A free and user-friendly software protocol for the quantification of microfauna swimming behavior.

Characterizing swimming behavior can provide a holistic assessment of the health, physiology and ecology of microfaunal species when done in conjunction with measuring other biological parameters. However, tracking and quantifying microfauna swimming behavior using existing automated tools is often difficult due to the animals' small size or transparency, or because of the high cost, expertise, or labor needed for the analysis. To address these issues, we created a cost-effective, user-friendly protocol for behavior analysis that employs the free software packages HitFilm and ToxTrac along with the R package 'trajr' and used the method to quantify the behavior of rotifers. This protocol can be used for other microfaunal species for which investigators may face similar issues in obtaining measurements of swimming behavior.

Environmental factors controlling seasonal and spatial variability of zooplankton in thermokarst lakes along a permafrost gradient of Western Siberia.

Humic thermokarst lakes of permafrost peatlands in Western Siberia Lowland (WSL) are major environmental controllers of carbon and nutrient storage in inland waters and greenhouse gases emissions to the atmosphere in the subarctic. In contrast to sizable former research devoted to hydrochemical and hydrobiological (phytoplankton) composition, zooplankton communities of these thermokarst lakes and thaw ponds remain poorly understood, especially along the latitudinal gradient, which is a perfect predictor of permafrost zones. To fill this gap, 69 thermokarst lakes of the WSL were sampled using unprecedented spatial coverage, from continuous to sporadic permafrost zone, in order to assess zooplankton (Cladocera, Copepoda, Rotifera) diversity and abundance across three main open water physiological seasons (spring, summer and autumn). We aimed at assessing the relationship of environmental factors (water column hydrochemistry, nutrients, and phytoplankton parameters) with the abundance and diversity of zooplankton. A total of 74 zooplankton species and taxa were detected, with an average eight taxa per lake/pond. Species richness increased towards the north and reached the maximum in the continuous permafrost zone with 13 species found in this zone only. In contrast, the number of species per waterbody decreased towards the north, which was mainly associated with a decrease in the number of cladocerans. Abundance and diversity of specific zooplankton groups strongly varied across the seasons and permafrost zones. Among the main environmental controllers, Redundancy Analysis revealed that water temperature, lake area, depth, pH, Dissolved Inorganic and Organic Carbon and CO2 concentrations were closely related to zooplankton abundance. Cladocerans were positively related to water temperature during all seasons. Copepods were positively related to depth and lake water pH in all seasons. Rotifers were related to different factors in each season, but were most strongly associated with DOC, depth, CH4, phytoplankton and cladoceran abundance. Under climate warming scenario, considering water temperature increase and permafrost boundary shift northward, one can expect an increase in the diversity and abundance of cladocerans towards the north which can lead to partial disappearance of copepods, especially rare calanoid species.

Molecular insight into the binding properties of marine algogenic dissolved organic matter for polybrominated diphenyl ethers and their combined effect on marine zooplankton.

Polybrominated diphenyl ethers (PBDEs) are widespread in marine ecosystems, despite the limits placed on several congeners, and pose a threat to marine organisms. Many coexisting factors, especially dissolved organic matter (DOM), affect the environmental behavior and ecological risk of PBDEs. Since blooms frequently occur in coastal waters, we used algogenic DOM (A-DOM) from the diatom Skeletonem costatum and examined the interaction of A-DOM with 2,2',4,4'-tetrabromodiphenyl ether (BDE-47). Moreover, their combined effect on the rotifer Brachionus plicatilis was analyzed. During the stationary period, A-DOM had more proteins than polysaccharides, and 7 extracellular proteins were identified. A-DOM fluorescence was statically quenched by BDE-47, and amide, carbonyl, and hydroxyl groups in A-DOM were involved. Molecular docking analysis showed that all 5 selected proteins of A-DOM could spontaneously bind with BDE-47 and that hydrophobic interactions, van der Waals forces and pi-bond interactions existed. The reproductive damage, oxidative stress and inhibition of mitochondrial activity induced by BDE-47 in rotifers were relieved by A-DOM addition. Transcriptomic analysis further showed that A-DOM could activate energy metabolic pathways in rotifers and upregulate genes encoding metabolic detoxification proteins and DNA repair. Moreover, A-DOM alleviated the interference effect of BDE-47 on lysosomes, the extracellular matrix pathway and the calcium signaling system. Alcian blue staining and scanning electron microscopy showed that A-DOM aggregates were mainly stuck to the corona and cuticular surface of the rotifers; this mechanism, rather than a real increase in uptake, was the reason for enhanced bioconcentration. This study reveals the complex role of marine A-DOM in PBDEs bioavailability and enhances the knowledge related to risk assessments of PBDE-like contaminants in marine environments.

