longer exposure (96 h), genes involved in the endocrine, detoxification, stress, and immune response pathways were altered. Moreover, fipronil at 96 h increased CAT and GST activity at 0.01 µg L-1 and AChE at the highest concentrations. The results demonstrate that even low environmentally relevant fipronil concentrations can modulate the molecular response of several cellular pathways in C. riparius after short-term exposure. These results bring new information about the underlying response of fipronil and its mode of action on a key aquatic invertebrate. Despite no effects on mortality, strong modulation at the suborganismal level emphasizes the advantage of biomarkers as early damage responses and the harmful impact of this pesticide on freshwater organisms. Environ Toxicol Chem 2024;43:405-417. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Microplastics from agricultural mulch films: Biodegradation and ecotoxicity in freshwater systems.

The application of bio-based biodegradable mulch films in agriculture has raised environmental concerns regarding their potential impacts on adjacent freshwater ecosystems. This study investigated the biodegradation of microplastics derived from a bio-based biodegradable mulch (bio-MPs) and its acute and chronic ecotoxicity considering relevant scenarios (up to 200 and 250 mg/kg of sediment, using pristine and/or UV-aged particles), using the fungus Penicillium brevicompactum and the dipteran Chironomus riparius as model organisms, respectively, due to their ecological relevance in freshwater environments. Fourier-transform infrared spectroscopy analysis suggested changes in the fungus's carbohydrate reserves and bio-MP degradation through the appearance of low molecular weight esters throughout a 28 day biodegradation test. In a short-term exposure (48 h), C. riparius larvae exposed to pristine or UV-aged bio-MPs had up to 2 particles in their gut. Exposure to pristine bio-MPs decreased larval aerobic metabolism (<20 %) and increased neurotransmission (>15 %), whereas exposure to UV-aged bio-MPs activated larval aerobic metabolism (>20 %) and increased antioxidant defences (catalase activity by >30 % and glutathione-s-transferase by >20 %) and neurotransmission (>30 %). Longer-term (28-d) exposure to UV-aged bio-MPs did not affect larval survival and growth nor the dipteran's emergence but increased male numbers (>30 %) at higher concentrations. This study suggests that the selected agricultural bio-based mulch film is prone to biodegradation by a naturally occurring fungus. However, there is a potential for endocrine disruption in the case of prolonged exposures to UV-aged microplastics. This study emphasises the importance of further research to elucidate the potential ecological effects of these plastic products, to ensure effective management practices, and to establish new regulations governing their use.

Characterization and expression of heat shock and immune genes in natural populations of Prodiamesa olivacea (Diptera) exposed to thermal stress.

This paper characterizes the heat stress response (HSR) and explores the impact of temperatures on the immune response of larvae from two chironomid species, Prodiamesa olivacea and Chironomus riparius. Genes involved in crucial metabolic pathways were de novo identified in P. olivacea: Hsp27, Hsp60, Hsp70, Hsc70, Cdc37, and HSF for the heat stress response (HSR) and TOLL, PGRP, C-type lectin, and JAK/hopscotch for the immune system response (ISR). Quantitative real-time PCR was used to evaluate the expression levels of the selected genes in short-term treatments (up to 120') at high temperatures (35 °C and 39 °C). Exposing P. olivacea to elevated temperatures resulted in HSR induction with increased expression of specific heat shock genes, suggesting the potential of HSPs as early indicators of acute thermal stress. Surprisingly, we found that heat shock represses multiple immune genes, revealing the antagonist relation between the heat shock response and the innate immune response in P. olivacea. Our results also showed species-dependent gene responses, with more significant effects in P. olivacea, for most of the biomarkers studied, demonstrating a higher sensitivity in this species to environmental stress conditions than that of C. riparius. This work shows a multi-species approach that enables a deeper understanding of the effects of heat stress at the molecular level in aquatic dipterans.

Subfossil Chironomid Assemblages as Indicators of Remedial Efficacy in the Historically Contaminated St. Lawrence River at Cornwall, Ontario.

