Whole-genome sequencing illuminates multifaceted targets of selection to humic substances in Eurasian perch.

Extreme environments are inhospitable to the majority of species, but some organisms are able to survive in such hostile conditions due to evolutionary adaptations. For example, modern bony fishes have colonized various aquatic environments, including perpetually dark, hypoxic, hypersaline and toxic habitats. Eurasian perch (Perca fluviatilis) is among the few fish species of northern latitudes that is able to live in very acidic humic lakes. Such lakes represent almost "nocturnal" environments; they contain high levels of dissolved organic matter, which in addition to creating a challenging visual environment, also affects a large number of other habitat parameters and biotic interactions. To reveal the genomic targets of humic-associated selection, we performed whole-genome sequencing of perch originating from 16 humic and 16 clear-water lakes in northern Europe. We identified over 800,000 single nucleotide polymorphisms, of which >10,000 were identified as potential candidates under selection (associated with >3000 genes) using multiple outlier approaches. Our findings suggest that adaptation to the humic environment may involve hundreds of regions scattered across the genome. Putative signals of adaptation were detected in genes and gene families with diverse functions, including organism development and ion transportation. The observed excess of variants under selection in regulatory regions highlights the importance of adaptive evolution via regulatory elements, rather than via protein sequence modification. Our study demonstrates the power of whole-genome analysis to illuminate the multifaceted nature of humic adaptation and provides the foundation for further investigation of causal mutations underlying phenotypic traits of ecological and evolutionary importance.

Humic-acid-driven escape from eye parasites revealed by RNA-seq and target-specific metabarcoding.

BACKGROUND: Next generation sequencing (NGS) technologies are extensively used to dissect the molecular mechanisms of host-parasite interactions in human pathogens. However, ecological studies have yet to fully exploit the power of NGS as a rich source for formulating and testing new hypotheses. METHODS: We studied Eurasian perch (Perca fluviatilis) and its eye parasite (Trematoda, Diplostomidae) communities in 14 lakes that differed in humic content in order to explore host-parasite-environment interactions. We hypothesised that high humic content along with low pH would decrease the abundance of the intermediate hosts (gastropods), thus limiting the occurrence of diplostomid parasites in humic lakes. This hypothesis was initially invoked by whole eye RNA-seq data analysis and subsequently tested using PCR-based detection and a novel targeted metabarcoding approach. RESULTS: Whole eye transcriptome results revealed overexpression of immune-related genes and the presence of eye parasite sequences in RNA-seq data obtained from perch living in clear-water lakes. Both PCR-based and targeted-metabarcoding approach showed that perch from humic lakes were completely free from diplostomid parasites, while the prevalence of eye flukes in clear-water lakes that contain low amounts of humic substances was close to 100%, with the majority of NGS reads assigned to Tylodelphys clavata. CONCLUSIONS: High intraspecific diversity of T. clavata indicates that massively parallel sequencing of naturally pooled samples represents an efficient and powerful strategy for shedding light on cryptic diversity of eye parasites. Our results demonstrate that perch populations in clear-water lakes experience contrasting eye parasite pressure compared to those from humic lakes, which is reflected by prevalent differences in the expression of immune-related genes in the eye. This study highlights the utility of NGS to discover novel host-parasite-environment interactions and provide unprecedented power to characterize the molecular diversity of cryptic parasites.

Role of potentially toxic cyanobacteria in crustacean zooplankton diet in a eutrophic lake.

The coexistence of potentially toxic bloom-forming cyanobacteria (CY) and generally smaller-sized grazer communities has raised the question of zooplankton (ZP) ability to control harmful cyanobacterial blooms and highlighted the need for species-specific research on ZP-CY trophic interactions in naturally occurring communities. A combination of HPLC, molecular and stable isotope analyses was used to assess in situ the importance of CY as a food source for dominant crustacean ZP species and to quantify the grazing on potentially toxic strains of Microcystis during bloom formation in large eutrophic Lake Peipsi (Estonia). Aphanizomenon, Dolichospermum, Gloeotrichia and Microcystis dominated bloom-forming CY, while Microcystis was the major genus producing cyanotoxins all over the lake. Grazing studies showed that CY, and especially colonial CY, formed a significant, and also preferred component of algae ingested by the cladocerans Bosmina spp. and Daphnia spp. while this was not the case for the more selective calanoid copepod Eudiaptomus gracilis. Molecular analyses confirmed the presence of CY, including Microcystis, in ZP guts. Further analyses using qPCR targeting cyanobacterial genus-specific mcyE synthase genes indicated that potentially toxic strains of Microcystis can be ingested directly or indirectly by all the dominant crustacean grazers. However, stable isotope analyses indicated that little, if any, assimilation from ingested bloom-forming CY occurred. The study suggests that CY, and particularly Microcystis with both potentially toxic and non-toxic strains, can be widely ingested by cladoceran grazers during a bloom event with implications for control of CY abundance and for transfer of CY toxins through the food web.