The response of zooplankton network indicators to winter water warming using shallow artificial reservoirs as model case study.

To predict the most likely scenarios, the consequences of the rise in water surface temperature have been studied using various methods. We tested the hypothesis that winter water warming significantly alters the importance and nature of the relationships in zooplankton communities in shallow reservoirs. These relationships were investigated using network graph analysis for three thermal variants: warm winters (WW), moderate winters (MW) and cold winters (CW). The CW network was the most cohesive and was controlled by eutrophic Rotifera and Copepoda, with a corresponding number of positive and negative interspecific relationships. An increase in water temperature in winter led to a decrease in the centrality of MW and WW networks, and an increase in the importance of species that communicated with the highest number of species in the subnetworks. The WW network was the least cohesive, controlled by psammophilous and phytophilous rotifers, and littoral cladocerans. Adult copepods were not identified in the network and the importance of antagonistic relationships decreased, indicating that the WW network structure was weak and unstable. This study can serve as a model for generalisations of zooplankton community response to the disappearance of long winter periods of low temperatures, as predicted in global climate change projections.

Metabolic pathways of methylmercury in rotifer Brachionus plicatilis.

Methylmercury (MeHg) readily accumulates in aquatic organisms while transferring and amplifying in the aquatic food chains. This study firstly explores the in vivo accumulation sites and metabolic regulation of MeHg in the rotifer Brachionus plicatilis by aggregation-induced emission fluorogen (AIEgen) and metabolomics. Fluorescent image analysis by AIEgen showed that MeHg in B. plicatilis mainly occured in the ciliary corona, esophagus, mastax, stomach and intestine in the direct absorption group. In the other group, where B. plicatilis were indirectly supplied with MeHg via food intake, the accumulation of MeHg in the rotifer occurred in the ciliary corona, various digestive organs, and the pedal gland. However, the MeHg accumulated in the rotifer is difficult to metabolize outside the body. Metabolomics analysis showed that the significant enrichment of ABC transporters was induced by the direct exposure of rotifers to dissolved MeHg. In contrast, exposure of rotifers to MeHg via food intake appeared to influence carbon, galactose, alanine, aspartate and glutamate metabolisms. Besides, the disturbed biological pathways such as histidine metabolism, beta-alanine metabolism and pantothenate and CoA biosynthesis in rotifers may be associated with L-aspartic acid upregulation in the feeding group. The significant enrichment of ABC transporters and carbon metabolism in rotifers may be related to the accumulation of MeHg in the intestine of rotifers. In both pathways of MeHg exposure, the arginine biosynthesis and metabolism of rotifers were disturbed, which may support the hypothesis that rotifers produce more energy to resist MeHg toxicity. This study provides new insight into the accumulation and toxicity mechanisms of MeHg on marine invertebrates from the macro and micro perspectives.

Inhibitory Effects of Antipsychotic Chlorpromazine on the Survival, Reproduction and Population Growth Other Than Neurotransmitters of Zooplankton in Light of Global Warming.

Global warming and environmental pollution have created a unique combination of abiotic and biotic stresses to zooplankton. However, little information is available on the effects of antipsychotic drugs commonly used to treat psychosis, such as chlorpromazine (CPZ), on non-target aquatic organisms in light of global warming. This study investigated how dopamine concentrations (DAC), acute toxicity and chronic toxicity of Brachionus calyciflorus changed in response to CPZ and gradually increasing temperatures. The results showed that the concentration range of rotifer DAC was 1.06~2.51 ng/g. At 18, 25 and 32 °C, the 24 h LC50 was 1.795, 1.242 and 0.833 mg/L, respectively. Compared to the control, exposure to CPZ significantly decreased life expectancy at hatching, the net reproduction rate, generation time, population growth rate and dopamine concentration of B. calyciflorus in all three temperatures (p < 0.05). The toxicity of CPZ to rotifers was increased by high temperature. These findings indicated that CPZ is highly toxic to rotifers, displaying high ecological risks to aquatic ecosystems.