Long-term data are required to quantify the impacts of historic industrial pollution and subsequent remedial action on the nearshore benthic community in the St. Lawrence River Area of Concern at Cornwall, Ontario. Specifically, high-quality temporal records are needed to understand changes in benthic invertebrate assemblages in response to multiple possible drivers including industrial pollution, environmental heterogeneity, and climate warming. We compare long-term records of subfossil chironomid assemblages and geochemical variables among sediment cores from two Cornwall sites with differing pollution histories and a minimally disturbed downstream reference site. Chironomids were functionally absent from the Cornwall sediment cores when mercury and zinc concentrations were elevated. As metal concentrations decreased in more recent sediment intervals, chironomid abundance and the relative abundance of pollution-sensitive taxa increased. Recently deposited sediment in all three sediment cores display increased relative abundance of warm-water, macrophyte-associated taxa. We conclude that these temporal changes in chironomid assemblages provide evidence for ecological recovery for both of the impacted sites, consistent with the objectives of the current management strategy. These findings advance our understanding of industrial impacts on fluvial chironomid ecology, directly inform local management strategies, and further develop the application of chironomids as bioindicators for contaminated sediments.

Investigating the impact of climate change on the lake ecosystem during the late Holocene using a sedimentary record from the southern Arabian Desert, Yemen.

Lake systems respond physically, chemically, and biologically to hydro-climatic change and variability, and these responses are documented in the sediments. Individual proxies and lacustrine environments may respond to climate variations in a nonlinear way, making it difficult to determine the direction and extent of a climatic shift. Here we investigate the response of lake ecosystem to climatic and environmental changes using a suite of paleo-proxies including ostracods, chironomids, and n-alkanes distribution from paleolake 'Gayal el Bazal (Yemen)'. A previous study from this site has provided a continuous, and high-resolution dataset providing an understanding of precipitation during the last ca 1200 years, particularly during Medieval Climate Anomaly (MCA) and Little Ice Age (LIA). However, the response of the lake ecosystem to these changing hydro-climate conditions, including water-level, salinity, and productivity, remains unknown. The n-alkanes dataset shows that during pluvial interval such as the MCA, the lake experienced an increase in nutrient input resulting in enhanced aquatic productivity. Concurrently, ostracods assemblage displays an increased abundance of swimmer species (like Bradleytriebella lineata and Fabaeomiscandona cf. breuili), suggesting an indirect response between ostracods and climate shifts. The chironomid community during the MCA interval is dominated by taxa belonging to the subfamilies of Chironomini, suggesting a warm, shallow, productive environment with macrophyte vegetation. The LIA interval is marked by increased abundance of higher-chain length n-alkanes, suggesting increased contribution from higher plants. Furthermore, ostracod distribution revealed increased abundance of non-swimmer species like Vestalenula cylindrica., which thrive under saline conditions in the lake. Changes in abundances of Tanytarsini during the LIA interval, which are associated with higher oxygen levels, suggest changes in lake productivity. As a result, the overall patterns in biological indicators reveal that their individual abundance and species/tribe distribution fluctuates in response to changes in the climate and hydrological conditions.

Vibrio cholerae Invasion Dynamics of the Chironomid Host Are Strongly Influenced by Aquatic Cell Density and Can Vary by Strain.

