2023, Schwarzenberger, Anke, Bartolin, Patrick, Wacker, Alexander

Changes in day-length entrain the endogenous clock of organisms leading to complex responses to photoperiod. In long-lived organisms experiencing several seasons this response of the clock to photoperiod is phenotypically plastic. However, short-lived organisms often experience a single season without pronounced changes in day-length. For those, a plastic response of the clock to different seasons would not necessarily be adaptive. In aquatic ecosystems, zooplankton species like Daphnia live only for some weeks, i.e. one week up to ca. two months. However, they often show a succession of clones that are seasonally adapted to environmental changes. Here, we found that 16 Daphnia clones per each of three seasons ( = 48 clones) from the same pond and year differed in clock gene expression with a homogenous gene expression pattern in ephippia-hatched spring clones and a bimodal expression pattern in summer and autumn populations indicating an ongoing adaptation process. We clearly demonstrate that spring clones were adapted to a short, and summer clones to a long photoperiod. Furthermore, we found that gene expression of the melatonin-synthesis enzyme AANAT was always lowest in summer clones. In the Anthropocene, Daphnia’s clock might be disturbed by light-pollution and global warming. Since Daphnia is a key-organism in trophic carbon transfer, a disruption of its clock rhythm would be devastating for the stability of freshwater ecosystems. Our results are an important step in understanding the adaptation of Daphnia’s clock to environmental changes.

2022, Weiss, Linda C., Schwarzenberger, Anke, Kruppert, Sebastian

2021-02, Schwarzenberger, Anke, Handke, Natascha H., Romer, Tina, Wacker, Alexander

Nearly all organisms show daily and seasonal physiological and behavioural responses that are necessary for their survival. Often these responses are controlled by the rhythmic activity of an endogenous clock that perceives day length. Day length differs not only between seasons but also along latitudes, with different seasonal day lengths between the north and the south. Both seasonal and latitudinal differences in day length are discussed to be perceived/processed by the endogenous clock. Some species are distributed over a wide range of latitudes; it should be highly adaptive for these species to be able to time physiological responses (e.g. migration behaviour and diapause) according to the organisms’ respective photoperiod, i.e. their respective seasonal and latitudinal day length. The mediator of day length is the indoleamine hormone melatonin which is synthesized by melatonin-producing enzymes (AANAT and HIOMT). These enzymes are in turn controlled by an endogenous clock. The ubiquitous aquatic keystone organism Daphnia possess clock and melatonin synthesis genes that are rhythmically expressed over 24 hours. We were able to show that the 24 -h rhythm of D. magna’s clock persists in constant darkness and is thus truly circadian. In one particular photoperiod, all D. magna clones produced a similar melatonin concentration due to a fixed AANAT activity. However, we have demonstrated that clones originating from different latitudes are adapted to their respective photoperiod by showing a geographic cline in clock and downstream melatonin synthesis gene expression. These findings hint at the problem locally adapted organisms face when they are forced to leave their respective photoperiod, e.g. because of climate change-driven range-expansion. If such a species is incapable of adjusting its endogenous clock to an unknown photoperiod, it will likely become extinct.

2020, Schwarzenberger, Anke, Chen, Luxi, Weiss, Linda C.

Diapause is a mechanism necessary for survival in arthropods. Often diapause induction and resurrection is light-dependent and therefore dependent on the photoperiod length and on the number of consecutive short-days. In many organisms, including the microcrustacean Daphnia magna, one functional entity with the capacity to measure seasonal changes in day-length is the circadian clock. There is a long-standing discussion that the circadian clock also controls photoperiod-induced diapause. We tested this hypothesis in D. magna, an organism which goes into a state of suspended animation with the shortening of the photoperiod. We measured gene expression of clock genes in diapause-destined embryos of D. magna in the initiation, resting and resurrection phases and checked it against gene expression levels of continuously developing embryos. We demonstrate that some genes of the clock are differentially expressed during diapause induction but not during its maintenance. Furthermore, the photoreceptor gene cry2 and the clock-associated gene brp are highly expressed during induction and early diapause, probably in order to produce excess mRNA to prepare for immediate resurrection. After resurrection, both types of embryos show a similar pattern of gene expression during development. Our study contributes significantly to the understanding of the molecular basis of diapause induction, maintenance and termination.

