The immune response of fairy shrimp Branchinella kugenumaensis against Edwardsiella anguillarum infections by de novo transcriptome analysis.

To explore the immune defense mechanisms of the ancient crustacean fairy shrimp (B.kugenumaensis) and uncover antibacterial-related gene resources, the present study analyzed the pathological changes in B. kugenumaensis infected with E. anguillarum. Differential gene expression changes between the infected and uninfected groups were investigated through comparative transcriptome sequencing to elucidate the molecular responses to the infection. Under transmission electron microscopy, the intestinal mucosal structure of B. kugenumaensis was damaged, the microvilli disappeared, the number of mitochondria and endoplasmic reticulum increased, mitochondria vacuolated and arranged disordered. The transcriptome data indicated that a total of 250,520,580 clean reads were assembled into 66,502 unigenes, with an average length of 789 bp and an N50 length of 1326 bp. Following bacterial infection, approximately 2678 differentially expressed genes (DEGs) were identified, with 1732 genes upregulated and 946 genes downregulated. The detected DEGs related to immune responses, particularly involving apoptosis, lysosome, autophagy, phagosome, and MAPK signaling pathways. Moreover, 9 immunity-related genes with different expressions were confirmed by using real-time quantitative PCR (RT-qPCR). This study first reports the pathogenicity of E. anguillarum on B. kugenumaensis and speculates that immune effectors such as lysozyme and lectin, as well as apoptosis, lysosome, and the MAPK signaling pathway, play crucial roles in the innate immunity of fairy shrimp. These findings deepen our understanding of fairy shrimp immune regulatory mechanisms and provide a theoretical foundation for disease prevention and control.

Frog hatchlings use early environmental cues to produce an anticipatory resource-use phenotype.

Developmental plasticity can occur at any life stage, but plasticity that acts early in development may give individuals a competitive edge later in life. Here, we asked if early (pre-feeding) exposure to a nutrient-rich resource impacts hatchling morphology in Mexican spadefoot toad tadpoles, Spea multiplicata. A distinctive carnivore morph can be induced when tadpoles eat live fairy shrimp. We investigated whether cues from shrimp--detected before individuals are capable of feeding--alter hatchling morphology such that individuals could potentially take advantage of this nutritious resource once they begin feeding. We found that hatchlings with early developmental exposure to shrimp were larger and had larger jaw muscles--traits that, at later stages, increase a tadpole's competitive ability for shrimp. These results suggest that early developmental stages can assess and respond to environmental cues by producing resource-use phenotypes appropriate for future conditions. Such anticipatory plasticity may be an important but understudied form of developmental plasticity.

A chromosome-level reference genome for the Versatile Fairy Shrimp, Branchinecta lindahli.

We present the novel reference genome of the Versatile Fairy Shrimp, Branchinecta lindahli. The Versatile Fairy Shrimp is a freshwater anostracan crustacean found across the western United States from Iowa to Oregon and from Alberta to Baja California. It is an ephemeral pool specialist, living in prairie potholes, irrigation ditches, tire treads, vernal pools, and other temporary freshwater wetlands. Anostracan fairy shrimp are facing global declines with 3 species in California on the Endangered Species list. This species was included in the California Conservation Genomics Project to provide an easily accessible reference genome, and to provide whole-genome resources for a generalist species, which may lead to new insights into Anostracan resiliency in the face of climate change. The final gapped genome comprises 15 chromosome-length scaffolds covering 98.63% of the 384.8 Mb sequence length, and an additional 55 unscaffolded contigs.

A draft reference genome of the Vernal Pool Fairy Shrimp, Branchinecta lynchi.