Cholera has been a human scourge since the early 1800s and remains a global public health challenge, caused by the toxigenic strains of the bacterium Vibrio cholerae. In its aquatic reservoirs, V. cholerae has been shown to live in association with various arthropod hosts, including the chironomids, a diverse insect family commonly found in wet and semiwet habitats. The association between V. cholerae and chironomids may shield the bacterium from environmental stressors and amplify its dissemination. However, the interaction dynamics between V. cholerae and chironomids remain largely unknown.  In this study, we developed freshwater microcosms with chironomid larvae to test the effects of cell density and strain on V. cholerae-chironomid interactions. Our results show that chironomid larvae can be exposed to V. cholerae up to a high inoculation dose (109 cells/mL) without observable detrimental effects. Meanwhile, interstrain variability in host invasion, including prevalence, bacterial load, and effects on host survival, was highly cell density-dependent. Microbiome analysis of the chironomid samples by 16S rRNA gene amplicon sequencing revealed a general effect of V. cholerae exposure on microbiome species evenness. Taken together, our results provide novel insights into V. cholerae invasion dynamics of the chironomid larvae with respect to various doses and strains. The findings suggest that aquatic cell density is a crucial driver of V. cholerae invasion success in chironomid larvae and pave the way for future work examining the effects of a broader dose range and environmental variables (e.g., temperature) on V. cholerae-chironomid interactions. IMPORTANCE Vibrio cholerae is the causative agent of cholera, a significant diarrheal disease affecting millions of people worldwide. Increasing evidence suggests that the environmental facets of the V. cholerae life cycle involve symbiotic associations with aquatic arthropods, which may facilitate its environmental persistence and dissemination. However, the dynamics of interactions between V. cholerae and aquatic arthropods remain unexplored. This study capitalized on using freshwater microcosms with chironomid larvae to investigate the effects of bacterial cell density and strain on V. cholerae-chironomid interactions. Our results suggest that aquatic cell density is the primary determinant of V. cholerae invasion success in chironomid larvae, while interstrain variability in invasion outcomes can be observed under specific cell density conditions. We also determined that V. cholerae exposure generally reduces species evenness of the chironomid-associated microbiome. Collectively, these findings provide novel insights into V. cholerae-arthropod interactions using a newly developed experimental host system.

Chironomus calligraphus Goeldi, 1905 (Diptera: Chironomidae) Larvae: Feeding Habits, Growth, and Maturation.

Chironomus calligraphus Goeldi, 1905 has a flexible life cycle with several generations per year, which can be modified by the quality and type of food. To categorize the functional feeding group of the species, food preference was evaluated in larval instars III and IV, through laboratory experiments and gut content analysis. To evaluate the influence of the type of food on the duration of the life cycle growth and maturation, experiments were carried out. Instar III preferred conditioned leaves and animal food, while instar IV preferred algae and fine particulate organic matter. Gut contents of instar IV collected from streams showed increased consumption of fine particulate organic matter than other items. All these observations allowed us to assign the species to the gatherer collector group. The duration of the life cycle varied between the different types of foods, being the conditioned leaves, animal food, and algae the items that caused a greater growth in the larvae and a faster passage to the pupal instar. Our results suggest that the opportunistic feeding behavior of C. calligraphus allows it to use several different foods, but the quality of the foods influences the length of the life cycle and this contributes to its plasticity.

Effects of wildfire ashes on aquatic invertebrates: First molecular approach on Chironomus riparius larvae.

The wildfire magnification in recent years has raised increasing concern about their adverse impacts on the environment. Wildfires are recognized as an important source of diffuse pollution for the nearby aquatic systems being potentially toxic to aquatic life. Albeit previous studies with wildfire runoff/ashes observed effects in aquatic organisms, to date, different severity origins of ashes and their impact at the sub-organismal level on aquatic biota have not been assessed. In this work, the molecular response of Chironomus riparius exposed to wildfire with low (LS) and high (HS) severity ashes from burnt Pine plantations was evaluated by employing an array of 42 genes related to crucial metabolic pathways by Real time-PCR. IV instar larvae were exposed for 72 h to aqueous extract of ashes (12.5 %, 25 %, 50 %, 75 % and 100 %) prepared from LS and HS ashes. Mn, Zn, and Pb were the metals found at highest concentration in both ash extracts, for HS notable Cd, Mn and Cr presence. From the 42 genes studied only 4 were not altered (22 genes modulated their response by LS and 38 genes in the case of HS) showing the opposite response at 100% with downregulated by LS and upregulated by HS. The 12.5 %, 25 %, 100 % HS and 25 % LS were the main modulators, confirmed by the integrative biomarkers response (IBR). Remarkable genotoxicity was generated by ashes even activating the apoptosis response, and endocrine disruption observed could modify the development. Moreover, detoxification and stress response were strongly activated, limiting the organism's future response to external aggressions. The employment of this novelty approach with molecular tools act as early alarm signal preventing greater damages. Overall, wildfire ashes showed to be a significant environmental disruptor to C. riparius even at lower concentration and the short exposure time employed, emphasizing the strong impact of wildfires on aquatic systems.

A paleolimnological context of ecological vulnerability for the freshwater ecosystems of Sable Island National Park Reserve, Canada.

