A living bdelloid rotifer from 24,000-year-old Arctic permafrost.

In natural, permanently frozen habitats, some organisms may be preserved for hundreds to tens of thousands of years. For example, stems of Antarctic moss were successfully regrown from an over millennium-old sample covered by ice for about 400 years1. Likewise, whole campion plants were regenerated from seed tissue preserved in relict 32,000-year-old permafrost2, and nematodes were revived from the permafrost of two localities in northeastern Siberia, with source sediments dated over 30,000 years BP3. Bdelloid rotifers, microscopic multicellular animals, are known for their ability to survive extremely low temperatures4. Previous reports suggest survival after six to ten years when frozen between -20° to 0°C4-6. Here, we report the survival of an obligate parthenogenetic bdelloid rotifer, recovered from northeastern Siberian permafrost radiocarbon-dated to ∼24,000 years BP. This constitutes the longest reported case of rotifer survival in a frozen state. We confirmed the finding by identifying rotifer actin gene sequences in a metagenome obtained from the same sample. By morphological and molecular markers, the discovered rotifer belongs to the genus Adineta, and aligns with a contemporary Adineta vaga isolate collected in Belgium. Experiments demonstrated that the ancient rotifer withstands slow cooling and freezing (∼1°C min-1) for at least seven days. We also show that a clonal culture can continuously reproduce in the laboratory by parthenogenesis.

A rotifer-derived paralytic compound prevents transmission of schistosomiasis to a mammalian host.

Schistosomes are parasitic flatworms that infect over 200 million people, causing the neglected tropical disease, schistosomiasis. A single drug, praziquantel, is used to treat schistosome infection. Limitations in mass drug administration programs and the emergence of schistosomiasis in nontropical areas indicate the need for new strategies to prevent infection. It has been known for several decades that rotifers colonizing the schistosome's snail intermediate host produce a water-soluble factor that paralyzes cercariae, the life cycle stage infecting humans. In spite of its potential for preventing infection, the nature of this factor has remained obscure. Here, we report the purification and chemical characterization of Schistosome Paralysis Factor (SPF), a novel tetracyclic alkaloid produced by the rotifer Rotaria rotatoria. We show that this compound paralyzes schistosome cercariae and prevents infection and does so more effectively than analogous compounds. This molecule provides new directions for understanding cercariae motility and new strategies for preventing schistosome infection.

The effects of environmental factors and geographic distance on species turnover in an agriculturally dominated river network.

Both environmental and geographic factors interact to structure the metacommunities in river networks, but the importance of these factors is difficult to distinguish. We used six aquatic taxonomic groups to test the relationship between environmental and geographic factors and their effect on species turnover patterns in an agriculturally dominated river (Chaohu Lake Basin, China). The relationships between three dissimilarity indices and geographic distance were assessed using the Mantel test while considering the differences in environmental factors between sites. Then, we employed a variation partitioning method to distinguish the isolated and combined effects of environmental and geographic distance on species turnover. There were significant relationships between environmental distance and species turnover in all groups. All organisms except periphytic diatoms were significantly correlated with two geographic (Euclidean and network) distances when the Chao dissimilarity index was considered. The results suggest that the strength of the correlations changed with environmental and geographic distances and with the aquatic community. The communities displayed more complex relationships with the distance measures when different dissimilarity (Jaccard, Chao, and Bray-Curtis dissimilarity) indices were considered. Nevertheless, aquatic communities are strongly influenced by both environmental and geographic distance, and the former has a stronger effect than the latter.

Microplankton dynamics of a coastal lagoon, Chilika: interactive effect of environmental parameters on microplankton groups.

