Molecular and morphological characterisation of larvae of the genus Diamesa Meigen, 1835 (Diptera: Chironomidae) in Alpine streams (Ötztal Alps, Austria).

Diamesa species (Diptera, Chironomidae) are widely distributed in freshwater ecosystems, and their life cycles are closely linked to environmental variables such as temperature, water quality, and sediment composition. Their sensitivity to environmental changes, particularly in response to pollution and habitat alterations, makes them valuable indicators of ecosystem health. The challenges associated with the morphological identification of larvae invoke the use of DNA barcoding for species determination. The mitochondrial cytochrome oxidase subunit I (COI) gene is regularly used for species identification but faces limitations, such as similar sequences in closely related species. To overcome this, we explored the use of the internal transcribed spacers (ITS) region in addition to COI for Diamesa larvae identification. Therefore, this study employs a combination of molecular markers alongside traditional morphological identification to enhance species discrimination. In total, 129 specimens were analysed, of which 101 were sampled from a glacier-fed stream in Rotmoostal, and the remaining 28 from spring-fed streams in the neighbouring valleys of Königstal and Timmelstal. This study reveals the inadequacy of utilizing single COI or ITS genes for comprehensive species differentiation within the genus Diamesa. However, the combined application of COI and ITS markers significantly enhances species identification resolution, surpassing the limitations faced by traditional taxonomists. Notably, this is evident in cases involving morphologically indistinguishable species, such as Diamesa latitarsis and Diamesa modesta. It highlights the potential of employing a multi-marker approach for more accurate and reliable Diamesa species identification. This method can be a powerful tool for identifying Diamesa species, shedding light on their remarkable adaptations to extreme environments and the impacts of environmental changes on their populations.

Energy status of chironomid larvae (Diptera: Chironomidae) from high alpine rivers (Tyrol, Austria).

Chironomids (non-biting midges) inhabit almost every wet or semi-wet continental environment on Earth with probably 10,000 different species. Species occurrence and composition are undoubtedly limited by environmental harshness and food availability being reflected in their energy stores. Most animals store energy as glycogen and lipid. They enable the animals to survive adverse situations and to continue growth, development, and reproduction. This general statement is also true for insects and also particularly true for chironomid larvae. The rationale behind this research was, that probably any stress, any environmental burden, and any harmful influence increases the energy requirement of individual larvae depleting energy stores. We developed new methods to measure the glycogen and lipid content in small tissue samples. Here we show how to apply these methods to single chironomid larvae to demonstrate their energy stores. We compared different locations of the high Alpine rivers along harshness gradient densely populated and dominated by chironomid larvae. All samples show very low energy stores without any major differences. We found glycogen concentrations below 0.01% of dry weight (DW) and lipid concentrations below 5% of DW irrespective of the specific sampling point. These values are among the lowest ever observed in chironomid larvae. We demonstrate that individuals living in extreme environment are stressed leading to reduced energy stores in their bodies. This appears to be a general feature of high altitude regions. Our results provide new insights and a better understanding of population and ecological dynamics in harsh mountainous areas, also in view of a changing climate.

Metallomics reveals a persisting impact of cadmium on the evolution of metal-selective snail metallothioneins.

The tiny contribution of cadmium (Cd) to the composition of the earth's crust contrasts with its high biological significance, owing mainly to the competition of Cd with the essential zinc (Zn) for suitable metal binding sites in proteins. In this context it was speculated that in several animal lineages, the protein family of metallothioneins (MTs) has evolved to specifically detoxify Cd. Although the multi-functionality and heterometallic composition of MTs in most animal species does not support such an assumption, there are some exceptions to this role, particularly in animal lineages at the roots of animal evolution. In order to substantiate this hypothesis and to further understand MT evolution, we have studied MTs of different snails that exhibit clear Cd-binding preferences in a lineage-specific manner. By applying a metallomics approach including 74 MT sequences from 47 gastropod species, and by combining phylogenomic methods with molecular, biochemical, and spectroscopic techniques, we show that Cd selectivity of snail MTs has resulted from convergent evolution of metal-binding domains that significantly differ in their primary structure. We also demonstrate how their Cd selectivity and specificity has been optimized by the persistent impact of Cd through 430 million years of MT evolution, modifying them upon lineage-specific adaptation of snails to different habitats. Overall, our results support the role of Cd for MT evolution in snails, and provide an interesting example of a vestigial abiotic factor directly driving gene evolution. Finally, we discuss the potential implications of our findings for studies devoted to the understanding of mechanisms leading to metal specificity in proteins, which is important when designing metal-selective peptides.

Cadmium Pathways in Snails Follow a Complementary Strategy between Metallothionein Detoxification and Auxiliary Inactivation by Phytochelatins.

Metal detoxification is crucial for animals to cope with environmental exposure. In snails, a pivotal role in protection against cadmium (Cd) is attributed to metallothioneins (MTs). Some gastropod species express, in a lineage-specific manner, Cd-selective MTs devoted exclusively to the binding and detoxification of this single metal, whereas other species of snails possess non-selective MTs, but still show a high tolerance against Cd. An explanation for this may be that invertebrates and in particular snails may also synthetize phytochelatins (PCs), originally known to be produced by plants, to provide protection against metal or metalloid toxicity. Here we demonstrate that despite the fact that similar mechanisms for Cd inactivation exist in snail species through binding of the metal to MTs, the actual detoxification pathways for this metal may follow different traits in a species-specific manner. In particular, this depends on the detoxification capacity of MTs due to their Cd-selective or non-specific binding features. In the terrestrial slug Arion vulgaris, for example, Cd is solely detoxified by a Cd-selective MT isoform (AvMT1). In contrast, the freshwater snail Biomphalaria glabrata activates an additional pathway for metal inactivation by synthesizing phytochelatins, which compensate for the insufficient capacity of its non-selective MT system to detoxify Cd. We hypothesize that in other snails and invertebrate species, too, an alternative inactivation of the metal by PCs may occur, if their MT system is not Cd-selective enough, or its Cd loading capacity is exhausted.