We present the reference genome of the Vernal Pool Fairy Shrimp Branchinecta lynchi. This branchiopod crustacean is endemic to California's freshwater ephemeral ponds. It faces enormous habitat loss and fragmentation as urbanization and agriculture have fundamentally changed the vernal pool landscape over the past 3 centuries. The assembled genome consists of 22 chromosome-length scaffolds that account for 96.85% of the total sequence. One hundred and ninety-five unscaffolded contigs comprise the rest of the genome's 575.6 Mb length. The genome is substantially complete with a BUSCO score of 90.0%. There is no immediately identifiable sex chromosome, typical for this class of organism. This new resource will permit researchers to better understand the adaptive capacity of this imperiled species, as well as answer lingering questions about anostracan physiology, sex determination, and development.

The immune response of fairy shrimp Streptocephalus sirindhornae against bacterial black disease by de novo transcriptome analysis.

To enhance genomic resources and to understand the molecular immune mechanisms underlying the response of fairy shrimp (Streptocephalus sirindhornae) to pathogens, we first performed a comparative gene transcription analysis from Aeromonas hydrophila-immunized shrimp and from a control group through RNA sequencing. Meanwhile, the differentially expressed genes (DEGs) were investigated, and a total of 46,958,894 clean reads were obtained and then assembled into 73,297 unigenes with an average length of 993 bp and an N50 of 1,458 bp. Unigenes were annotated by comparison with the NR/NT/KO/SwissProt/PFAM/GO and KOG databases, and 28,198 unigenes (38.47%) were annotated in at least one database. After a bacterial challenge, 143 and 287 genes were identified as markedly up- or downregulated, respectively, and 345 were associated with 142 pathways, including the classic immune-related apoptosis, toll-like receptor and MAPK signaling pathways. Moreover, ten differently expressed immune-related genes were confirmed by using quantitative real-time PCR. This study characterized a gene expression pattern for normal and Aeromonas hydrophila-immunized S. sirindhornae for the first time and shed new light on its molecular mechanisms, thus enabling the future efforts of disease control programs for this valuable aquaculture species.

Thermal limits and preferences of large branchiopods (Branchiopoda: Anostraca and Spinicaudata) from temporary wetland arid zone systems.

Large branchiopods are specialist crustaceans adapted for life in temporary, thermally dynamic wetland ecosystems. Certain large branchiopod species are, however, restricted to specific temporary wetland types, exemplified by their physico-chemical and hydroperiod characteristics. Here, we contrasted the thermal preference and critical thermal maxima (CTmax) and minima (CTmin) of southern African anostracans and spinicaudatans found exclusively in either temporary rock-pool or pan wetland types. We hypothesized that environment of origin would be a good predictor of thermal preference and critical thermal limits. To test this, Branchiopodopsis tridens (Anostraca) and Leptestheria brevirostris (Spinicaudata) were collected from rock-pool habitats, while Streptocephalus cafer (Anostraca) and a Gondwanalimnadia sp. (Spinicaudata) were collected from pan habitats. In contrast to our hypothesis, taxonomic relatedness was a better predictor of CTmax and temperature preference than environment of origin. Spinicaudatans were significantly more tolerant of high temperatures than anostracans, with L. brevirostris and Gondwanalimnadia sp. median CTmax values of 45.1 °C and 44.1 °C, respectively, followed by S. cafer (42.8 °C) and B. tridens (41.4 °C). Neither environment or taxonomic relatedness were good predictors of CTmin trends, with B. tridens (0.9 °C) and Gondwanalimnadia sp. (2.1 °C) having the lowest median CTmin values, followed by L. brevirostris (3.4 °C) and S. cafer (3.6 °C). On the contrary, temperature preferences differed according to taxa, with spinicaudatans significantly preferring higher temperatures than anostracans. Leptestheria brevirostris and Gondwanalimnadia sp. both spent most time at temperatures 30-32 °C, S. cafer at 18-20 °C and B. tridens at 21-23 °C. Constrained thermal traits reported here suggest that the studied anostracans might be more susceptible to projected climatic warming than the spinicaudatans, irrespective of habitat type, however, these taxa may also compensate through phenotypic plasticity.

Ecotoxicological characteristics and ecological risk assessment of trace elements in the bottom sediments of the Roznów reservoir (Poland).