Predicting the effects of winter water warming in artificial lakes on zooplankton and its environment using combined machine learning models.

This work deals with the consequences of climate warming on aquatic ecosystems. The study determined the effects of increased water temperatures in artificial lakes during winter on predicting changes in the biomass of zooplankton taxa and their environment. We applied an innovative approach to investigate the effects of winter warming on zooplankton and physico-chemical factors. We used a modelling scheme combining hierarchical clustering, eXtreme Gradient Boosting (XGBoost) and SHapley Additive exPlanations (SHAP) algorithms. Under the influence of increased water temperatures in winter, weight- and frequency-dominant Crustacea taxa such as Daphnia cucullata, Cyclops vicinus, Cryptocyclops bicolor, copepodites and nauplii, and the Rotifera: Polyarthra longiremis, Trichocerca pusilla, Keratella quadrata, Asplanchna priodonta and Synchaeta spp. tend to decrease their biomass. Under the same conditions, Rotifera: Lecane spp., Monommata maculata, Testudinella patina, Notholca squamula, Colurella colurus, Trichocerca intermedia and the protozoan species Centropyxis acuelata and Arcella discoides with lower size and abundance responded with an increase in biomass. Decreases in chlorophyll a, suspended solids and total nitrogen were predicted due to winter warming. Machine learning ensemble models used in innovative ways can contribute to the research utility of studies on the response of ecological units to environmental change.

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Human activities have severely polluted environments during industrialization. Rotifers are commonly used as model species for ecotoxicological assays. The sensitivities of ecotoxicological indices of rotifers highly depend on different pollutants. We summarized available ecotoxicological indices of rotifers, and reviewed their sensitivity to commonly investigated pollutants. Under the stress of heavy metals, the life-table demographic parameters, physiological and biochemical indices generally show high sensitivity. In response to persistent organic pollutants, sexual indices generally show higher sensitivity than asexual ones. Some studies showed that swimming speed revealed higher sensitivity after exposure to several pollutants compared with life-table demographic and population growth indices. We suggested to consider swimming speed of rotifer as an alternative index for ecotoxicological assays. Overall, this study would provide a guideline for rotifer ecotoxicological studies in the future.

Body size variability across habitats in the Brachionus plicatilis cryptic species complex.

The body size response to temperature is one of the most recognizable but still poorly understood ecological phenomena. Other covarying environmental factors are frequently invoked as either affecting the strength of that response or even driving this pattern. We tested the body size response in five species representing the Brachionus plicatilis cryptic species complex, inhabiting 10 brackish ponds with different environmental characteristics. Principal Component Analysis selected salinity and oxygen concentration as the most important factors, while temperature and pH were less influential in explaining variation of limnological parameters. Path analysis showed a positive interclonal effect of pH on body size. At the interspecific level, the size response was species- and factor-dependent. Under the lack of a natural thermo-oxygenic relationship, the negative response of size to temperature, expected according to 'size-to-temperature response' rules, disappeared, but a positive response of size to oxygen, expected according to predictions selecting oxygen as a factor actually driving these rules, remained. Our results confirm the crucial role of oxygen in determining the size-to-temperature patterns observed in the field.

Sex ratio in the mother's environment affects offspring population dynamics: maternal effects on population regulation.

Classic population regulation theories usually concern the influence of immediate factors on current populations, but studies investigating the effect of parental environment factors on their offspring populations are scarce. The maternal environments can affect offspring life-history traits across generations, which may affect population dynamics and be a mechanism of population regulation. In cyclical parthenogens, sexual reproduction is typically linked with dormancy, thereby providing a negative feedback to population growth. In this study, we manipulated population sex ratios in the mother's environment to investigate whether this factor affected future population dynamics by regulating offspring sexual reproduction in the rotifer Brachionus calyciflorus. Compared with females in male-biased environments, those in female-biased environments produced fewer mictic (sexual) offspring, and their amictic (asexual) offspring also produced a lower proportion of mictic females at a gradient of population densities. Moreover, populations that were manipulated under male-biased conditions showed significantly smaller population sizes than those under female-biased conditions. Our results indicated that in cyclical parthenogens, mothers could adjust the sexual reproduction of their offspring in response to the current population sex ratio, thus providing fine-scale regulation of population dynamics in addition to population density.

