Resolving the intra-specific succession within Cochlodinium polykrikoides populations in southern Korean coastal waters via use of quantitative PCR assays

While the toxic dinoflagellate Cochlodinium polykrikoides is known to form blooms that are maintained for extended periods, the genetic differentiation of these blooms are currently unknown. To assess this, we developed a real-time PCR assay to quantify C. polykrikoides at the intra-specific level, and applied this assay to field samples collected in Korean coastal waters from summer through fall. Assays were successfully developed to target the large-subunit ribosomal RNA region of the three major ribotypes of C. polykrikoides: Philippines, East Asian, and American/Malaysian. Significant linear relationships (r² ≥ 0.995) were established between C_t and the log of the copy number for each ribotype qPCR assay. Using these assays, C. polykrikoides blooms in Korean coastal waters were found to be comprised of Philippines and East Asian ribotypes but not the American/Malaysian ribotype. The Philippines ribotype was found to be highly abundant during summer bloom initiation and peak, whereas the East Asian ribotype became the dominant ribotype in the fall. As such, this newly developed qPCR assay can be used to quantify the cryptic ecological succession of sub-populations of C. polykrikoides during blooms that light microscopy and previously developed qPCR assays cannot resolve.     

Phylogenetic relationships of Cochlodinium polykrikoides Margalef (Gymnodiniales,Dinophyceae) from the Mediterranean Sea and the implications of its global biogeography

Although the diversity of dinoflagellates has been intensively studied in several locations in the Mediterranean Sea since the 1950s, it is only during the last two decades that the morphotype of the toxic unarmoured dinoflagellate Cochlodinium polykrikoides Margalef has been detected, coinciding with its apparent worldwide expansion in marine coastal waters. In this study, vegetative cells of C. polykrikoides morphotype from the Catalan coast (NW Mediterranean Sea) were detected and isolated, and the DNA from collected cells was sequenced. While in the Mediterranean Sea, detections are scarce and C. polykrikoides is consistently present at low concentrations, we reported exceptional blooms of this species, in which the maximum abundance reached 2 × 10⁴ cells L⁻¹. Partial LSU rDNA region sequences showed that most C. polykrikoides populations from the Catalan coast formed a new differentiated ribotype, but others were included within the ‘Philippines’ ribotype, demonstrating their coexistence in the Mediterranean Sea. Thus, the current biogeographic nomenclature of the ribotypes is likely to be invalid with respect to the available information from populations comprising the ‘Philippines’ ribotype. The phylogeny suggests the existence of cryptic species that should be evaluated for species-level status. Accordingly, the ribotype determination must be carefully evaluated for all detections and bloom events, since accurate characterization of the morphology, ecophysiology and distribution of the ribotypes are not well resolved.     

The effects of major environmental factors and nutrient limitation on growth and encystment of planktonic dinoflagellate Akashiwo sanguinea

The bloom-forming dinoflagellate Akashiwo sanguinea is commonly observed in estuarine and coastal waters around the world. Annually recurrent blooms of this species have been observed in the coastal waters of China, particularly in the Sishili Bay, Yantai since 2004. However, limited studies have been conducted on the recurrence mechanism of A. sanguinea other than periodical monitoring of its population dynamics and associated environmental variables. Thus, to further explore the bloom and succession mechanisms of A. sanguinea in the field, we studied the effects of major nutritional components on the growth and encystment of A. sanguinea, as well as the effects of key environmental factors on the growth of A. sanguinea through a series of laboratory trials. Our results indicated that A. sanguinea was able to grow well within the temperature range of 20–25 °C, salinity range of 20 - 30, with the maximum laboratory irradiance of 78.14 μE m⁻² s⁻¹, and was able to survive and grow in low nutrient. However, lower concentrations of nutrients (e.g., nitrate, phosphate) and higher ammonium exerted different degrees of limiting effects on the growth of A. sanguinea, and induced 2.3–21.24% of vegetative cells to form resting cysts simultaneously in laboratory cultures. On the other hand, very limited or no cyst formation was observed in nutrient-replete or extremely low nutrient cultures, indicating the threshold effect of nutritional stress on the encystment of A. sanguinea. The physiological strategy of encystment of A. sanguinea in nutrient-limiting environment facilitates the survival and succession of A. sanguinea species in fluctuating seawaters, and provides seed sources for reoccurring algal blooms under favorable environmental conditions.     