The bottom sediments in catchment areas behind dams play a significant role in water ecosystems. On the other hand, the structure of sediments makes them a natural geosorbent, in which pollutants introduced to the aquatic environment accumulate. The use of biotests is recognised as an important approach for the assessment of the quality of bottom sediments, as the chemical analysis of sediment samples alone does not provide evidence of the impact of contaminants on biota. The aim of the study was to apply the chemical and ecological indices to determine the potential risk posed by trace elements in the bottom sediments and to evaluate sediment toxicity using organisms belonging to two taxonomic groups, i.e., plants (Phytotoxkit) and crustaceans (Rapidtoxkit). The 46 sediment samples were taken from the Roznów Dam Reservoir in Southern Poland. The mean concentration of the trace elements in the sediments was 5.22 mg As; 0.26 mg Cd; 63.23 mg Cr; 28.65 mg Cu; 37.11 mg Ni; 11.15 mg Pb; 69.69 mg Zn and 0.09 mg Hg ∙ kg-1 d.m. The mean probable effect concentration quotient (PECq) value among different sampling sites ranged between 0.04 and 0.33 suggested moderate potential toxicity to the biological communities in bottom sediments. The Ni was potentially the most toxic element for biota in the Roznów Reservoir. The sensitivity of organisms formed the following order: Thamnocephalus platyurus >Lepidium sativum >Sinapis alba >Sorghum saccharatum. For the plants, the stimulating effect of bottom sediments on root growth was often indicated, while a toxic effect was demonstrated for T. platyurus in 80% of the samples. However, the correlation analysis and PCA results showed that trace elements that originated from similar sources were associated to the toxicity of sediments towards T. platyurus, while ecotoxicity for plants could not be explained by the content of trace elements in bottom sediments. T. platyurus is a good indicator for predicting the toxicity of bottom sediments from the Roznów Reservoir. However, our study found that both chemical and ecotoxicological analyses are important for a comprehensive evaluation of the quality of bottom sediments.

Physicochemical and Toxicological Assay of Leachate from Malt Spent Rootlets Biochar.

The aim of this study was to characterize the leachate derived from biochar produced from malt spent rootlets (MSR) and to evaluate the required washing level in order to provide water free from inorganic substances. MSR biochar was placed in a column and subjected to six serial washes with distilled water, and the leachate was analysed for main anions and heavy metals. The 1st wash aliquot contained increased levels of mainly phosphates (980 mg/L) and chlorides (760 mg/L), and lower levels of nitrates, sulfates, fluoride and bromide, which were decreased over washes. Zero concentrations were observed after three washes for most anions. The increased levels of Zn, Be, Cs, Mn, V and Se determined in the 1st wash aliquot were eliminated in the successive washes. The toxic potency of each wash aliquot, determined by the use of the fairy shrimp Thamnocephalus platyurus showed that the 1st and 2nd MSR biochar leachates were toxic with 4.52 and 1.46 toxic units (TU), respectively, followed by a significant elimination of toxicity after further washes.

Climate change jeopardizes the persistence of freshwater zooplankton by reducing both habitat suitability and demographic resilience.

BACKGROUND: Higher temperatures and increased environmental variability under climate change could jeopardize the persistence of species. Organisms that rely on short windows of rainfall to complete their life-cycles, like desert annual plants or temporary pool animals, may be particularly at risk. Although some could tolerate environmental changes by building-up banks of propagules (seeds or eggs) that buffer against catastrophes, climate change will threaten this resilience mechanism if higher temperatures reduce propagule survival. Using a crustacean model species from temporary waters, we quantified experimentally the survival and dormancy of propagules under anticipated climate change and used these demographic parameters to simulate long term population dynamics. RESULTS: By exposing propagules to present-day and projected daily temperature cycles in an 8 month laboratory experiment, we showed how increased temperatures reduce survival rates in the propagule bank. Integrating these reduced survival rates into population models demonstrated the inability of the bank to maintain populations; thereby exacerbating extinction risk caused by shortened growing seasons. CONCLUSIONS: Overall, our study demonstrates that climate change could threaten the persistence of populations by both reducing habitat suitability and eroding life-history strategies that support demographic resilience.