Microplankton population of Asia's largest coastal lagoon Chilika was studied for five major groups, bacillariophyceae, cyanophyceae, chlorophyceae, dinophyceae, rotifera, and tintinninae. The study reported presence of 233 species of microplankton whose average annual abundance was 1631 cells/l. The physicochemical parameters contributing to the spatio-temporal fluctuations in microplankton diversity, abundance, and community structure were identified as salinity, pH, DO, nitrate, and silicate. Salinity, transparency, depth, and silicate most explained the abundance of bacillariophyceae; nitrate, pH, and DO influenced cyanophyceae; salinity, transparency, and chlorophyll concentration influenced chlorophyceae; salinity, depth, and water temperature influenced dinophyceae; salinity, free CO2, and nitrate-influenced rotifers, whereas salinity, pH, DO, and depth influenced tintinnids. Biotic-abiotic relationships revealed particular preference of environmental conditions at species level in groups like bacillariophyceae, cyanophyceae, and dinophyceae. Although the lagoon is shallow, bacillariophyceae-environment interaction showed depth can be a critical factor for species like Aulocoseira sp., Amphipleura sp., and Rhophalodia sp. Species of dinoflagellates like Dinophysis caudata, Noctiluca scintillans, and Protoperidinium proliferated in lower level of silicate. Unlike other cyanophyceae species Streptococcus sp., Chroococcus sp., Diplococcus sp., Aphanocapsa sp., and Gloeocapsa sp. were negatively influenced by nitrate concentration. The study provides better scope for ecological management of the lagoon with respect to conserving biodiversity and hydrological quality of the ecosystem.

Rapid Fabrication of Chip-Based Physiometers for Neurobehavioral Toxicity Assays Using Rotifers Brachionus calyciflorus.

An increased interest in implementations of Lab-on-a-Chip (LOC) technologies for in-situ analysis of multicellular metazoan model organisms and their embryonic stages demands development of new prototyping techniques. Due to size of multicellular organisms the fabrication of soft-lithography molds requires features with high aspect ratios as well as deposition of layers with significant thicknesses. This makes them time consuming and difficult to fabricate using conventional photolithography techniques. In this work we describe development of a rapid technique capable of generating thick films achieved with high viscosity SU-8 and used in fabricating master templates for high aspect ratio micro- and mesofluidic devices. The cost effective and rapid method eliminated the need for multiple spin coating cycles as well as edge bead artifacts while preserving low surface roughness and superior surface uniformity. Due to elimination of spin coating steps, typically constrained to clean room facilities, the new method allows to significantly reduce microfabrication costs. We have utilized the prototyping technique to develop proof-of-concept chip-based devices capable of effectively caging freshwater rotifers Brachionus calyciflorus for high-definition video-microscopy analysis. The combination of time-resolved video-microscopy and chip-based physiometers enabled us to demonstrate new applications for neurobehavioral assays utilizing non-invasive sub-lethal end-points.

Freeze-thaw revival of rotifers and algae in a desiccated, high-elevation (5500 meters) microbial mat, high Andes, Perú.

This is the first study of the highest elevation cyanobacteria-dominated microbial mat yet described. The desiccated mat was sampled in 2010 from an ephemeral rock pool at 5500 m above sea level in the Cordillera Vilcanota of southern Perú. After being frozen for 6 years at -20 °C in the lab, pieces of the mat were sequenced to fully characterize both the 16 and 18S microbial communities and experiments were conducted to determine if organisms in the mat could revive and become active under the extreme freeze-thaw conditions that these mats experience in the field. Sequencing revealed an unexpectedly diverse, multi-trophic microbial community with 16S OTU richness comparable to similar, seasonally desiccated mats from the Dry Valleys of Antarctica and low elevation sites in the Atacama Desert region. The bacterial community of the mat was dominated by phototrophs in the Cyanobacteria (Nostoc) and the Rhodospirillales, whereas the eukaryotic community was dominated by predators such as bdelloid rotifers (Philodinidae). Microcosm experiments showed that bdelloid rotifers in the mat were able to come out of dormancy and actively forage even under realistic field conditions (diurnal temperature fluctuations of -12 °C at night to + 27 °C during the day), and after being frozen for 6 years. Our results broaden our understanding of the diversity of life in periodically desiccated, high-elevation habitats and demonstrate that extreme freeze-thaw cycles per se are not a major factor limiting the development of at least some members of these unique microbial mat systems.