Morphology,phylogeny and azaspiracid profile of Azadinium poporum (Dinophyceae) from the China Sea

Azadinium poporum is a small dinoflagellate from the family Amphidomataceae which is known for the production potential of azaspiracid toxins. A. poporum has been recorded from European and Korean waters. Here we present the first report of its occurrence along the coast of China. Morphology of Chinese A. poporum is similar to those from Europe and Korea. Several stalked pyrenoids surrounded by a starch sheath were revealed with light microscopy and confirmed by transmission electron microscopy. Among 25 strains from the China Sea we identified two distinct ribotypes (referred to as ribotypes B and C). ITS sequences of strains within the same ribotype are identical, whereas ribotype B and C differ from each other at 11 positions (98.3% similarity). A. poporum ribotypes B and C type differ from European strains (referred to as ribotype A) at 16 and 15 positions (97.5% and 97.7% similarity). The ITS region pairwise distance within A. poporum ranged from 0.017 to 0.022. Among all three ribotypes, no hemi-compensatory based changes were found within helix III of ITS indicating that they are conspecific. Azaspiracid profiles were analyzed for six strains and turned out to be unexpectedly diverse. Whereas no AZAs could be detected for one strain, another strain was found to contain a m/z 348 fragment type AZA previously found in a Korean Isolate and traces of two other unknown AZAs of higher masses. A third strain produced a novel AZA with a molecular mass of 871 Da. Three strains were found to contain considerable amounts of toxic AZA-2 as the sole AZA, a finding that might elegantly explain the detection of AZA-2 in sponges in the Sea of Japan and which underline the risk potential of A. poporum blooms with subsequent shellfish intoxication episodes for the Asian Pacific.     

The dinoflagellate Akashiwo sanguinea will benefit from future climate change: The interactive effects of ocean acidification,warming and high irradiance on photophysiology and hemolytic activity

Due to global climate change, marine phytoplankton will likely experience low pH (ocean acidification), high temperatures and high irradiance in the future. Here, this work report the results of a batch culture experiment conducted to study the interactive effects of elevated CO₂, increased temperature and high irradiance on the harmful dinoflagellate Akashiwo sanguinea, isolated at Dongtou Island, Eastern China Sea. The A. sanguinea cells were acclimated in high CO₂ condition for about three months before testing the responses of cells to a full factorial matrix experimentation during a 7-day period. This study measured the variation in physiological parameters and hemolytic activity in 8 treatments, representing full factorial combinations of 2 levels each of exposure to CO₂ (400 and 1000 μatm), temperature (20 and 28 °C) and irradiance (50 and 200 μmol photons m⁻² s⁻¹). Sustained growth of A. sanguinea occurred in all treatments, but high CO₂ (HC) stimulated faster growth than low CO₂ (LC). The pigments (chlorophyll a and carotenoid) decreased in all HC treatments. The quantum yield (F_v/F_m) declined slightly in all high-temperature (HT) treatments. High irradiance (HL) induced the accumulation of ultraviolet-absorbing compounds (UV_abc) irrespective of temperature and CO₂. The hemolytic activity in the LC treatments, however, declined when exposed to HT and HL, but HC alleviated the adverse effects of HT and HL on hemolytic activity. All HC and HL conditions and the combinations of high temperature*high light (HTHL) and high CO₂*high temperature*high light (HCHTHL) positively affected the growth in comparison to the low CO₂*low temperature*low light (LCLTLL) treatment. High temperature (HT), high light (HL) and a combination of HT*HL, however, negatively impacted hemolytic activity. CO₂ was the main factor that affected the growth and hemolytic activity. There were no significant interactive effects of CO₂*temperature*irradiance on growth, pigment, F_v/F_m or hemolytic activity, but there were effects on P_m, α, and E_k. If these results are extrapolated to the natural environment, it can be hypothesized that A. sanguinea cells will benefit from the combination of ocean acidification, warming, and high irradiance that are likely to occur under future climate change. It is assumed that faster growth and higher hemolytic activity and UV_abc of this species will occur under future conditions compared with those the current CO₂ (400 μatm) and temperature (20 °C) conditions.     

Bloom formation potential in the harmful dinoflagellate Akashiwo sanguinea: Clues from movement behaviors and growth characteristics

We measured the growth rates and swimming behaviors of recently isolated strains of the dinoflagellate Akashiwo sanguinea to investigate to what degree growth and motility could contribute to the formation of in situ blooms. To quantify the effect of variation in in situ conditions on population growth rate, we applied two temperature treatments (10 °C and 20 °C) and measured growth in still conditions and on a shaker table, to emulate mild turbulence. To quantify the importance of intra-strain variability and trait variation in the species growth potential and vertical distribution, we included six strains isolated from a spatially and temporally extensive bloom on the US West Coast. Overall, as reported previously, A. sanguinea was observed to tolerate conditions amounting to a broad ecological niche with intra-specific variability further broadening tolerable conditions. In agreement with prior observations of slow growth rates of the species, average growth rates across all strains increased significantly from 0.12 d⁻¹ (±0.03) at 10 °C to 0.28 d⁻¹ (±0.13) at 20 °C in still conditions. Contrary to prior reports, mild turbulence had neutral or positive effects on most strains’ growth rates, with one strain only able to grow on the shaker table. Growth rates in mild turbulence were higher than in still conditions and increased from 0.15 d⁻¹ (±0.01) at 10 °C to 0.43 d⁻¹ (± 0.04) at 20 °C. There was significant intra-strain variation in growth rates (>50% coefficient of variation) and movement behaviors. All strains had both up and down swimming fractions, leading to predictions of vertically patchy distributions, rather than surface aggregations. Slow growth rates and dispersive swimming behaviors suggest in situ mortality must be low and tolerance of seasonally varying water temperatures lead to accumulation and persistence of cells over months and kilometers. Estimates of in situ loss rates are a critical but missing component of identifying the bloom formation mechanisms of this species.     