Protected areas require long-term monitoring to understand the influence and extent of ecosystem stress to inform management and conservation decisions. As long-term data are not always available, paleolimnological methods offer a way of extending our knowledge of past environmental conditions necessary to use as context for remediation. Here, we examine four sediment cores and additional surface sediments from 14 ponds located on Sable Island National Park Reserve Canada (SINPR), where long-term ecological changes and vulnerability to disturbance are not well defined. We develop a paleolimnological approach to assessing environmental vulnerability through the use of biological indicators (Diptera: Chironomidae), where shifts in the environment are inferred by shifts in chironomid assemblages over time. Analysis of surface sediments show four distinct assemblage types reflecting four different habitat conditions; primarily represented by the presence of Glyptotendipes, Chironomus, Microtendipes, and Dicrotendipes. Differences in habitat conditions through time based on these results are then compared to biostratigraphic analysis of sediment cores from four of the ponds. We found that two ponds had large shifts in chironomids assemblages that were associated with changes in habitat over time, while two others that were not as exposed to the influence of erosion and influx of sand dunes did not. Our findings established a baseline of historical change in SINPR, broadening the scope of long-term monitoring, which is essential for defining goals for management and conservation of the ecological integrity of Sable Island.

First complete mitogenome of Axarus fungorum (Albu, 1980) from Guizhou Province, China (Diptera, Chironomidae).

Axarus fungorum (Albu, 1980) exhibits certain adaptations to different aquatic environments, appearing as an important evaluation element for freshwater quality monitoring. In this study, complete mitogenome of A. fungorum was provided for the first time to define the systematic and phylogenetic history of this taxon. The whole mitogenome is 15,696 bp long with high A + T content that consists of 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and a noncoding control region. ML analysis showed support for monophyly of Chironominae and close relationship between A. fungorum and Chironomus generic genera.

Exposure of Midge Larvae (Chironomus riparius) to Graphene Oxide Leads to Development Alterations.

Despite the fast-growing use and production of graphene-based nanomaterials (GBMs), data concerning their effects on freshwater benthic macroinvertebrates are scarce. This study aims to investigate the effects of graphene oxide (GO) on the midge Chironomus riparius. Mortality, growth inhibition, development delay and teratogenicity, assessed using mentum deformity analysis, were investigated after a 7-day static exposure of the first instar larvae under controlled conditions. The collected data indicated that the survival rate was not impacted by GO, whereas chronic toxicity following a dose-dependent response occurred. Larval growth was affected, leading to a significant reduction in larval length (from 4.4 to 10.1%) in individuals reaching the fourth instar at any of the tested concentrations (from 0.1 to 100 mg/L). However, exposure to GO is not associated with an increased occurrence of mouthpart deformities or seriousness in larvae. These results highlight the suitability of monitoring the larval development of C. riparius as a sensitive marker of GO toxicity. The potential ecological consequences of larval size decrease need to be considered for a complete characterization of the GO-related environmental risk.

Deciphering the Biological Enigma-Genomic Evolution Underlying Anhydrobiosis in the Phylum Tardigrada and the Chironomid Polypedilum vanderplanki.

Anhydrobiosis, an ametabolic dehydrated state triggered by water loss, is observed in several invertebrate lineages. Anhydrobiotes revive when rehydrated, and seem not to suffer the ultimately lethal cell damage that results from severe loss of water in other organisms. Here, we review the biochemical and genomic evidence that has revealed the protectant molecules, repair systems, and maintenance pathways associated with anhydrobiosis. We then introduce two lineages in which anhydrobiosis has evolved independently: Tardigrada, where anhydrobiosis characterizes many species within the phylum, and the genus Polypedilum, where anhydrobiosis occurs in only two species. Finally, we discuss the complexity of the evolution of anhydrobiosis within invertebrates based on current knowledge, and propose perspectives to enhance the understanding of anhydrobiosis.

On the delay between water temperature and invertebrate community response to warming climate.