Colonization of maritime glacier ice by bdelloid Rotifera.

Very few animal taxa are known to reside permanently in glacier ice/snow. Here we report the widespread colonization of Icelandic glaciers and ice fields by species of bdelloid Rotifera. Specimens were collected within the accumulation zones of Langjökull and Vatnajökull ice caps, among the largest European ice masses. Rotifers reached densities up to ∼100 individuals per liter-equivalent of glacier ice/snow, and were freeze-tolerant. Phylogenetic analyses indicate that glacier rotifers are polyphyletic, with independent ancestries occurring within the Pleistocene. Collectively, these data identify a previously undescribed environmental niche for bdelloid rotifers and suggest their presence in comparable habitats worldwide.

Predation and transport of persistent pathogens in GAC and slow sand filters: a threat to drinking water safety?

Zooplankton has been shown to transport internalized pathogens throughout engineered drinking water systems. In this study, experimental measurements from GAC and SSF filtration tests using high influent concentrations of Cryptosporidium (1.3 × 10(6) and 3.3 × 10(4) oocysts L(-1)) and Giardia (4.8 × 10(4) cysts L(-1)) are presented and compared. A predation and transport conceptual model was developed to extrapolate these results to environmental conditions of typical (oo)cyst concentrations in surface water in order to predict concentrations of internalized (oo)cysts in filtered water. Pilot test results were used to estimate transport and survival ratios of internalized (oo)cysts following predation by rotifers in the filter beds. Preliminary indications of lower transport and survival ratios in SSF were found as compared with GAC filters. A probability of infection due to internalized (oo)cysts in filtered water was calculated under likeliest environmental conditions and under a worst-case scenario. Estimated risks under the likeliest environmental scenario were found to fall below the tolerable risk target of 10(-4) infections per person per year. A discussion is presented on the health significance of persistent pathogens that are internalized by zooplankton during granular filtration processes and released into treated water.

Efficacy of binary combinations of botanical pesticides for rotifer elimination in microalgal cultivation.

Binary interactions of celangulin, matrine and toosendanin against the rotifer Brachionus plicatilis were studied. Types of interactions (antagonism, synergism and addition) were dependent on the biocides themselves and their ratios in combinations. Mixtures of matrine/toosendanin mainly produced addition owing to their similar modes of action aiming at the nervous system. Combinations of celangulin mixed with matrine or toosendanin at 1:9 exhibited synergism, which is attributed to the interference of matrine or toosendanin with the detoxification enzymes of celangulin. Both the synergistic combinations were inappropriate for rotifer extermination in Isochrysis sp. cultivation owing to the high phytotoxicity resulting from the absence of cell walls. However, the celangulin/toosendanin (1:9) mixture decreased rotifer reproduction without damaging cells of Chlorella and Nannochloropsis sp. Application of frequent, low doses of celangulin/toosendanin (1:9) mixture also reduced the dosage of biocides, thereby reducing the cost of exterminating rotifers, and indicating a considerable practical application in microalgal cultivation.

Bdelloid rotifer, Philodina species in the breeding containers of Aedes aegypti and Aedes albopictus.

The vector mosquitoes of dengue and chikungunya fever, Aedes aegypti and Aedes albopictus have adapted to feed on humans and undergo larval and pupal development in natural and artificial freshwater collections. Although several studies reported, still, much information is required to understand the successful survival of Aedes mosquitoes in small temporary containers. In an investigation conducted in the chikungunya affected areas of Kerala state, India, the presence of Bdelloid rotifer, Philodina in 95% of breeding habitats of Ae. aegypti and Ae. albopictus was recorded. The role of Philodina in the breeding containers was investigated. It was found that while in control the number of Philodina was found increasing in the water sample during the study period of seven days, the number found decreased in the containers with larvae of Aedes. The gut content analysis also confirmed the presence of the rotating wheel, corona of Philodina in some of the specimen suggests its role as major larval food.