Newly discovered role of the heterotrophic nanoflagellate Katablepharis japonica,a predator of toxic or harmful dinoflagellates and raphidophytes

Heterotrophic nanoflagellates are ubiquitous and known to be major predators of bacteria. The feeding of free-living heterotrophic nanoflagellates on phytoplankton is poorly understood, although these two components usually co-exist. To investigate the feeding and ecological roles of major heterotrophic nanoflagellates Katablepharis spp., the feeding ability of Katablepharis japonica on bacteria and phytoplankton species and the type of the prey that K. japonica can feed on were explored. Furthermore, the growth and ingestion rates of K. japonica on the dinoflagellate Akashiwo sanguinea—a suitable algal prey item—heterotrophic bacteria, and the cyanobacteria Synechococcus sp., as a function of prey concentration were determined. Among the prey tested, K. japonica ingested heterotrophic bacteria, Synechococcus sp., the prasinophyte Pyramimonas sp., the cryptophytes Rhodomonas salina and Teleaulax sp., the raphidophytes Heterosigma akashiwo and Chattonella ovata, the dinoflagellates Heterocapsa rotundata, Amphidinium carterae, Prorocentrum donghaiense, Alexandrium minutum, Cochlodinium polykrikoides, Gymnodinium catenatum, A. sanguinea, Coolia malayensis, and the ciliate Mesodinium rubrum, however, it did not feed on the dinoflagellates Alexandrium catenella, Gambierdiscus caribaeus, Heterocapsa triquetra, Lingulodinium polyedra, Prorocentrum cordatum, P. micans, and Scrippsiella acuminata and the diatom Skeletonema costatum. Many K. japonica cells attacked and ingested a prey cell together after pecking and rupturing the surface of the prey cell and then uptaking the materials that emerged from the ruptured cell surface. Cells of A. sanguinea supported positive growth of K. japonica, but neither heterotrophic bacteria nor Synechococcus sp. supported growth. The maximum specific growth rate of K. japonica on A. sanguinea was 1.01 d⁻¹. In addition, the maximum ingestion rate of K. japonica for A. sanguinea was 0.13 ng C predator⁻¹d⁻¹ (0.06 cells predator⁻¹d⁻¹). The maximum ingestion rate of K. japonica for heterotrophic bacteria was 0.019 ng C predator⁻¹d⁻¹ (266 bacteria predator⁻¹d⁻¹), and the highest ingestion rate of K. japonica for Synechococcus sp. at the given prey concentrations of up to ca. 10⁷ cells ml⁻¹ was 0.01 ng C predator⁻¹d⁻¹ (48 Synechococcus predator⁻¹d⁻¹). The maximum daily carbon acquisition from A. sanguinea, heterotrophic bacteria, and Synechococcus sp. were 307, 43, and 22%, respectively, of the body carbon of the predator. Thus, low ingestion rates of K. japonica on heterotrophic bacteria and Synechococcus sp. may be responsible for the lack of growth. The results of the present study clearly show that K. japonica is a predator of diverse phytoplankton, including toxic or harmful algae, and may also affect the dynamics of red tides caused by these prey species.     

Developmental temperature responses of Chrysoperla agilis (Neuroptera: Chrysopidae), a member of the European carnea cryptic species group