Do organic matter metrics included in lake surveillance monitoring in Europe provide a broad picture of brownification and enrichment with oxygen consuming substances?

Organic matter (OM) has numerous geochemical and ecological functions in inland waters and can affect water quality. Different parameters of aquatic OM are measured with various methods as no single analytical tool can provide definitive structural or functional information about it. In the present paper we review different OM metrics used in the European Union (EU) lake surveillance monitoring programmes and assess their suitability to provide sufficient data about the brownification and enrichment with oxygen consuming substances in European lakes. In the EU Water Framework Directive (WFD), metrics of OM are not mandatory physico-chemical parameters, but only recommended parameters to characterize water transparency, oxygenation conditions or acidification status. Our analysis shows that, as lake OM is monitored under the WFD in only 14 countries, no Europe-wide conclusions on the situation regarding brownification and organic enrichment can be drawn based on these data. Applied parameters in lake surveillance monitoring programmes are biochemical oxygen demand (BOD), chemical oxygen demand (COD), total organic carbon (TOC), dissolved organic carbon (DOC), water colour (WCol), and yellow substance. Different national OM metrics used avoid getting a broad picture of lake OM concentration changes in Europe over the last decades. Furthermore, our results demonstrate that the possibilities to convert different OM parameters to each other are limited because empirical relationships between them are region-specific. OM sensors for continuous measurements and remote sensing surveys could improve the effectiveness of lake OM monitoring, especially its temporal and spatial representativeness. It would be highly suggested to include in lake monitoring programmes also methods (e.g. absorbance or fluorescence spectroscopy) allowing to characterize the composition of OM as it influences strongly the biogeochemical role of OM in lakes.

Algal Diet of Small-Bodied Crustacean Zooplankton in a Cyanobacteria-Dominated Eutrophic Lake.

Small-bodied cladocerans and cyclopoid copepods are becoming increasingly dominant over large crustacean zooplankton in eutrophic waters where they often coexist with cyanobacterial blooms. However, relatively little is known about their algal diet preferences. We studied grazing selectivity of small crustaceans (the cyclopoid copepods Mesocyclops leuckarti, Thermocyclops oithonoides, Cyclops kolensis, and the cladocerans Daphnia cucullata, Chydorus sphaericus, Bosmina spp.) by liquid chromatographic analyses of phytoplankton marker pigments in the shallow, highly eutrophic Lake Võrtsjärv (Estonia) during a seasonal cycle. Copepods (mainly C. kolensis) preferably consumed cryptophytes (identified by the marker pigment alloxanthin in gut contents) during colder periods, while they preferred small non-filamentous diatoms and green algae (identified mainly by diatoxanthin and lutein, respectively) from May to September. All studied cladoceran species showed highest selectivity towards colonial cyanobacteria (identified by canthaxanthin). For small C. sphaericus, commonly occuring in the pelagic zone of eutrophic lakes, colonial cyanobacteria can be their major food source, supporting their coexistence with cyanobacterial blooms. Pigments characteristic of filamentous cyanobacteria and diatoms (zeaxanthin and fucoxanthin, respectively), algae dominating in Võrtsjärv, were also found in the grazers' diet but were generally avoided by the crustaceans commonly dominating the zooplankton assemblage. Together these results suggest that the co-occurring small-bodied cyclopoid and cladoceran species have markedly different algal diets and that the cladocera represent the main trophic link transferring cyanobacterial carbon to the food web in a highly eutrophic lake.

From bacteria to piscivorous fish: estimates of whole-lake and component-specific metabolism with an ecosystem approach.

The influence of functional group specific production and respiration patterns on a lake's metabolic balance remains poorly investigated to date compared to whole-system estimates of metabolism. We employed a summed component ecosystem approach for assessing lake-wide and functional group-specific metabolism (gross primary production (GPP) and respiration (R)) in shallow and eutrophic Lake Võrtsjärv in central Estonia during three years. Eleven functional groups were considered: piscivorous and benthivorous fish; phyto-, bacterio-, proto- and metazooplankton; benthic macroinvertebrates, bacteria and ciliates; macrophytes and their associated epiphytes. Metabolism of these groups was assessed by allometric equations coupled with daily records of temperature and hydrology of the lake and measurements of food web functional groups biomass. Results revealed that heterotrophy dominated most of the year, with a short autotrophic period observed in late spring. Most of the metabolism of the lake could be attributed to planktonic functional groups, with phytoplankton contributing the highest share (90% of GPP and 43% of R). A surge of protozooplankton and bacterioplankton populations forming the microbial loop caused the shift from auto- to heterotrophy in midsummer. Conversely, the benthic functional groups had overall a very small contribution to lake metabolism. We validated our ecosystem approach by comparing the GPP and R with those calculated from O2 measurements in the lake. Our findings are also in line with earlier productivity studies made with 14C or chlorophyll a (chl-a) based equations. Ideally, the ecosystem approach should be combined with diel O2 approach for investigating critical periods of metabolism shifts caused by dynamics in food-web processes.