Widespread secondary contact and new glacial refugia in the halophilic rotifer Brachionus plicatilis in the Iberian Peninsula.

Small aquatic organisms harbour deep phylogeographic patterns and highly structured populations even at local scales. These patterns indicate restricted gene flow, despite these organisms' high dispersal abilities, and have been explained by a combination of (1) strong founder effects due to rapidly growing populations and very large population sizes, and (2) the development of diapausing egg banks and local adaptation, resulting in low effective gene flow, what is known as the Monopolization hypothesis. In this study, we build up on our understanding of the mitochondrial phylogeography of the halophilic rotifer Brachionus plicatilis in the Iberian Peninsula by both increasing the number of sampled ponds in areas where secondary contact is likely and doubling sample sizes. We analyzed partial mitochondrial sequences of 252 individuals. We found two deep mitochondrial DNA lineages differing in both their genetic diversity and the complexity of their phylogeographic structure. Our analyses suggest that several events of secondary contact between clades occurred after their expansion from glacial refugia. We found a pattern of isolation-by-distance, which we interpret as being the result of historical colonization events. We propose the existence of at least one glacial refugium in the SE of the Iberian Peninsula. Our findings challenge predictions of the Monopolization hypothesis, since coexistence (i.e., secondary contact) of divergent lineages in some ponds in the Iberian Peninsula is common. Our results indicate that phylogeographic structures in small organisms can be very complex and that gene flow between diverse lineages after population establishment can indeed occur.

Genome size evolution at the speciation level: the cryptic species complex Brachionus plicatilis (Rotifera).

BACKGROUND: Studies on genome size variation in animals are rarely done at lower taxonomic levels, e.g., slightly above/below the species level. Yet, such variation might provide important clues on the tempo and mode of genome size evolution. In this study we used the flow-cytometry method to study the evolution of genome size in the rotifer Brachionus plicatilis, a cryptic species complex consisting of at least 14 closely related species. RESULTS: We found an unexpectedly high variation in this species complex, with genome sizes ranging approximately seven-fold (haploid '1C' genome sizes: 0.056-0.416 pg). Most of this variation (67%) could be ascribed to the major clades of the species complex, i.e. clades that are well separated according to most species definitions. However, we also found substantial variation (32%) at lower taxonomic levels--within and among genealogical species--and, interestingly, among species pairs that are not completely reproductively isolated. In one genealogical species, called B. 'Austria', we found greatly enlarged genome sizes that could roughly be approximated as multiples of the genomes of its closest relatives, which suggests that whole-genome duplications have occurred early during separation of this lineage. Overall, genome size was significantly correlated to egg size and body size, even though the latter became non-significant after controlling for phylogenetic non-independence. CONCLUSIONS: Our study suggests that substantial genome size variation can build up early during speciation, potentially even among isolated populations. An alternative, but not mutually exclusive interpretation might be that reproductive isolation tends to build up unusually slow in this species complex.

Combined effects of heavy metal (Hg) concentration and algal (Chlorella vulgaris) food density on the population growth of Brachionus calyciflorus (Rotifera: Brachionidae).

The combined effects of two food levels (0.5 x 10(6) and 1.5 x 10(6) cells ml(-1) of Chlorella vulgaris) and five concentrations (0, 0.000625, 0.00125, 0.0025, 0.005 mg l(-1) of HgCl2) of mercury on the population growth of the rotifer Brachionus calyciflorus was evaluated. The growth experiments were conducted for 18 days at 23+/-1 degrees C under continuous fluorescent illumination. For each food level - heavy metal combination, we maintained 3 replicates. Our data showed that regardless of food level, increase in the heavy metal concentration in the medium resulted in decreased population growth of B. calyciflorus. At any given heavy metal concentration, B. calyciflorus grown under higher food levels had higher population abundance. The rate of population increase was significantly influenced by both the heavy metal concentration and the algal level. The highest population growth rate (0.435+/-0.003 per day) was observed in controls at 1.5 x 10(6) cells ml(-1). The results of this study were discussed in relation to the protective role of algal density against heavy metal toxicity.