Acute ecotoxicity bioassay using Dendrocephalus brasiliensis: alternative test species for monitoring of contaminants in tropical and subtropical freshwaters.

In International guidelines for standard ecotoxicological bioassays, Daphnia magna is the most applied microcrustacea for assessing toxicity of different pollutants. However, in research realized in tropical and subtropical areas, autochthonous species must be prioritized because they are adapted to the specificities of ecosystems. In this sense, the present study aimed to assess and compare (with D. magna) the sensitivity of the tropical species Dendrocephalus brasiliensis as alternative test species for monitoring of contaminants in tropical and subtropical freshwaters, by carrying out acute toxicity tests with different pollutants. According results, D. brasiliensis presented EC50-48 h values lower than D. magna for all substances tested, indicating higher sensitivity of the tropical organism in relation to the temperate organism. Furthermore, comparing the results obtained with data from other studies, D. brasiliensis is more sensitive to the chemicals tested than D. magna and has similar sensitivity to Pseudosida ramosa and Ceriodaphnia dubia, common species in tropical areas. In view of this, we suggest that D. brasiliensis can be used as alternative test species for monitoring of contaminants in tropical and subtropical freshwaters.

Occurrence of Dendrocephalus brasiliensis Pesta, 1921 (Crustacea, Anostraca) in the Caras river, southern Ceara, Brazil.

Occurrence of Dendrocephalus brasiliensis Pesta, 1921 (Crustacea, Anostraca) in the Caras river, southern Ceara, Brazil. The specimens were collected in March and April 2014. The new occurrence extends the distribution and update area of occupancy of the species, which is characterized by a specific habitat: temporary lakes.

Wnt repertoire and developmental expression patterns in the crustacean Thamnocephalus platyurus.

Wnt genes are a family of conserved glycoprotein ligands that play a role in a wide variety of cell and developmental processes, from cell proliferation to axis elongation. There are 13 Wnt subfamilies found among metazoans. Eleven of these appear conserved in arthropods with a pattern of loss during evolution of as many as six subfamilies among hexapods. Here we report on Wnt genes in the branchiopod crustacean, Thamnocephalus platyurus, including the first documentation of the expression of the complete Wnt gene family in a crustacean. Our results suggest fewer Wnt genes were retained in Thamnocephalus than in the related crustacean, Daphnia, although the Thamnocephalus Wnt repertoire is larger than that found in insects. We also find an intriguing pattern of staggered expression of Wnts-an anterior-posterior stagger within the posterior growth zone and a dorsal-ventral stagger in the developing segments-suggesting a potential for subfunctionalization of Wnts in these regions.

Investigating the Toxicity of the Aeruginosin Chlorosulfopeptides by Chemical Synthesis.

Harmful algal blooms are becoming more prevalent all over the world, and identification and mechanism-of-action studies of the responsible toxins serve to protect ecosystems, livestock, and humans alike. In this study, the chlorosulfopeptide aeruginosin 828A, which rivals the well-known toxin microcystin LR in terms of crustacean toxicity, has been synthesized for the first time. Furthermore, three congeners with different permutations of the chloride and sulfate groups were prepared, thereby enabling toxicity studies without the risk of contamination by other natural toxins. Toxicity assays with the sensitive crustacean Thamnocephalus platyurus demonstrated that the introduction of a sulfate group leads to pronounced toxicity, and NMR spectroscopic evidence suggests that the chloride substituent modulates the conformation, which in turn might influence protease inhibition.

Preparation of Fluorescent Microcystin Derivatives by Direct Arginine Labelling and Their Biological Evaluation.

