Insect cold-tolerance and lipidome: Membrane lipid composition of two chironomid species differently adapted to cold.

Cell-membrane fluidity is a fundamental parameter in cold resistance. It is regulated by a fine tuning of lipid composition, usually involving a great chemical diversity among head-groups, chain lengths, and degree of unsaturation. To give new insights on Alpine chironomid cold adaptation, we analysed the lipid membrane composition of Diamesa tonsa and Pseudodiamesa branickii, two species known to have different cold-tolerance, stronger in the former. Membrane lipid composition was analysed by NMR and HPLC-MS in larvae under natural (4 °C) and laboratory conditions (30 min at - 4 °C). In both species the major class of membrane lipids were phosphatidylethanolamine (PE), reaching 93% in D. tonsa and 80% in P. branickii, followed by a minor relative amount of phosphatidylcholine (PC). Phospholipids (PL) acyl chains were highly unsaturated given the presence of a relevant amount of polyunsaturated fatty acid (PUFA), among which a high proportion of ω-3 chains. This study demonstrated that these species have a similar lipidome (e.g. relevant amount of PUFA and predominance of PE), but with relevant differences on which to base different membrane fluidity: (i) a higher unsaturation index and chain length of both PE and PC and a higher ratio PE/PC ratio in D. tonsa than in P. branickii; (ii) the absence of modifications in the lipid composition in D. tonsa under sub-zero temperature. These differences might support the different cold-tolerance of the two species. In fact, we suggest that the high PE/PC ratio and the low sterols content (as in D. tonsa) could be involved in the formation of highly deformable membranes increasing their capacity to survive freezing. Interestingly, LC-MS analysis of D. tonsa lipidome revealed a new class of lipids that we named 'PpC', absent in P. branickii, that is worth investigating.

Glacier shrinkage driving global changes in downstream systems.

Glaciers cover ∼10% of the Earth's land surface, but they are shrinking rapidly across most parts of the world, leading to cascading impacts on downstream systems. Glaciers impart unique footprints on river flow at times when other water sources are low. Changes in river hydrology and morphology caused by climate-induced glacier loss are projected to be the greatest of any hydrological system, with major implications for riverine and near-shore marine environments. Here, we synthesize current evidence of how glacier shrinkage will alter hydrological regimes, sediment transport, and biogeochemical and contaminant fluxes from rivers to oceans. This will profoundly influence the natural environment, including many facets of biodiversity, and the ecosystem services that glacier-fed rivers provide to humans, particularly provision of water for agriculture, hydropower, and consumption. We conclude that human society must plan adaptation and mitigation measures for the full breadth of impacts in all affected regions caused by glacier shrinkage.

VectAbundance: a spatio-temporal database of Aedes mosquitoes observations.

Modelling approaches play a crucial role in supporting local public health agencies by estimating and forecasting vector abundance and seasonality. However, the reliability of these models is contingent on the availability of standardized, high-quality data. Addressing this need, our study focuses on collecting and harmonizing egg count observations of the mosquito Aedes albopictus, obtained through ovitraps in monitoring and surveillance efforts across Albania, France, Italy, and Switzerland from 2010 to 2022. We processed the raw observations to obtain a continuous time series of ovitraps observations allowing for an extensive geographical and temporal coverage of Ae. albopictus population dynamics. The resulting post-processed observations are stored in the open-access database VectAbundance.This initiative addresses the critical need for accessible, high-quality data, enhancing the reliability of modelling efforts and bolstering public health preparedness.

Thermal stress induces HSP70 proteins synthesis in larvae of the cold stream non-biting midge Diamesa cinerella Meigen.