Tempo-spatial variations of zooplankton communities in relation to environmental factors and the ecological implications: A case study in the hinterland of the Three Gorges Reservoir area, China.

To expand the knowledge on the tempo-spatial patterns of zooplankton and the key modulated factors in urban aquatic ecosystem, we investigated zooplankton and water quality from April 2018 to January 2019 in the hinterland of the Three Gorges Reservoir area, Wanzhou City of China. The results indicated that water quality indicated by the trophic state index (TSI) reached a state of mesotrophication to light eutrophication in the Yangtze River, and a state of moderate- to hyper- eutrophication in its tributaries. Based on the biomass of zooplanktons, Asplanchna priodonta was the most common specie in April; Encentrum sp., Filinia cornuta and Epiphanes senta were the most noticeable species in summer; Cyclopoida Copepodid, Sinocalanus dorrii and Philodina erythrophthalma became the dominant species in winter. Generally, rotifers prevailed in April and August, and copepods became the most popular in January. According to canonical correspondence analysis, nitrate, temperature (T), ammonia, water level and permanganate index (CODMn) significantly influenced the community structure of zooplankton (p < 0.05). The dominant species shifts of zooplankton were partly associated with nutrient level (nitrate and ammonia) under periodic water level fluctuations. Rotifers and protozoans were characterized as high T adapted and CODMn-tolerant species comparing with cladocerans and copepods. The ratio of microzooplankton to mesozooplankton (Pmicro/meso) has presented a strongly positive relationship with T (p < 0.001), as well as Pmicro/meso and CODMn (p < 0.001). It implied that zooplankton tended to miniaturize individual size via species shift under high T and/or CODMn conditions induced by global warming and human activities. The information hints us that climate change and human activities are likely to produce fundamental changes in urban aquatic ecosystem by reorganizing biomass structure of the food web in future.

The evolution of convex trade-offs enables the transition towards multicellularity.

The evolutionary transition towards multicellular life often involves growth in groups of undifferentiated cells followed by differentiation into soma and germ-like cells. Theory predicts that germ soma differentiation is facilitated by a convex trade-off between survival and reproduction. However, this has never been tested and these transitions remain poorly understood at the ecological and genetic level. Here, we study the evolution of cell groups in ten isogenic lines of the unicellular green algae Chlamydomonas reinhardtii with prolonged exposure to a rotifer predator. We confirm that growth in cell groups is heritable and characterized by a convex trade-off curve between reproduction and survival. Identical mutations evolve in all cell group isolates; these are linked to survival and reducing associated cell costs. Overall, we show that just 500 generations of predator selection were sufficient to lead to a convex trade-off and incorporate evolved changes into the prey genome.

Application of heavy-ion-beam irradiation to breeding large rotifer.

In larviculture facilities, rotifers are generally used as an initial food source, while a proper size of live feeds to connect rotifer and Artemia associated with fish larval growth is needed. The improper management of feed size and density induces mass mortality and abnormal development of fish larvae. To improve the survival and growth of target larvae, this study applied carbon and argon heavy-ion-beam irradiation in mutation breeding to select rotifer mutants with larger lorica sizes. The optimal irradiation conditions of heavy-ion beam were determined with lethality, reproductivity, mutant frequency, and morphometric characteristics. Among 56 large mutants, TYC78, TYC176, and TYA41 also showed active population growth. In conclusion, (1) heavy-ion-beam irradiation was defined as an efficient tool for mutagenesis of rotifers and (2) the aforementioned 3 lines that have larger lorica length and active population growth may be used as a countermeasure of live feed size gap during fish larviculcure.

Effects of bromate on life history parameters, swimming speed and antioxidant biomarkers in Brachionus calyciflorus.