2022-11-07, Schwarzenberger, Anke

The plethora of cyanobacterial toxins are an enormous threat to whole ecosystems and humans. Due to eutrophication and increases in lake temperatures from global warming, changes in the distribution of cyanobacterial toxins and selection of few highly toxic species/ strains are likely. Globally, one of the most important grazers that controls cyanobacterial blooms is Daphnia, a freshwater model organism in ecology and (eco)toxicology. Daphnia–cyanobacteria interactions have been studied extensively, often focusing on the interference of filamentous cyanobacteria with Daphnia’s filtering apparatus, or on different nutritional constraints (the lack of essential amino acids or lipids) and grazer toxicity. For a long time, this toxicity only referred to microcystins. Currently, the focus shifts toward other deleterious cyanotoxins. Still, less than 10% of the total scientific output deals with cyanotoxins that are not microcystins; although these other cyanotoxins can occur just as frequently and at similar concentrations as microcystins in surface water. This review discusses the effects of different cyanobacterial toxins (hepatotoxins, digestive inhibitors, neurotoxins, and cytotoxins) on Daphnia and provides an elaborate and up-to-date overview of specific responses and adaptations of Daphnia. Furthermore, scenarios of what we can expect for the future of Daphnia–cyanobacteria interactions are described by comprising anthropogenic threats that might further increase toxin stress in Daphnia.

2021-06, Schwarzenberger, Anke, Ilić, Maja, von Elert, Eric

Cyanobacterial blooms often produce different classes and chemical variants of toxins such as dietary protease inhibitors (PIs) that affect the keystone grazer Daphnia. However, it has been shown that Daphnia populations are able to locally adapt to frequently occurring dietary PIs by modulating their digestive proteases. Up until now, local adaptation has exclusively been tested by making use of single cyanobacterial strains and by measuring average population tolerance. In contrast, we measured juvenile somatic growth rates and egg numbers of several individual clones per each of three different D. magna populations that have previously been found to be either tolerant or sensitive to the Microcystis strain BM25. Clones from the three D. magna populations were either treated with BM25 that produces three different protease inhibitor variants of the class of Ahp-cyclodepsipeptides or another Microcystis strain that produces two other Ahp-cyclodepsipeptide variants. Subsequently, the population growth was calculated as mean of the single-clone growth rates. Both tolerant populations (which originate from ponds with a cyanobacterial history) proved to be similarly tolerant to both Microcystis strains. However, single genotypes of the populations differed in their response to the different strains. Both the tolerant and the sensitive populations contained both sensitive and tolerant genotypes but in different proportions. Furthermore, the genotypes from the sensitive population showed a higher variance in response to one or both strains. Trade-offs between somatic growth rate and clutch size were found in one of the tolerant populations that originated from a pond where cyanobacteria were frequent in the past but completely absent since the pond's restoration. Because of those intra-population difference, we conclude that the tolerant populations were putatively selected by different Ahp-cyclodepsipeptide variants in the past and that all populations still possess the potential to adapt to other environmental conditions by genotype frequency shifts.

2020-10-21, Schwarzenberger, Anke, Kurmayer, Rainer, Martin-Creuzburg, Dominik

The harmful bloom-forming cyanobacterium Planktothrix is commonly considered to be nutritionally inadequate for zooplankton grazers, resulting in limited top-down control. However, interactions between Planktothrix and zooplankton grazers are poorly understood. The food quality of Planktothrix is potentially constrained by morphological properties (i.e., filament formation), the production of harmful secondary metabolites, and a deficiency in essential lipids (i.e., primarily sterols). Here, we investigated the relative significance of toxin production (microcystins, carboxypeptidase A inhibitors, protease inhibitors) and sterol limitation for the performance of Daphnia feeding on one Planktothrix rubescens and one P. agardhii wild-type/microcystin knock-out mutant pair. Our data suggest that the poor food quality of both Planktothrix spp. is due to deleterious effects mediated by various harmful secondary metabolites and that the impact of sterol limitation is partially or completely superimposed by toxicity. The significance of the different factors seems to depend on the metabolite profile of the considered Planktothrix strain and the Daphnia clone that is used for the experiments. The toxin-responsive gene expression (transporter genes, gpx, and trypsin) and enzyme activity patterns revealed strain-specific food quality constraints and that Daphnia is capable of modulating its physiological responses according to the ingested Planktothrix strain. Future studies need to consider that Planktothrix–grazer interactions are simultaneously modulated by multiple factors to improve our understanding of top-down influences on Planktothrix bloom formation.