Laboratory experiments on the cold stenothermal midge Diamesa cinerella (Diptera, Chironomidae) were performed to study the relationship between increasing temperature and heat shock proteins (HSP70) expression at translational level (Western blotting). Thermotolerance of IV instar larvae collected in nature at 1.5-4.3°C during seasons was analyzed through short-term (1 h at ten different temperatures from 26°C to 35°C) and long-term (1-14 h at 26°C and 1-4 h at 32°C) heat shocks. A high thermotolerance was detected (LT50=30.9-32.8°C and LT100=34.0-37.8°C). However, survival decreased consistently with increasing exposure time, especially at higher temperature (LTime50=7.64 h at 26°C and LTime50=1.73 h at 32°C). The relationship between such heat resistance and HSP70 expression appeared evident because a relationship between HSP70 level and larval survival rate was generally found. A heat shock response (HSR) was consistent only in the summer larvae. The absence of HSR in the other populations coupled with even higher amounts of HSP70 than in summer, led us to hypothesize that other macromolecules and other adaptive mechanisms, apart from biochemical ones, are involved in the response of D. cinerella larvae to high temperature. Altogether these results stressed how in this midge the HSP70 protein family confers resistance against cold, being detected under natural conditions in control larvae collected in all seasons, but also against warm under experimental heat shocks. These results give new insights into possible responses to climate changes in freshwater insects within the context of global warming.

Metal enrichment in ice-melt water and uptake by chironomids as possible legacy of World War One in the Italian Alps.

Relics of World War One (WW1) were buried in alpine glaciers around 100 years ago. Today, these are emerging from the ice due to widespread glacier retreat, and are in direct contact with glacial meltwater-fed streams. To address a possible emergent contamination, we quantified major and trace elements (M-TEs) by mass spectrometry in water and larvae of Diamesa zernyi from three glacial streams fed by glaciers differently impacted by the Italian Austro-Hungarian war, in the Adamello-Presanella mountain range (Italian Alps): Lares and Presena, the two main battlefields, and Amola, 8 km from the front. M-TEs in stream water were interpreted using the crustal enrichment factor (EFc) while larval uptake was quantified by adopting the bioaccumulation factor (BAF). Despite low M-TEs concentrations in the water, in a range between 1 ng L-1 (Ag, Ta) and 1-2 mg L-1 (Al, Fe, Mg), low to moderate enrichments (10 ≥ EFc≥ 6) were observed for Sb and U in Presena and for Ag, As, Bi, Cd, Li, Mo, Pb, Sb and U in Lares. In addition, M-TE mass concentrations in larvae were up to ninety thousand times higher than in water, from 20 to 50 ng g-1 dry weight (d.w.; for Bi, Sb, Ta, Tl) to 1-4 mg g-1 d.w. (for Al, Fe, Na, and Mg). Larvae from Lares accumulated the largest amount of metals and metalloids, including those mostly used in the manufacture of artillery shells (As, Cu, Ni, Pb, Sb; BAFs from 375 to about 11,500). This was expected as most of the WW1 battles in this mountain range were fought on the Lares glacier, where the greatest number of war relics are emerging. These results provide preliminary evidence of water contamination and bioaccumulation of metals and metalloids by glacial fauna as a possible legacy of WW1 in the Alps.

Glacier foreland insect uptake synthetic compounds: an emerging environmental concern.

Pesticides, synthetic fragrances and polycyclic aromatic hydrocarbons contaminated two glacier-fed streams (Amola, Mandrone) and one spring (Grostè) in the Italian Alps. Ten compounds (chlorpyrifos (CPY), chlorpyrifos-methyl (CPY-m), galaxolide (HHCB), tonalide (AHTN), fluorene (Flu), phenanthrene (Phen), anthracene (Ant), fluoranthene (Fl), pyrene (Pyr), benzo[a]anthracene (BaA)) accumulated in aquatic larvae of chironomids (Diamesa steinboecki, D. latitarsis, D. bertrami, D. tonsa, D. zernyi, Pseudokiefferiella parva, Orthocladiinae) and tipulids. Their tissue concentrations (detected by gas chromatography coupled with mass spectrometry) ranged from 1.1 ± 0.1 ng/g d.w. (= dry weight) (CPY-m in D. tonsa from Amola) to 68.0 ± 9.1 ng/g d.w. (Pyr in D. steinboecki from Mandrone). HHCB, AHTN, and CPY, with one exception, were accumulated by all aquatic insects. Six compounds (CPY, CPY-m, HHCB, AHTN, Fl, Pyr) also contaminated carabids (Nebria germarii, N. castanea, N. jockischii) predating adults of merolimnic insects. Their tissue concentrations ranged from 1.1 ± 0.3 ng/g d.w. (CPY-m in N. germarii from Mandrone) to 84.6 ± 0.3 ng/g d.w. (HHCB in N. castanea from Grostè). HHCB and AHTN were accumulated by all Nebria species. Intersite and interspecies differences were observed, which might be attributed to different environmental contamination levels. There was a stronger similarity between species from the same site than among the same species from different sites, suggesting that uptake is not species specific. At all sites, the concentration of xenobiotics was higher in larvae than in water and comparable or higher in carabids than in larvae from the same site, suggesting trophic transfer by emerging aquatic insects to their riparian predators.

