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Pauls S.U.,Biodiversity and Climate Research Center | Alp M.,Leibniz Institute of Freshwater Ecology and Inland Fisheries | Alp M.,CNRS Biological Evolution and Diversity Laboratory | Balint M.,Biodiversity and Climate Research Center | And 14 more authors.
Freshwater Biology | Year: 2014

Summary: Molecular genetic techniques have been used in freshwater biology for more than 30 years. Early work focussed on studies of population structure, systematics and taxonomy. More recently, the range of studies has broadened to include ecology and adaptation. Advances in analytical methods and in technology (e.g. next-generation sequencing) and decreasing costs of data production ensure that the field will continue to develop and broaden in scope. At least three factors make the application of molecular techniques to freshwater biology exciting. First, the highly variable nature of many aquatic habitats makes them excellent models for the study of environmental change on ecological and evolutionary time scales. Second, the mature state of the field of freshwater biology provides an extensive foundation of ecological knowledge of freshwater organisms and their distinct adaptations. Third, the methodological advances allow researchers to focus more on merging molecular and ecological research and less on designing studies around technical limitations. We identified eight research areas in freshwater biology in which the integration of molecular and ecological approaches provides exceptional opportunities. The list is not exhaustive, but considers a broad range of topics and spans the continuum from basic to applied research. The areas identified use a combination of natural, experimental and in silico approaches. With advancing molecular techniques, freshwater biology is in an unusually strong position to link the genetic basis and ecological importance of adaptations across a wide range of taxa, ecosystems and spatiotemporal scales. Our aim was to identify opportunities for the integration of molecular and ecological approaches, to motivate greater collaboration and crossover, and to promote exploitation of the synergies of bridging ecological and evolutionary freshwater research. © 2014 John Wiley & Sons Ltd.


Bernabol P.,University of Modena and Reggio Emilia | Bernabol P.,University of Trento | Rebecchi L.,University of Modena and Reggio Emilia | Jousson O.,University of Trento | And 2 more authors.
Cell Stress and Chaperones | Year: 2011

To better understand the physiological capability of cold-stenothermal organisms to survive high-temperature stress, we analyzed the thermotolerance limits and the expression level of hsp70 genes under temperature stress in the alpine midge Pseudodiamesa branickii (Diptera Chironomidae). A lethal temperature (LT100) of 36°C and a lethal temperature 50% (LT50) of 32.2°C were found for the coldstenothermal larvae after short-term shocks (1 h). Additional experiments revealed that the duration of the exposure negatively influenced survival, whereas a prior exposure to a less severe high temperature generated an increase in survival. To investigate the molecular basis of this high thermotolerance, the expression of the hsp70 gene family was surveyed via semi-quantitative reverse transcription-polymerase chain reaction analysis in treated larvae. The constitutive (hsc70) and inducible (hsp70) forms were both analyzed. Larvae of P. branickii showed a significant up-regulation of inducible hsp70 gene with increasing temperatures and an over-expression of both hsp70 and hsc70 by increasing the time of exposure. Different from that was shown in many cold-stenothermal Antarctic organisms, P. branickii was able to activate hsp70 genes transcription (equal to heat shock response) in response to thermal stress. Finally, the unclear relationship between hsp70 expression and survival led us to surmise that genes other than hsp70 and other processes apart from the biochemical processes might generate the high thermaltolerance of P. branickii larvae. These results and future high-throughput studies at both the transcriptome and proteome level will improve our ability to predict the future geographic distribution of this species within the context of global warming. © 2010 Cell Stress Society International.


PubMed | Section of Invertebrate Zoology and Hydrobiology, CNR Institute of Neuroscience, Center for Integrative Biology and Fondazione Bruno Kessler
Type: | Journal: Journal of insect physiology | Year: 2015

In stressed organisms, strategic proteins are selectively translated even if the global process of protein synthesis is compromised. The determination of protein concentrations in tissues of non-model organisms (thus with limited genomic information) is challenging due to the absence of specific antibodies. Moreover, estimating protein levels quantifying transcriptional responses may be misleading, because translational control mechanisms uncouple protein and mRNAs abundances. Translational control is increasingly recognized as a hub where regulation of gene expression converges to shape proteomes, but it is almost completely overlooked in molecular ecology studies. An interesting approach to study translation and its control mechanisms is the analysis of variations of gene-specific translational efficiencies by quantifying mRNAs associated to ribosomes. In this paper, we propose a robust and streamlined pipeline for purifying ribosome-associated mRNAs and calculating global and gene-specific translation efficiencies from non-model insects species. This method might found applications in molecular ecology to study responses to environmental stressors in non-model organisms.