2022-02-14, Cremer, Ricarda, Wacker, Alexander, Schwarzenberger, Anke

Cryptochromes are evolutionary ancient blue-light photoreceptors that are part of the circadian clock in the nervous system of many organisms. Cryptochromes transfer information of the predominant light regime to the clock which results in the fast adjustment to photoperiod. Therefore, the clock is sensitive to light changes and can be affected by anthropogenic Artificial Light At Night (ALAN). This in turn has consequences for clock associated behavioral processes, e.g., diel vertical migration (DVM) of zooplankton. In freshwater ecosystems, the zooplankton genus Daphnia performs DVM in order to escape optically hunting predators and to avoid UV light. Concomitantly, Daphnia experience circadian changes in food-supply during DVM. Daphnia play the keystone role in the carbon-transfer to the next trophic level. Therefore, the whole ecosystem is affected during the occurrence of cyanobacteria blooms as cyanobacteria reduce food quality due to their production of digestive inhibitors (e.g., protease inhibitors). In other organisms, digestion is linked to the circadian clock. If this is also the case for Daphnia, the expression of protease genes should show a rhythmic expression following circadian expression of clock genes (e.g., cryptochrome 2). We tested this hypothesis and demonstrated that gene expression of the clock and of proteases was affected by ALAN. Contrary to our expectations, the activity of one type of proteases (chymotrypsins) was increased by ALAN. This indicates that higher protease activity might improve the diet utilization. Therefore, we treated D. magna with a chymotrypsin-inhibitor producing cyanobacterium and found that ALAN actually led to an increase in Daphnia’s growth rate in comparison to growth on the same cyanobacterium in control light conditions. We conclude that this increased tolerance to protease inhibitors putatively enables Daphnia populations to better control cyanobacterial blooms that produce chymotrypsin inhibitors in the Anthropocene, which is defined by light pollution and by an increase of cyanobacterial blooms due to eutrophication.

2021-05, Schwarzenberger, Anke, Martin-Creuzburg, Dominik

Cyanobacterial blooms are an omnipresent and well-known result of eutrophication and climate change in aquatic systems. Cyanobacteria produce a plethora of toxic secondary metabolites that affect humans, animals and ecosystems. Many cyanotoxins primarily affect the grazers of phytoplankton, e.g., Daphnia. The neurotoxin anatoxin-α has been reported world-wide; despite its potency, anatoxin-α and its effects on Daphnia have not been thoroughly investigated. Here, we investigated the effects of the anatoxin-α-producing Tychonema on life-history parameters and gene expression of nicotine-acetylcholine receptors (NAR), the direct targets of anatoxin-α, using several D. magna clones. We used juvenile somatic growth rates as a measure of fitness and analyzed gene expression by qPCR. Exposure to 100% Tychonema reduced the clones’ growth rates and caused an up-regulation of NAR gene expression. When 50% of the food consisted of Tychonema, none of the clones were reduced in growth and only one of them showed an increase in NAR gene expression. We demonstrate that this increased NAR gene expression can be maternally transferred and that offspring from experienced mothers show a higher growth rate when treated with 50% Tychonema compared with control offspring. However, the addition of further (anthropogenic) stressors might impair Daphnia’s adaptive responses to anatoxin-α. Especially the presence of certain pollutants (i.e., neonicotinoids), which also target NARs, might reduce Daphnia’s capability to cope with anatoxin-α.

2020-02-07, Schwarzenberger, Anke, Hasselmann, Martin, von Elert, Eric

Due to the combined effects of global warming and eutrophication, the frequency of deleterious cyanobacterial blooms in freshwater ecosystems has increased. In line with this, local adaptation of the aquatic keystone herbivore Daphnia to cyanobacteria has received major attention. Besides microcystins, the most frequent cyanobacterial secondary metabolites in such blooms are protease inhibitors (PIs). Recently, it has been shown that a protease gene showed copy number variation between four D. magna populations that differed in tolerance to PIs. From that study, we chose two distinct populations of D. magna which had or had not coexisted with cyanobacteria in the past. By calculating FST values, we found that the two populations were genetically more distant in the protease loci than in neutral loci. Population genetic tests applied to the tolerant population revealed that positive selection was most probably acting on the gene loci of the digestive protease CT448 and CT802. We conclude that the selection of digestive proteases and subsequent reduction in copy number is the molecular basis of evolutionary changes leading to local adaptation to PIs.