The baking industries and disinfection of tap water released a considerable amount of bromate into surface water, which has been reported as a carcinogenic compound to mammals. Rotifers play an important role in freshwater ecosystems and are model organisms to assess environmental contamination. In the present study, the effects of different concentrations (0.001, 0.01, 0.1, 1, 10, 100 and 200 mg/L) of bromate on the life-table and population growth parameters were investigated in the rotifer Brachionus calyciflorus. The results showed that the 24-h LC50 of bromate to B. calyciflorus was 365.29 mg/L (95%Cl: 290.37-480.24). Treatments with 0.01, 10 and 200 mg/L bromate shorten the reproductive period. High levels of bromate (100 and 200 mg/L) significantly decreased net reproductive rate, intrinsic rate of population increase, life span, mictic rate of B. calyciflorus. To investigate the underlying mechanisms, swimming speed and antioxidative biomarkers were compared between bromate treatments and the control. The results showed that glutathione (GSH) and malondialdehyde (MDA) contents, total superoxide dismutase (T-SOD) and peroxidase (POD) activities decreased significantly in response to bromate exposure and the reasons required further investigation. Treatments with 0.001-200 mg/L bromate all significantly reduced swimming linear speed to rotifer larvae and treatments with 100-200 mg/L bromate significantly suppressed the swimming linear speed of adult rotifer. These changes would reduce filtration of algal food and could explain the decreased survival and reproduction. Overall, bromate may not show acute toxicity to rotifers, but still have potential adverse effects on rotifer behavior, which may then influence the community structure in aquatic ecosystems.

Gene expression in diapausing rotifer eggs in response to divergent environmental predictability regimes.

In unpredictable environments in which reliable cues for predicting environmental variation are lacking, a diversifying bet-hedging strategy for diapause exit is expected to evolve, whereby only a portion of diapausing forms will resume development at the first occurrence of suitable conditions. This study focused on diapause termination in the rotifer Brachionus plicatilis s.s., addressing the transcriptional profile of diapausing eggs from environments differing in the level of predictability and the relationship of such profiles with hatching patterns. RNA-Seq analyses revealed significant differences in gene expression between diapausing eggs produced in the laboratory under combinations of two contrasting selective regimes of environmental fluctuation (predictable vs unpredictable) and two different diapause conditions (passing or not passing through forced diapause). The results showed that the selective regime was more important than the diapause condition in driving differences in the transcriptome profile. Most of the differentially expressed genes were upregulated in the predictable regime and mostly associated with molecular functions involved in embryo morphological development and hatching readiness. This was in concordance with observations of earlier, higher, and more synchronous hatching in diapausing eggs produced under the predictable regime.

Progress on the usage of the rotifer Brachionus plicatilis in marine ecotoxicology: A review.

The rotifer, Brachionus plicatilis, is a widely used model species in marine ecotoxicology for evaluating pollutions, toxins, and harmful algae. In this paper, the marine ecotoxicology of Brachionus plicatilis was reviewed, including toxicity measurements of harmful algae species and environmental stresses. In addition, marine pollution involving pesticides, heavy metals, drugs, petroleum, and petrochemicals were addressed. Methods for measuring toxicity were also discussed. The standard acute lethal assay and the chronic population dynamics test were indicated as common methods of toxicity evaluating using B. plicatilis. Research on other biomarkers, such as behaviour, enzyme activity, or gene expression, are also reported here, with potential applications for fast detection or the scientific exploration of underlying molecular mechanisms. It is suggested that the methods selected should reflect the experimental purpose. Additionally, series assays should be conducted for comprehensive evaluation of ecotoxicity as well as to elucidate the correct mechanisms. Genetic methods, such as transcriptomics, were suggested as useful tools for exploring the toxicity mechanism using the rotifer B. plicatilis.

Functional groups of rotifers and an exotic species in a tropical shallow lake.

In freshwater environments the rotifer group may be divided into microphagous and raptorial species regarding their feeding patterns, and such guilds differently interact with other community components. Here, we analyzed the influence of cladocerans, cyclopoid nauplii, temperature, food resources and an exotic species on rotifer guilds, based on weekly samplings for 1 year. We have identified rotifer species and their trophi types in order to separate them into the raptorial and microphagous functional groups. The ratio raptorial:microphagous rotifers (Guild ratio, GR) was used in interaction analyses with cladocerans, nauplii, temperature, food resources and the exotic species Kellicottia bostoniensis. Correlations between total rotifers and food (phytoplankton carbon) and temperature were negative and significant, therefore, these factors did not lead to the increase of rotifer community. On the other hand, microphagous rotifers had opposing relation to cladoceran densities, as GR values showed that they became predominant when cladoceran populations declined. The use of density-based GR was adequate, with similar results compared to biomass-based studies regarding interactions with other organisms. Furthermore, we have found no invasive characteristics for the exotic microphagous rotifer, Kellicottia bostoniensis, and it seems to be outcompeted by the native microphagous species.