Lencioni V.,Section of Invertebrate Zoology and Hydrobiology | Grazioli V.,Section of Invertebrate Zoology and Hydrobiology | Grazioli V.,University of Milan | Rossaro B.,University of Milan | And 2 more authors.
Science of the Total Environment | Year: 2016

Copper (Cu) and azadirachtin (AZA-A + B) are pesticides allowed in organic agriculture whose environmental risk and toxicity for aquatic wildlife is only partially known. Reverse Transcription Polymerase Chain Reaction was used to assess the molecular effect of acute and short-term exposure (3, 24 h) of Cu (0.01, 0.05, 1, 10, 25 mg l-1) and AZA-A + B (0.2, 0.3, 0.4, 0.5, 1 mg l-1) on the expression of five candidate genes (hsp70, hsc70, hsp40, hsp10 and cyP450) in a non-target species, Chironomus riparius. Fourth-instar larvae were collected from a mountain stream polluted by agricultural land run-off. All genes were responsive to both pesticides but each gene had a specific response to the different experimental concentrations and exposure times. A few similarities in transcriptional profiling were observed, such as a linear concentration-dependent response of hsp70 after 24 h of exposure (at ≥1 mg l-1 of Cu and ≥0.2 mg l-1 of AZA-A + B) and an up-regulation regardless of the concentration of hsc70 after 24 h of exposure (at ≥0 mg l-1 of Cu and ≥0.2 mg l-1 of AZA-A + B and the up-regulation of hsp70 after 3 h of exposure at ~LC50 (Cu-LC50 = 26.1 ± 2.5 mg l-1, AZA-A + B-LC50 = 1.1 ± 0.2 mg l-1). According to the results, hsp40, hsp10 and cyP450 may be defined as pesticide-dependent (i.e., hsp40 and hsp10 seemed to responded mainly to AZA-A + B and cyP450 to Cu), while hsc70 as time-dependent regardless of the pesticide (i.e., hsc70 responded only after 24 h of treatment with Cu and AZA-A + B). This study gives new insights on the potential role of the C. riparius's hsps and cyP450 genes as sensitive biomarkers for freshwater monitoring. © 2016 Elsevier B.V.


Grazioli V.,University of Milan | Rossaro B.,University of Milan | Parenti P.,University of Milan Bicocca | Giacchini R.,University of Milan Bicocca | Lencioni V.,Section of Invertebrate Zoology and Hydrobiology
Journal of Limnology | Year: 2016

The metabolic effects of low oxygen content on alcohol-dehydrogenase (ADH) activity and hemoglobin (Hb) concentration were investigated in IV-instar larvae of Chironomus riparius (Diptera: Chironomidae) from an Italian stream. Two series of short-term (48 h) experiments were carried out: exposure to (1) progressive hypoxia (95 to 5% of oxygen saturation) and (2) anoxia (at <5% of oxygen saturation). In (1), Hb amount increased with increasing oxygen depletion up to a critical value of oxygenation (about 70% of oxygen saturation). Below this percentage, the Hb amount declined to values comparable with those present in the control. The respiration rate (R) remained almost constant at oxygen saturation >50% and decreased significantly only after 48 h of treatment (= <5% of oxygen saturation) reaching values <100 μmolO2 gAFDW–1 h–1. ADH activity showed two phases of growth, within the first 14 h and over 18 h of exposure. Overall, we inferred that i) Hb might function as short-term oxygen storage, enabling animals to delay the onset of anaerobiosis; and ii) alcoholic fermentation co-occurs for a short time with aerobic respiration, becoming the prevalent metabolic pathway below 5% of oxygen saturation (<1 mg L–1). These considerations were supported also by results from anoxia exposure (2). In such condition, larvae were visibly stressed, becoming immobile after few minutes of incubation, and ADH reached higher values than in the hypoxia treatment (2.03±0.15 UADH mg prot–1). Overall, this study showed a shift from aerobic to anaerobic activity in C. riparius larvae exposed to poorly oxygenated water with an associated alteration of ADH activity and the Hb amount. Such metabolites might be valid candidate biomarkers for the environmental monitoring of running waters. © 2016, Page Press Publications. All rights reserved.


PubMed | Section of Invertebrate Zoology and Hydrobiology, CNR Institute of Biophysics and University of Milan
Type: | Journal: The Science of the total environment | Year: 2016

Copper (Cu) and azadirachtin (AZA-A+B) are pesticides allowed in organic agriculture whose environmental risk and toxicity for aquatic wildlife is only partially known. Reverse Transcription Polymerase Chain Reaction was used to assess the molecular effect of acute and short-term exposure (3, 24h) of Cu (0.01, 0.05, 1, 10, 25mgl(-1)) and AZA-A+B (0.2, 0.3, 0.4, 0.5, 1mgl(-1)) on the expression of five candidate genes (hsp70, hsc70, hsp40, hsp10 and cyP450) in a non-target species, Chironomus riparius. Fourth-instar larvae were collected from a mountain stream polluted by agricultural land run-off. All genes were responsive to both pesticides but each gene had a specific response to the different experimental concentrations and exposure times. A few similarities in transcriptional profiling were observed, such as a linear concentration-dependent response of hsp70 after 24h of exposure (at 1mgl(-1) of Cu and 0.2mgl(-1) of AZA-A+B) and an up-regulation regardless of the concentration of hsc70 after 24h of exposure (at 0mgl(-1) of Cu and 0.2mgl(-1) of AZA-A+B and the up-regulation of hsp70 after 3h of exposure at ~LC50 (Cu-LC50=26.12.5mgl(-1), AZA-A+B-LC50=1.10.2mgl(-1)). According to the results, hsp40, hsp10 and cyP450 may be defined as pesticide-dependent (i.e., hsp40 and hsp10 seemed to responded mainly to AZA-A+B and cyP450 to Cu), while hsc70 as time-dependent regardless of the pesticide (i.e., hsc70 responded only after 24h of treatment with Cu and AZA-A+B). This study gives new insights on the potential role of the C. ripariuss hsps and cyP450 genes as sensitive biomarkers for freshwater monitoring.