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.

Synorthocladiusfedericoi sp. nov., a new species occurring in the middle basin of the Adige River, northern Italy (Diptera, Chironomidae, Orthocladiinae).

An adult male Synorthocladius was collected in the middle basin of the Adige River in the city of Verona, northern Italy. A combination of atypical characters for the genus signalled a new species. Synorthocladiusfedericoi sp. nov. is here diagnosed and described. The new species is known only from its type locality and is presumed to be a local biogeographical representative of the Italian Pre-Alps. An emended generic diagnosis, a key to known Synorthocladius from Europe and comments on the taxonomic position of the new species are given.

Can DNA barcoding be used to identify closely related Clunio Haliday, 1855 species (Diptera: Chironomidae, Orthocladiinae)?

DNA barcoding based on a fragment of mitochondrial Cytochrome C Oxidase subunit 1 gene (COI) was applied to the two chironomids Clunio balticus Heimbach (690 base pairs) and C. ponticus Michailova (691 base pairs). The two species differed by one deletion in the nucleotide sequence Adenine. However, the 658-nucleotide long sequences of DNA from the mitochondrial Cytochrome C Oxidase subunit 1 gene (COI) of C. balticus and C. ponticus were identical upon comparison. Further, they compared with homologous sequences for C. marinus Holiday and C. tsushimensis Tokunaga from the Barcode of Life (BOLD) database and the results plotted as a weighted graph, where C. tsushimensis, C. marinus and C. balticus C. ponticus formed three almost equidistant groups. From this, we established that the genetic distance between the respective COI sequences of C. balticus and C. ponticus is minimal, indicating a close relationship between the species indicative of recent common origin. However, the comparative analysis between C. tsushimensis, C. marinus, C. balticus and C. ponticus showed a wider divergence in their respective nucleotide sequences. Overall, our results emphasized that the COI region does not work well as a DNA barcode to identify species within the Clunio genus. Either longer sequences or a multifaceted methodological approach, including morphology, cytogenetic and ecology is needed to distinguish some members of Clunio genus.

Molecular biomarkers as tool for early warning by chlorpyrifos exposure on Alpine chironomids.

Pesticides used in agriculture can be transported at a medium-high distance due to the drift effect, reaching even remote areas as mountain regions, glaciers, and snow cover. With the melting process, pesticides enter freshwater glacier ecosystems, becoming a threat to wildlife fauna, mainly dominated by Diptera Chironomidae. Chlorpyrifos (CPF), as one of the most commonly used pesticides in alpine vineyards and apple orchards, is frequently detected in icemelt waters. We selected as target species, larvae of the cold stenothermal chironomid Diamesa zernyi, collected in two glacier-fed streams (Presena and Amola) in the Italian Alps. Firstly, a de novo transcriptome was obtained, and secondly, a gene array was designed to study the molecular response of a wild population of D. zernyi exposed to three sub-lethal CPF concentrations corresponding to 1/100 LC10 (0.011 µg/L), 1/10 LC10 (0.11 µg/L), and LC10 (1.1 µg/L), for 24 h. The sub-organismal response was evaluated by Real-Time Polymerase Chain Reaction (RT-PCR), employing 40 genes related to essential metabolic routes as future candidates for biomarkers in wildlife chironomids. After 24 h, the endocrine system (E75, E93, EcR, and Met), detoxification response (GSTO3, GSTS1), and stress response (hsp75, hsp83, HYOU1) were altered. CPF seems to act as an endocrine disruptor and could lead to defective larval development, disrupted cellular homeostasis through heat shock proteins (HSPs) alteration (defective protein folding and mitochondrial functions), as well as oxidative damage (confirmed by increased GST expression). For the first time, molecular studies detected early alarm signals in wildlife in glacier environments. Our findings confirm the high environmental risk of CPF affecting aquatic insect metabolism and raise the level of concern about this pesticide in high altitude water bodies, generally considered pristine. Furthermore, this study emphasizes the incipient need to use non-model organisms for the evaluation of natural ecosystems. We also highlight the demand for research into new molecular biomarkers, and the importance of including molecular approaches in toxicology evaluations to detect the early adverse effects of pollutants.