Distinct pathways for zinc metabolism in the terrestrial slug Arion vulgaris.

In most organisms, the concentration of free Zn2+ is controlled by metallothioneins (MTs). In contrast, no significant proportions of Zn2+ are bound to MTs in the slug, Arion vulgaris. Instead, this species possesses cytoplasmic low-molecular-weight Zn2+ (LMW Zn) binding compound that divert these metal ions into pathways uncoupled from MT metabolism. Zn2+ is accumulated in the midgut gland calcium cells of Arion vulgaris, where they associate with a low-molecular-weight ligand with an apparent molecular mass of ~ 2,000 Da. Mass spectrometry of the semi-purified LMW Zn binding compound combining an electrospray ion source with a differential mobility analyser coupled to a time-of-flight mass spectrometer revealed the presence of four Zn2+-containing ion signals, which arise from disintegration of one higher MW complex resulting in an ion-mobility diameter of 1.62 nm and a molecular mass of 837 Da. We expect that the novel Zn2+ ion storage pathway may be shared by many other gastropods, and particularly species that possess Cd-selective MT isoforms or variants with only very low affinity to Zn2+.

Metal binding functions of metallothioneins in the slug Arion vulgaris differ from metal-specific isoforms of terrestrial snails.

Arion vulgaris is a land-living European slug belonging to the gastropod clade of Stylommatophora. The species is known as an efficient pest organism in vegetable gardening and horticulture, which may in part be the consequence of its genetically based innate immunity, along with its high ability to withstand toxic metal stress by intracellular detoxification. Like many species of terrestrial snails, slugs possess a distinct capacity for Cd accumulation in their midgut gland, where the metal is stored and inactivated, conferring to these animals an increased metal tolerance. Although midgut gland Cd fractions in slugs have been shown to be variably allocated between different metal-binding protein pools, depending on the level of environmental metal contamination, a true metallothionein (MT) was so far never characterized from slugs. Instead, the Cd binding proteins identified so far were described as Metallothionein-like proteins (MTLPs). In the present study, the slug A. vulgaris was used as a model organism, in order to verify the presence of true MTs in experimentally metal-exposed slugs. We wanted to find out if these suggested slug MTs have similar metal binding properties and metal-selective features like those previously reported from helicid snails. To this aim, two MT isoform genes (AvMT1 and AvMT2) were characterized from midgut gland extracts and localized in the cells of this tissue. The AvMT1 and AvMT2 proteins were purified and partially sequenced, and their metal-binding features analysed after recombinant expression. Eventually, we wanted to understand if and by how much the metal binding features of the two MT isoforms of A. vulgaris may be related, owing to their reciprocal amino acid sequence similarities, to the binding properties of metal-specific MTs from terrestrial snails.

Background fish feminization effects in European remote sites.

Human activity has spread trace amounts of chemically stable endocrine-disrupting pollutants throughout the biosphere. These compounds have generated a background level of estrogenic activity that needs to be assessed. Fish are adequate sentinels for feminization effects as male specimens are more sensitive than humans to exogenous estrogenic compounds. High mountain lakes, the most distant environments of continental areas, only receive semi-volatile compounds from atmospheric deposition. We analyzed the expression levels of estrogen-regulated genes in male fish from these mountain lakes in Europe. Incipient feminization involving expression of estrogen receptor and zona radiata genes revealed a widespread diffuse estrogenic impact. This effect was correlated with the concentrations of some organochlorine compounds in fish and was consistent with the persistent occurrence of these tropospheric pollutants in the most remote planet regions. These results should be of general concern given the increasing endocrine disruption effects in human populations.

Physiological response to persistent organic pollutants in fish from mountain lakes: analysis of CYP1A gene expression in natural populations of Salmo trutta.

Cytochrome p450 1A (CYP1A) gene expression in fish liver increases upon exposure to a variety of chemical compounds, including organochlorine compounds (OCs) and polycyclic aromatic hydrocarbons (PAHs). To use this physiological response as a marker of environmental impact, we developed and validated a set of primers to quantify CYP1A expression by qRT-PCR in the brown trout, Salmo trutta. These primers were used to explore the natural variability of CYP1A expression in 8 isolated populations (65 samples) from European remote lakes, in a geographical distribution encompassing the Tyrolean Alps, Pyrenees, Rila, Tatras, and Norwegian and Scottish mountains. CYP1A expression values varied more than 2 orders of magnitude among samples, with strong variations within each population. CYP1A expression values were significantly elevated in Tatras and Pyrenees fish populations, whereas the lowest median values were found in populations from the Tyrolean Alps and Rila. These values correlated with the content of different environmentally relevant pollutants in the sediments of the lakes harboring each fish population, particularly with HCB and 4,4'-DDE contents. To our knowledge, this works represents a first report of a physiological response linked to persistent organic pollutants in fish from mountain lakes.