Microcystin is the most prevalent toxin produced by cyanobacteria and poses a severe threat to livestock, humans and entire ecosystems. We report the preparation of a series of fluorescent microcystin derivatives by direct arginine labelling of the unprotected peptides at the guanidinium side chain. This new method allows a simple late-stage diversification strategy for native peptides devoid of protecting groups under mild conditions. A series of fluorophores were conjugated to microcystin-LR in good to very good yield. The fluorescent probes displayed biological activity comparable to that of unlabelled microcystin, in both phosphatase inhibition assays and toxicity tests on the crustacean Thamnocephalus platyurus. In addition, we demonstrate that the fluorescent probes penetrated Huh7 cells. Whole-animal imaging was performed on T. platyurus: labelled compound was mainly observed in the digestive tract.

Is distribution of cold stenotherms constrained by temperature? The case of the Arctic fairy shrimp (Branchinecta paludosa O.F. Müller 1788).

Small water bodies in cold climate respond fast to global warming, and species adapted to such habitats may be valuable indicators for climate change. We investigated the geographical and physiological temperature limits of the Arctic fairy shrimp (Branchinecta paludosa), which is common in cold water arctic ponds, but at present retracts its range in alpine areas along its southern outreach of Norway. Seasonal logging of water temperatures along an altitudinal transect revealed an upper temperature limit of 12.7°C for its presence, which closely matched a calculated upper temperature limit of 12.9°C throughout its entire Norwegian range. Field data hence point to cold stenotherm features, which would be consistent with its Arctic, circumpolar distribution. Lab experiments, on the other hand, revealed a linear increase in respiration over 10-20°C. When fed ad libitum somatic growth increased with temperature, as well, without negative physiological impacts of higher temperatures. The absence of Branchinecta paludosa in ponds warmer than 13°C could still be due to a mismatch between temperature dependent metabolism and limited energy supply in these ultraoligotrophic water bodies. We discuss the concept of cold stenothermy in this context, and the impacts of regional warming on the future distribution of the Arctic fairy shrimp.

The microbiotest battery as an important component in the assessment of snowmelt toxicity in urban watercourses--preliminary studies.

The aim of the study was to use a battery of biotests composed of producers (Selenastrum capricornutum, Sorghum saccharatum, Lepidium sativum, and Sinapis alba), consumers (Thamnocephalus platyurus), and decomposers (Tetrahymena thermophila) to evaluate the toxicity of snowmelt and winter storm water samples. The toxicity of the samples collected in the winter period December to February (2010-2011), in one of the largest agglomerations in Poland, the city of Lodz, was compared to that of storm water samples taken under similar conditions in June. The most toxic snowmelt samples were found to be high acute hazard (class IV), while the remaining samples were rated as slight acute hazard (class II). L. sativum (in the Phytotox test) was the most sensitive test organism, giving 27 % of all toxic responses, followed by S. capricornutum with 23 % of all responses. T. thermophila was the least sensitive, with only 2 % of all toxic responses. The greatest range of toxicity was demonstrated by samples from the single family house catchment: no acute hazard (class I) to high acute hazard (class IV).

LETHAL EFFECTS OF PESTICIDES ON THE NAUPLII FAIRY SHRIMP, BRANCHINELLA THAILANDENSIS.

During agricultural activities, large amounts of insecticides and herbicides are used for controlling pests. The overspraying of pesticides may contaminate freshwater bodies, as well as having a significant effect on aquatic animals. To determine the potential risk of pesticides, the acute toxicity of insecticides and herbicides on the nauplii fairy shrimp, Branchinella thailandensis were evaluated. The organisms were exposed to different concentrations of insecticides (chlorpyrifos and carbaryl), and herbicides (paraquat and 2,4-D) for 24 h, after which the LC50 values were compared. The acute toxicity tests determined that the LC50 values of chlorpyrifos and carbaryl after 24 h were 0.00186 mg/L and 0.21 mg/L, respectively. For herbicides, paraquat and 2,4-D, the LC50 values were 0.11 mg/L and 27.91 mg/L. Therefore, chlorpyrifos insecticides were found to be more toxic than herbicides (2,4-D and paraquat) with regard to the nauplii fairy shrimp. The high toxicity of pesticides has a long-term, and potentially damaging effect on aquatic organisms in the application areas.