A Novel Allele Specific Polymerase Chain Reaction (AS-PCR) Assay to Detect the V1016G Knockdown Resistance Mutation Confirms Its Widespread Presence in Aedes albopictus Populations from Italy.

Polymerase chain reaction (PCR)-based genotyping of mutations in the voltage-sensitive sodium channel (vssc) associated with resistance to pyrethroid insecticides is widely used and represents a potential early warning and monitoring system for insecticide resistance arising in mosquito populations, which are vectors of different human pathogens. In the secondary vector Aedes albopictus-an Asian species that has invaded and colonized the whole world, including temperate regions-sequencing of domain II of the vssc gene is still needed to detect the V1016G mutation associated with pyrethroid resistance. In this study we developed and tested a novel allele-specific PCR (AS-PCR) assay to genotype the V1016G mutation in this species and applied it to the analysis of wild populations from Italy. The results confirm the high accuracy of the novel AS-PCR and highlight frequencies of the V1016G allele as >5% in most sampling sites, with peaks of 20-45% in coastal touristic sites where pyrethroid treatments are extensively implemented, mostly for mosquito nuisance reduction. The high frequency of this mutation observed in Italian Ae. albopictus populations should serve as a warning bell, advocating for increased monitoring and management of a phenomenon which risks neutralizing the only weapon today available to counteract (risks of) arbovirus outbreaks.

A long noncoding RNA acts as a post-transcriptional regulator of heat shock protein (HSP70) synthesis in the cold hardy Diamesa tonsa under heat shock.

Cold stenothermal insects living in glacier-fed streams are stressed by temperature variations resulting from glacial retreat during global warming. The molecular aspects of insect response to environmental stresses remain largely unexplored. The aim of this study was to expand our knowledge of how a cold stenothermal organism controls gene expression at the transcriptional, translational, and protein level under warming conditions. Using the chironomid Diamesa tonsa as target species and a combination of RACE, qPCR, polysomal profiling, western blotting, and bioinformatics techniques, we discovered a new molecular pathway leading to previously overlooked adaptive strategies to stress. We obtained and characterized the complete cDNA sequences of three heat shock inducible 70 (hsp70) and two members of heat-shock cognate 70 (hsc70). Strikingly, we showed that a novel pseudo-hsp70 gene encoding a putative long noncoding RNA (lncRNA) which is transcribed during thermal stress, acting as a ribosome sponge to provide post-transcriptional control of HSP70 protein levels. The expression of the pseudo-hsp70 gene and its function suggest the existence of a new and unexpected mechanism to cope with thermal stress: lowering the pace of protein production to save energy and optimize resources for recovery.

Behavioural and biochemical alterations by chlorpyrifos in aquatic insects: an emerging environmental concern for pristine Alpine habitats.

This study aimed to assess how different concentrations of the insecticide chlorpyrifos (1.1, 5.24, 11, 52.4, 110, 262, 524 and 1100 ng L-1) affect the swimming behaviour of Diamesa zernyi larvae following exposure. A video tracking system was employed to analyse two swimming traits (total distance moved and average speed) of the larvae simultaneously after 3 days of exposure to the pesticide at 2 °C. The behavioural results were also interpreted according to biochemical responses to oxidative stress (OS) induced by chlorpyrifos, based on malondialdehyde (MDA) and protein carbonyl (PCC) content. Both distance and speed significantly decreased after 72 h of exposure to chlorpyrifos concentrations of ≥ 110 ng L-1, under which significant OS was detected as lipid peroxidation (level of MDA) and protein carbonylation (level of carbonyl). Analysis of altered swimming behaviour, along with MDA and carbonyl content, indicated that ≥ 110 ng L-1 contamination levels of the insecticide cause the organism to reallocate energy normally used for locomotor activity to repair cell damage, which might explain the strong impairment to locomotor performance. Locomotor performance is an ecologically relevant trait for elucidating the population dynamics of key species, with disturbance to this trait having long-term negative impacts on population and community structure. Therefore, chlorpyrifos insecticides represent a serious ecological risk for mountain aquatic species based on the detrimental effects observed in the current study, as the tested concentrations were those at which the insecticide is found in many Alpine rivers of Italy.