Lencioni V.,Section of Invertebrate Zoology and Hydrobiology | Bernabo P.,Section of Invertebrate Zoology and Hydrobiology | Bernabo P.,University of Trento | Latella L.,Section of Zoology
Journal of Thermal Biology | Year: 2010

Supercooling points (SCPs), lower lethal temperatures (LLTs), and the effect of short-term exposures (1min) to low temperatures were examined in the adults of two stenothermal leptodirin species, Neobathyscia mancinii and Neobathyscia pasai (Coleoptera, Cholevidae). Specimens were collected from two caves in the Venetian Prealps (NE-Italy). Inter-species comparison highlighted lower values of SCP in N. mancinii (-7.1±0.9°C) than in N. pasai (-6.4±0.3°C), with no significant intersexual differences in both species. N. pasai (LLT50±SE=-16.96±2.30°C; LLT100=-25.41°C) tolerated short exposures to subzero temperatures better than N. mancinii (LLT50±SE=-4.89±1.08°C; LLT100=-11.72°C). According to the mortality and cumulative proportion of individual freezing curves (CPIF), SCPs and LLT100, N. pasai may be defined as "strongly freeze tolerant", N. mancinii as "moderately freezing tolerant". Overall, these results may justify the different in-cave habitat selection showed by the two species (N. pasai was abundant close to the entrance where the temperature is variable whereas N. mancinii was confined to the internal part of the cave where the temperature is constant throughout the year), and suggest hypotheses on the effects of such habitat selection on freeze tolerance strategy adopted. Finally, they give new insights into possible responses to climate changes in cave dwelling species. © 2010 Elsevier Ltd.


Lencioni V.,Section of Invertebrate Zoology and Hydrobiology | Jousson O.,University of Trento | Guella G.,University of Trento | Bernabo P.,Section of Invertebrate Zoology and Hydrobiology | Bernabo P.,University of Trento
Physiological Entomology | Year: 2015

Insects inhabiting cold streams must either tolerate or avoid freezing to survive. The present study reports the strategy adopted by fourth-instar larvae of two chironomid species [Pseudodiamesa branickii (Nowicki) and Diamesa cinerella (Meigen)] overwintering in a glacial stream (in the Italian Alps). The cold adaptive potential of both species under acute cold stress is investigated down to -30°C. Supercooling points, lower lethal temperatures (LLTs), haemolymph thermal hysteresis, whole body content of sugars and polyols, and the expression of heat shock protein (HSP) genes (hsc70 and hsp70) expression are estimated. Comparable thermal hysteresis (>2°C) is measured in the two species, both of which accumulate glucose and sucrose as the main cryoprotectants. According to the supercooling points (=-6.37 and -6.85°C, respectively) and LLT100 (=-16.2 and -14.7°C, respectively), P. branickii and D. cinerella can both be considered as freeze tolerant. However, the cumulative proportion of individual freezing values and the LLT50 (-9.14 and -6.13°C, respectively) suggest that P. branickii is more cold hardy than D. cinerella, whereas the gene expression data (i.e. an absence of up-regulation of hsp70 in D. cinerella) suggest that D. cinerella is more cold hardy than P. branickii. These findings are discussed in relation to the validity of the different metabolic indicators for defining the level of cold hardiness of a species, even in relation to its cold stenothermy. The results are also discussed in relation to climate warming, which represents a serious threat for species from glacier-fed streams. © 2015 The Royal Entomological Society.


Lencioni V.,Section of Invertebrate Zoology and Hydrobiology | Bernabo P.,Section of Invertebrate Zoology and Hydrobiology | Cesari M.,Section of Invertebrate Zoology and Hydrobiology | Rebecchi L.,University of Modena and Reggio Emilia | Cesari M.,University of Modena and Reggio Emilia
Archives of Insect Biochemistry and Physiology | Year: 2013

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. © 2013 Wiley Periodicals, Inc.


PubMed | Section of Invertebrate Zoology and Hydrobiology
Type: | Journal: Insect science | Year: 2015

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 37C. Supercooling Point (SCP), Lower (LLTs) and Upper Lethal Temperatures (ULTs), and oxygen consumption rate were measured in overwintering young (I-II instar) and mature (III-IV instar) larvae from an Alpine glacier-fed stream. Both young and mature larvae were freezing tolerant (SCPs = -7.1C and -6.4C, respectively; LLT

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