The influence of soluble microbial products on microbial community composition: hypothesis of microbial community succession.

Soluble microbial products (SMP) are organic compounds produced by activated sludge microorganisms as they degrade substrates. They include by-products of microbial activity, death and lysis. The available literature does not reveal how SMP influence microbial community composition. In this regard, we microscopically studied changes in composition of microbial communities, especially protozoa and metazoa, under the influence of increased as well as reduced levels of SMP. The presence of SMP at high level significantly caused changes in microbial community composition. Microbial species shifted from attached ciliates (12-175 microm) to free-swimming and crawling ciliates (35-330 microm) and then invertebrates, which included rotifers (0.2-1 mm) and nematodes (1-50 mm). The shift of small-size microorganisms to large ones was observed as one of the most significant influences of SMP. Attached ciliates reappeared when we removed the SMP that had accumulated in the bioreactors - we have called this as the resurrection phenomenon of microorganisms. Such rapid changes in microbial community composition were not observed in the experiment with low concentration of SMP. Overall, the results suggest that accumulation of SMP is one of the intrinsic regulatory mechanisms that control viability and dormancy of microbial communities in activated sludge.

Transmission of specific groups of bacteria through water distribution system.

Microbial contamination of a water distribution system was examined. The number and the taxonomy of non-pigmented and pigmented heterotrophic bacteria (HB), number of bacteria (Pseudomonas sp., Enterococcus sp., Campylobacter sp., Yersinia sp., representatives of the Enterobacteriaceae, coagulase-positive staphylococci, and C. pefringens) in the bulk water phase, biomass of zoogloeal aggregates of bacteria, fungi, algae, protozoa and rotifers (ZABFAPR) (separated from the above on 5 microm pore size filters) and in pipe sediments was determined. An increased number of HB occurred at the sampling sites situated as close as 4.2 km to the Water Treatment Plant (WTP), and was especially significant at 10.3 km. It was shown that the main reservoir of hygienically relevant bacteria did not occur in the water phase which is monitored in routine control analyses carried out by the WTP laboratories, but in the ZABFAPR biomass not monitored so far.

Distribution and seasonal variability in the benthic eukaryotic community of Río Tinto (SW, Spain), an acidic, high metal extreme environment.

The eukaryotic community of the Río Tinto (SW, Spain) was surveyed in fall, winter and spring through the combined use of traditional microscopy and molecular approaches, including Denaturing Gradient Gel Electrophoresis (DGGE) and sequence analysis of 18S rRNA gene fragments. Eukaryotic assemblages of surface sediment biofilms collected in January, May and September 2002 were compared from 13 sampling stations along the river. Physicochemical data revealed extremely acidic conditions (the pH ranged from 0.9 to 2.5) with high concentrations of heavy metals, including up to 20 mg l(-1) Fe, 317 mg l(-1) Zn, 47 mg l(-1) As, 42 mg l(-1) Cd and 4 mg l(-1) Ni. In total, 20 taxa were identified, including members of the Bacillariophyta, Chlorophyta and Euglenophyta phyla as well as ciliates, cercomonads, amoebae, stramenopiles, fungi, heliozoans and rotifers. In general, total cell abundances were highest in fall and spring but decreased drastically in winter, and the sampling stations with the most extreme conditions showed the lowest number of cells, as well as the lowest diversity. Species diversity did not vary much during the year. Only the filamentous algae showed a dramatic seasonal change, since they almost disappeared in winter and reached the highest biomass during the summer. Principal Components Analysis (PCA) showed a high inverse correlation between pH and most of the heavy metals analyzed, as well as Dunaliella sp., while Chlamydomonas sp. was directly related to pH during May and September. Three heavy metals (Zn, Cu and Ni) remained separate from the rest and showed an inverse correlation with most of the species analyzed, except for Dunaliella sp.