Dragonfly biodiversity 90 years ago in an Alpine region: The Odonata historical collection of the MUSE (Trento, Italy).

BACKGROUND: Historical collections of natural science museums play a fundamental role in documenting environmental changes and patterns of biodiversity transformation. This considered, they should have a pivotal role to plan conservation and management actions.The MUSE - Science Museum of Trento is an Italian regional museum preserving about 5.5 million items (organised in 297 collections). About one million of them are invertebrates, 70% of which are of local origin, gathered in the collection "Miscellanea Invertebrati". Odonata account for a minor part of this collection; however, most of them are of local or regional relevance. A complete catalogue of this collection does not exist to date. NEW INFORMATION: The collection was studied in 2017-2018 and this contribution aims to present the Catalogue of the historic collection of Odonata of the MUSE - Museo delle Scienze of Trento (Italy).In all, 836 specimens of adult dragonflies and damselflies are found in the collection referring to an overall 56 species. The collection covers a period between 1924 and 1957 and refer to 74 defined localities, all located in northern Italy (most of them in Trentino - Alto Adige Region).The samples conserved in the collection are, for several species, the only indisputable confirmation of their former occurrence in that region.

Respiration rate and oxy-regulatory capacity in cold stenothermal chironomids.

The effects of temperature and oxygen saturation on the respiration rate of two cold stenothermal chironomids, Diamesa insignipes and Pseudodiamesa branickii were investigated. Fourth instar larvae were collected in winter in a glacio-rhithral stream (1300 m a.s.l., Alps, NE-Italy) and their respiration rate was measured with a Clark's electrode in the range 0-14 degrees C. The respiration rate was significantly higher in D. insignipes than in P. branickii at low temperatures (

Glacial influence and stream macroinvertebrate biodiversity under climate change: Lessons from the Southern Alps.

The aim of this work was to highlight the main ecological predictors driving invertebrate distribution in eight glacier-fed streams in the Southern Alps. Thirty-five sites belonging to four stream types were sampled monthly during the ablation season of one, two or three years between 1996 and 2014. Taxa from glacial (kryal and glacio-rhithral) and non-glacial (kreno-rhithral and lake outlet) sites were separated by canonical correspondence analysis (CCA) along a glacial influence gradient and a hydrological-altitudinal gradient. High glacial influence was associated mainly with low maximum water temperature (Tmax), high Glacial Index (calculated as a function of glacier area and distance from the glacier), and the abundance of Diamesa species (D. steinboecki, D. goetghebueri, D. zernyi, and D. latitarsis). Change-point analysis and Threshold Indicator Taxa Analysis confirmed the CCA results in identifying these Diamesa species as the taxa with the strongest preference for high percent glacier cover in the catchment (change point~30%) and low Tmax (change point~6°C). Temporal changes in community structure were highlighted in seven sites fed by glaciers under different retreat rates. Where the rate was faster and the remaining glacier smaller (≪1km2), the most cold-stenothermal kryal inhabitant, D. steinboecki, almost disappeared or survived only as brachypterous populations, whereas other Diamesinae (Pseudokiefferiella parva), Orthocladiinae (e.g. Eukiefferiella, Orthocladius), Limoniidae, Baetidae, Nemouridae, and non-insect taxa (e.g. Oligochaeta, Hydracarina) became more abundant. Upstream migration was observed in Diamesa spp. which conquered new stream reaches left free by the retreating glacier, and euriecious taxa which colonized reaches with ameliorated environmental conditions, no longer the exclusive habitat of Diamesa spp. Co-occurrence of stochastic and deterministic assembly processes seem to drive spatio-temporal changes in these invertebrate communities. Long-term ecological studies on the adaptive biology of kryal species will be useful to predict the fate of Alpine biodiversity.

Comparison of the behavioural effects of pharmaceuticals and pesticides on Diamesa zernyi larvae (Chironomidae).