Growth performance, phenotypic traits, and antioxidant responses of the rotifer Brachionus plicatilis under different proportions of Phaeocystis globosa.

Harmful Phaeocystis blooms disrupt seawater recreation and pose serious challenges to aquatic animals. The growth performance, phenotypic traits, and antioxidant responses of Brachionus plicatilis Müller to different proportions of Phaeocystis globosa were evaluated. B. plicatilis rotifers were exposed to cultures with Chlorella sp. and P. globosa alone and in mixtures of these two algae with proportions of 25%, 50%, and 75%. The total proportions of the two algae were maintained at 100%. Results showed that P. globosa inhibited the rotifer net reproduction rate, intrinsic growth rate, and finite rate of increase (P < 0.01). It induced the formation of defense phenotypic traits in terms of the increased posterolateral spine length and the reduced body length, swimming speed, and grazing rate of B. plicatilis (P < 0.001). Superoxide dismutase and catalase activities decreased, but the reactive oxygen species levels increased as the proportions of P. globosa increased (P < 0.01). The mixture of 50% Chlorella and 50% Phaeocystis positively affected the glutathione content, glutathione peroxidase activity, and generation time of rotifers (P < 0.01). Although P. globosa released toxicants with harmful effects on the growth performance of B. plicatilis, rotifers changed their antioxidant defense system and formed defense phenotypic traits in response to eutrophic conditions.

Temperature-dependent life history and transcriptomic responses in heat-tolerant versus heat-sensitive Brachionus rotifers.

Thermal stress response is an essential physiological trait that determines occurrence and temporal succession in nature, including response to climate change. We compared temperature-related demography in closely related heat-tolerant and heat-sensitive Brachionus rotifer species. We found significant differences in heat response, with the heat-sensitive species adopting a strategy of long survival and low population growth, while the heat-tolerant followed the opposite strategy. In both species, we examined the genetic basis of physiological variation by comparing gene expression across increasing temperatures. Comparative transcriptomic analyses identified shared and opposing responses to heat. Interestingly, expression of heat shock proteins (hsps) was strikingly different in the two species and mirrored differences in population growth rates, showing that hsp genes are likely a key component of a species' adaptation to different temperatures. Temperature induction caused opposing patterns of expression in further functional categories including energy, carbohydrate and lipid metabolism, and in genes related to ribosomal proteins. In the heat-sensitive species, elevated temperatures caused up-regulation of genes related to meiosis induction and post-translational histone modifications. This work demonstrates the sweeping reorganizations of biological functions that accompany temperature adaptation in these two species and reveals potential molecular mechanisms that might be activated for adaptation to global warming.

A demographic and evolutionary analysis of maternal effect senescence.

Maternal effect senescence-a decline in offspring survival or fertility with maternal age-has been demonstrated in many taxa, including humans. Despite decades of phenotypic studies, questions remain about how maternal effect senescence impacts evolutionary fitness. To understand the influence of maternal effect senescence on population dynamics, fitness, and selection, we developed matrix population models in which individuals are jointly classified by age and maternal age. We fit these models to data from individual-based culture experiments on the aquatic invertebrate, Brachionus manjavacas (Rotifera). By comparing models with and without maternal effects, we found that maternal effect senescence significantly reduces fitness for B. manjavacas and that this decrease arises primarily through reduced fertility, particularly at maternal ages corresponding to peak reproductive output. We also used the models to estimate selection gradients, which measure the strength of selection, in both high growth rate (laboratory) and two simulated low growth rate environments. In all environments, selection gradients on survival and fertility decrease with increasing age. They also decrease with increasing maternal age for late maternal ages, implying that maternal effect senescence can evolve through the same process as in Hamilton's theory of the evolution of age-related senescence. The models we developed are widely applicable to evaluate the fitness consequences of maternal effect senescence across species with diverse aging and fertility schedule phenotypes.