The microfauna communities and operational monitoring of an activated sludge plant in China.

The composition of the microfauna community in aeration tanks at the Baoding Sewage Treatment Plant in China was analysed each week from July 2002 to July 2003. The community composition of these microfauna populations was compared with effluent quality data recorded on the same days. A total of 94 species of ciliates, 40 species of amoebae and 13 species of large flagellates were identified in the 50 samples analysed. Numbers of metazoa including rotifers, nematodes, gastrotrichs and oligochaeta were also recorded. Although, Aspidisca cicada showed the highest mean abundance and was present in 98% of the samples, factor analysis revealed, among other things, that high Vorticella convallaria and Arcella hemisphaerica populations correlated with good performance of the treatment plant, while high numbers of Litonotus obtusus indicated poor conditions for settlement of sludge. These results show some agreement with an earlier study of sewage plants in Beijing, but analysis of more plants having a diversity of input components working under a range of different operating conditions should be performed to gain a general understanding of the value of indicator species for predicting the efficiency of activated sludge plants in China.

Dynamics of protozoan and metazoan communities in a full scale wastewater treatment plant by rotating biological contactors.

Performance of a full-scale wastewater treatment plant by rotating biological contactors (RBC) system was monitored during a year by physico-chemical and microbial characterisation. Six points along wastewater treatment were selected in the plant: three points along the water line (influent, sedimentation tank and effluent) and three points along RBC system (RBC1, RBC2 and RBC3). Although a large seasonal change in the values of physico-chemical parameters was observed, operation of the plant was optimal during all year (90% of removal in BOD5 and SS influent content). Microbial characterisation was approached by determining the structure and dynamics of protozoan and metazoan communities. Protozoa were the most abundant in all stages in the plant, heterotrophic flagellates being the most representative group in the water line and ciliates in the RBC system. The same seasonal preference was only observed for heterotrophic flagellates in the water line and green flagellates in the RBC system, both groups having highest abundances in summer and spring, respectively. Identification of ciliated protozoa populations rendered 58 species of ciliates in the plant. Most of these species are typical of aerobic wastewater treatment systems except three of them, which are cited for the first time in this type of ecosystems: Chaenea stricta, Holosticha mancoidea and Oxytricha lanceolata. Along the water line 34 species were identified, and half of them only appeared occasionally (once in all the study), while along the RBC system biofilms 55 species were observed, and the majority appeared permanently in this system. Our results indicate that the type of habitat, rather than the physico-chemical water parameters, was the primary factor in determining the different distribution of protozoan and metazoan communities in the plant. In RBC biofilms, the structure of ciliate protozoa community was found to be quite sensitive to changes in physico-chemical parameters, mainly to organic loading (BOD5) variations.

Ice shelf microbial ecosystems in the high arctic and implications for life on snowball earth.

The Ward Hunt Ice Shelf (83 degrees N, 74 degrees W) is the largest remaining section of thick (> 10 m) land-fast sea ice along the northern coastline of Ellesmere Island, Canada. Extensive meltwater lakes and streams occur on the surface of the ice and are colonized by photosynthetic microbial mat communities. This High Arctic cryo-ecosystem is similar in several of its physical, biological and geochemical features to the McMurdo Ice Shelf in Antarctica. The ice-mats in both polar regions are dominated by filamentous cyanobacteria but also contain diatoms, chlorophytes, flagellates, ciliates, nematodes, tardigrades and rotifers. The luxuriant Ward Hunt consortia also contain high concentrations (10(7)-10(8) cm-2) of viruses and heterotrophic bacteria. During periods of extensive ice cover, such as glaciations during the Proterozoic, cryotolerant mats of the type now found in these polar ice shelf ecosystems would have provided refugia for the survival, growth and evolution of a variety of organisms, including multicellular eukaryotes.