Several studies have indicated the presence of contaminants in Alpine aquatic ecosystems. Even if measured concentrations are far below those that cause acute effects, continuous exposure to sub-lethal concentrations may have detrimental effects on the aquatic species present in these remote environments. This may lead to a cascade of indirect effects at higher levels of the ecological hierarchy (i.e., the community). To improve the determination of ecologically relevant risk endpoints, behavioural alterations in organisms due to pollutants are increasingly studied in ecotoxicology. In fact, behaviour links physiological function with ecological processes, and can be very sensitive to environmental stimuli and chemical exposure. This is the first study on behavioural alteration in a wild population of an Alpine species. In the present study, a video tracking system was standardized and subsequently used to identify contaminant-induced behavioural alterations in Diamesa zernyi larvae (Diptera, Chironomidae). Diamesa zernyi larvae, collected in an Italian Alpine stream (Rio Presena, Trentino Region), were acclimated for 24 h and successively exposed to several aquatic contaminants (pesticides: chlorpyrifos, metolachlor, boscalid, captan; pharmaceuticals: ibuprofen, furosemide, trimethoprim) at concentrations corresponding to their Lowest Observed Effect Concentration (LOEC). After 24, 48, 72, and 96 h of exposure, changes in the distance moved, the average speed, and the frequency of body bends were taken to reflect contaminant- and time-dependent effects on larval behaviour. In general, metolachlor, captan, and trimethoprim tended to reduce all the endpoints under consideration, whereas chlorpyrifos, boscalid, ibuprofen, and furosemide seemed to increase the distances moved by the larvae. This could be related to the different mechanisms of action of the investigated chemicals. Independently of the contaminant, after 72 h a general slowing down of all the behavioural activities occurred. Finally, we propose a behavioural stress indicator to compare the overall behavioural effects induced by the various contaminants.

Functional diversity and community assembly of river invertebrates show globally consistent responses to decreasing glacier cover.

Global change threatens invertebrate biodiversity and its central role in numerous ecosystem functions and services. Functional trait analyses have been advocated to uncover global mechanisms behind biodiversity responses to environmental change, but the application of this approach for invertebrates is underdeveloped relative to other organism groups. From an evaluation of 363 records comprising >1.23 million invertebrates collected from rivers across nine biogeographic regions on three continents, consistent responses of community trait composition and diversity to replicated gradients of reduced glacier cover are demonstrated. After accounting for a systematic regional effect of latitude, the processes shaping river invertebrate functional diversity are globally consistent. Analyses nested within individual regions identified an increase in functional diversity as glacier cover decreases. Community assembly models demonstrated that dispersal limitation was the dominant process underlying these patterns, although environmental filtering was also evident in highly glacierized basins. These findings indicate that predictable mechanisms govern river invertebrate community responses to decreasing glacier cover globally.

Thermal survival limits of young and mature larvae of a cold stenothermal chironomid from the Alps (Diamesinae: Pseudodiamesa branickii [Nowicki, 1873]).

The threats posed by climate change make it important to expand knowledge concerning cold and heat tolerance in stenothermal species from habitats potentially threatened by temperature changes. Thermal limits and basal metabolism variations were investigated in Pseudodiamesa branickii (Diptera: Chironomidae) under thermal stress between -20 and 37 °C. Supercooling point (SCP), lower (LLTs) and upper lethal temperatures (ULTs), and oxygen consumption rate were measured in overwintering young (1st and 2nd instar) and mature (3rd and 4th instar) larvae from an Alpine glacier-fed stream. Both young and mature larvae were freezing tolerant (SCPs = -7.1 °C and -6.4 °C, respectively; LLT100 -20 °C) and thermotolerant (ULT50 = 31.7 ± 0.4, 32.5 ± 0.3, respectively). However, ontogenetic differences in acute tolerance were observed. The LLT50 calculated for the young larvae (= -7.4 °C) was almost equal to their SCP (= -7.1 °C) and the overlapping of the proportion of mortality curve with the CPIF curve highlighted that the young larvae are borderline between freezing tolerance and freezing avoidance. Furthermore, a lower ULT100 in the young larvae (of ca. 1 °C), suggests that they are less thermotolerant than mature larvae. Finally, young larvae exhibit a higher oxygen consumption rate (mgO2 /gAFDM/h) at any temperature tested and are overall less resistant to oxygen depletion compared to mature larvae at ≥10 °C. These findings suggest that mature larvae enter into a dormant state by lowering their basal metabolism until environmental conditions improve in order to save energy for life cycle completion during stressful conditions.