We evaluated the effect of global warming on invertebrate communities at high altitudes using data from the Careser system. We procured data on air temperature, which was obtained over 50 years at altitudes above 2600 m a.s.l., and data on water temperature, which was available for approximately 30 years. We sampled thrice in the past 20 years (2001, 2014, 2018) at three sampling sites (CR0-metakryal, CR1-hypokryal, CR2-glacio-rhithral) of the Careser glacier-fed stream and its main non-glacial tributary (CR1bis-krenal). Warmer climates were observed in the last decade compared to the 1980s, with a mean maximum summer air temperature (mTmax) increase of 1.7 °C at 2642 m a.s.l. and 1.8 °C at 2858 m a.s.l. Compared to air temperatures, the rise in water temperature was delayed by approximately 20 years; water mTmax started to increase in 2003, reaching 8.1 °C at 2642 m a.s.l. and 2.4 °C at 2858 m a.s.l in the year 2020. The invertebrate community exhibited a delayed response approximately 13 years from the water warming; there was a sequential increase in the number of taxa, Shannon diversity, and after 17 years, functional diversity. In the kryal sites, taxonomical and functional diversity changed more consistently than in the glacio-rhithral site in the same period, due to the arrival of taxa that were previously absent upstream and bearers of entirely new traits. Progressive taxonomical homogenisation was evident with decreasing glacial influence, mainly between glacio-rhithral and krenal sites. The numbers of Diamesa steinboecki, an insect that was adapted to the cold, declined in summer (water mTmax >6 °C and air mTmax >12 °C). This study highlights the mode and time of response of stream invertebrate communities to global warming in alpine streams and provides guidelines for analysing changes in the stream invertebrate communities of other glacial systems in alpine regions.

First report of the complete mitogenome of Tanypus punctipennis Meigen, 1818 (Diptera, Chironomidae) from Hebei Province, China.

Tanypus punctipennis Meigen, 1818 is an important bioindicator for freshwater ecosystems monitoring. Although COI barcode analyses have been performed on T. punctipennis, the mitogenome of this taxon has not been assembled and analyzed. Here, the complete mitogenome of T. punctipennis was sequenced and analyzed to confirm the systematic and phylogenetic history of this species. The mitogenome is 16,215 bp long with high A + T content, and consists of 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and a noncoding control region. The phylogenomic analysis supports monophyletic Tanypodinae and close relationship between T. punctipennis and Clinotanypus. Our results indicate that mitogenomes showed strong signals in phylogenetic reconstructions at the genus level of Tanypodinae.

Corrections and Additions to Descriptions of Some Species of the Subgenus Orthocladius s. str. (Diptera, Chironomidae, Orthocladiinae).

The larvae of some species of the subgenus Orthocladius s. str. (Diptera, Chironomidae) are here described for the first time with corrections and additions to the descriptions of adult males and pupal exuviae. The identification of larvae is generally not possible without association with pupal exuviae and/or adult males, so the descriptions here are based only on reared material or on pupae with the associated larval exuviae. Usually, Chironomidae larvae can be separated on the basis of morphometric characters, the most discriminant ones are: (1) the ratio between the width of median tooth of mentum (Dm) and the width of the first lateral tooth (Dl) = mental ratio (DmDl), (2) the ratio between the length of the first antennal segment (A1) and the combined length of segments 2-5 (A2-5) = antennal ratio (AR). The shape of mandible, maxilla, and other body parts are almost identical in all the species considered in this study. The larva of Orthocladius (Symposiocladius) lignicola is very characteristic and can be separated by the shape of mentum and the larvae of all the known species of Symposiocladius are characterized by the presence of large Lauterborn organs on antennae and of tufts of setae on abdominal segments. The larvae of Orthocladius (Orthocladius) oblidens and Orthocladius (Orthocladius) rhyacobius can be distinguished from other species basing on their large Dm and to each other by AR. A principal component analysis was carried out using 5 characters: (1) Dm, (2) Dl, (3) length of A1, (4) width of A1 (A1W), (5) combined length of segments 2-5 (A2-5). The most discriminant characters were Dm and A1, confirming that DmDl and AR can be used to separate species at larval stage, but the large superposition of morphometric characters in different species confirms that association with pupal exuviae is in any case needed to identify larvae. In future perspective, the development of reference DNA barcodes from specimens identified by specialists is recommended since possibly the best tool for larvae identification, but association of barcodes with morphotypes is in any case fundamental.