Chemical pollution and toxicity of water samples from stream receiving leachate from controlled municipal solid waste (MSW) landfill.

The present study was aimed to determine the impact of municipal waste landfill on the pollution level of surface waters, and to investigate whether the choice and number of physical and chemical parameters monitored are sufficient for determining the actual risk related to bioavailability and mobility of contaminants. In 2007-2012, water samples were collected from the stream flowing through the site at two sampling locations, i.e. before the stream׳s entry to the landfill, and at the stream outlet from the landfill. The impact of leachate on the quality of stream water was observed in all samples. In 2007-2010, high values of TOC and conductivity in samples collected down the stream from the landfill were observed; the toxicity of these samples was much greater than that of samples collected up the stream from the landfill. In 2010-2012, a significant decrease of conductivity and TOC was observed, which may be related to the modernization of the landfill. Three tests were used to evaluate the toxicity of sampled water. As a novelty the application of Phytotoxkit F™ for determining water toxicity should be considered. Microtox(®) showed the lowest sensitivity of evaluating the toxicity of water samples, while Phytotoxkit F™ showed the highest. High mortality rates of Thamnocephalus platyurus in Thamnotoxkit F™ test can be caused by high conductivity, high concentration of TOC or the presence of compounds which are not accounted for in the water quality monitoring program.

The in-vitro antibacterial effects of organic salts, chemical disinfectants and antibiotics against pathogens of black disease in fairy shrimp of Thailand.

The antibacterial effects of organic salts, chemical disinfectants and antibiotics were evaluated on cultures of Aeromonas hydrophila C03, Aeromonas sobria C26, A. sobria C29, Aeromonas caviae C24 and Acinetobacter sp. SH-94B, the pathogens that cause black disease found in fairy shrimps (Streptocephalus sirindhornae Sanoamuang et al. (2000) and Branchinella thailandensis Sanoamuang, Saengphan & Murugan) of Thailand. The minimal inhibitory concentrations (MICs) of organic salts (sodium chloride and potassium chloride) and antibiotics (oxytetracycline dihydrate, streptomycin sulphate, kanamycin monosulphate, chloramphenicol and ampicillin) were determined using the agar-dilution method. The effect of chemical disinfectants (sodium hypochlorite and chlorine dioxide) was evaluated by exposing bacteria to different concentrations of these chemicals for different periods of time. Interestingly, all strains were intrinsically resistant to 0.25-3% sodium chloride and potassium chloride. The effect of sodium hypochlorite was greater than that of chlorine dioxide, and 5-20 µg mL(-1) of sodium hypochlorite was sufficient to inhibit the growth of these bacteria, but the exposure time varied, depending on the bacterial species. Of the antibiotics tested, chloramphenicol and oxytetracycline dihydrate completely inhibited the selected strains. Chloramphenicol showed the highest antibacterial effect against all pathogenic species--the MIC and minimal bactericidal concentration (MBC) ranged from 0.8 to 3.1 µg mL(-1) from 3.1 to 6.25 µg mL(-1), respectively. To achieve control of black disease during cultivation of fairy shrimp, data derived from this study can be used as a basis for further toxicity tests in vivo.

A new species of Streptocephalus (Crustacea: Anostraca: Streptocephalidae) from the Western Ghats, India, with a key to the Asian species.

We present and describe the fairy shrimp Streptocephalus sahyadriensis sp. nov. from the Western Ghats of India. This species is most similar to S. simplex Gurney, 1906 and S. dichotomus Baird, 1860 sharing similar basic antennal appendage morphology. However, S. sahyadriensis sp. nov. differs in the form of the ornamentation on the peduncle and the arrangement and form of spines on the antennal appendage. The three species also have different egg surface morphologies. The Asian species of Streptocephalus are discussed and a key to species is provided.