Chrysoperla agilis Henry et al. is one of the five cryptic species of the carnea group found in Europe. Identification of these species is mainly based on the distinct mating signals produced by both females and males prior to copulation, although there are also morphological traits that can be used to distinguish among different cryptic species. Ecological and physiological cryptic species-specific differences may affect their potential as important biological agents in certain agroecosystems. To understand the effects of temperature on the life-history traits of C. agilis preimaginal development, adult longevity and reproduction were studied at seven temperatures. Temperature affected the development, survival and reproduction of C. agilis. Developmental time ranged from approximately 62 days at 15 °C to 15 days at 30 °C. Survival percentages ranged from 42% at 15 °C to 76% at 27 °C. One linear and five nonlinear models (Briere I, II, Logan 6, Lactin and Taylor) used to model preimaginal development were tested to describe the relationship between temperature and developmental rate. Logan 6 model fitted the data of egg to adult development best according to the criteria adopted for the model evaluation. The predicted lower developmental threshold temperatures were 11.4 °C and 11.8 °C (linear model), whereas the predicted upper threshold temperatures (Logan 6 model) were 36.6 and 36.9 °C for females and males, respectively. Adult life span, preoviposition period and lifetime cumulative oviposition were significantly affected by temperature. The effect of rearing temperature on the demographic parameters is well summarized with the estimated values of the intrinsic rate of increase (r_m) which ranged from 0.0269 at 15 °C to 0.0890 at 32 °C and the highest value recorded at 27 °C (0.1530). These results could be useful in mass rearing C. agilis and predicting its population dynamics in the field.     

Cryptic speciation in Protoceratium reticulatum (Dinophyceae): Evidence from morphological,molecular and ecophysiological data

The cosmopolitan, potentially toxic dinoflagellate Protoceratium reticulatum possesses a fossilizable cyst stage which is an important paleoenvironmental indicator. Slight differences in the internal transcribed spacer ribosomal DNA (ITS rDNA) sequences of P. reticulatum have been reported, and both the motile stage and cyst morphology of P. reticulatum display phenotypic plasticity, but how these morpho-molecular variations are related with ecophysiological preferences is unknown. Here, 55 single cysts or cells were isolated from localities in the Northern (Arctic to subtropics) and Southern Hemispheres (Chile and New Zealand), and in total 34 strains were established. Cysts and/or cells were examined with light microscopy and/or scanning electron microscopy. Large subunit ribosomal DNA (LSU rDNA) and/or ITS rDNA sequences were obtained for all strains/isolates. All strains/isolates of P. reticulatum shared identical LSU sequences except for one strain from the Mediterranean Sea that differs in one position, however ITS rDNA sequences displayed differences at eight positions. Molecular phylogeny was inferred using maximum likelihood and Bayesian inference based on ITS rDNA sequences. The results showed that P. reticulatum comprises at least three ribotypes (designated as A, B, and C). Ribotype A included strains from the Arctic and temperate areas, ribotype B included strains from temperate regions only, and ribotype C included strains from the subtropical and temperate areas. The average ratios of process length to cyst diameter of P. reticulatum ranged from 15% in ribotype A, 22% in ribotype B and 17% in ribotype C but cyst size could overlap. Theca morphology was indistinguishable among ribotypes. The ITS-2 secondary structures of ribotype A displayed one CBC (compensatory change on two sides of a helix pairing) compared to ribotypes B and C. Growth response of one strain from each ribotype to various temperatures was examined. The strains of ribotypes A, B and C exhibited optimum growth at 15 °C, 20 °C and 20–25 °C, respectively, thus corresponding to cold, moderate and warm ecotypes. The profiles of yessotoxins (YTXs) were examined for 25 strains using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). The parent compound yessotoxin (YTX) was produced by strains of ribotypes A and B, but not by ribotype C strains, which only produced the structural variant homoyessotoxin (homoYTX). Our results support the notion that there is significant intra-specific variability in Protoceratium reticulatum and the biogeography of the different ribotypes is consistent with specific ecological preferences.     

Diversity of moxifloxacin resistance during a nosocomial outbreak of a predominantly ribotype ARU 027 Clostridium difficile diarrhea

To characterize the extent and diversity of moxifloxacin resistance among Clostridium difficile isolates recovered during a predominantly Anaerobe Reference Unit (ARU) ribotype 027-associated nosocomial outbreak of antibiotic associated diarrhea we measured the susceptibility of 34 field isolates and 6 laboratory strains of C. difficile to moxifloxacin. We ribotyped the isolates as well as assaying them by PCR for the metabolic gene, gdh, and the virulence genes, tcdA, tcdB, tcdC, cdtA and cdtB. All the laboratory isolates, including the historical ARU 027 isolate Cd196, were susceptible to moxifloxacin (≤2 μg/mL). 13 field isolates were susceptible to ≤2 μg/mL. Five were resistant to from 4 to 12 μg/mL (moderate resistance); 16 were resistant to ≥16 μg/mL (high resistance). We sequenced the quinolone resistance determining regions of gyrA (position 71-460) and gyrB (position 1059-1448) from two susceptible laboratory strains, all five isolates with moderate resistance and two highly resistant isolates. Two highly resistant isolates (Pitt 40, ribotype ARU 027 and Pitt 33, ribotype ARU 001) had the same C245T (Thr₈₂ΔIle) mutation. No other changes were seen. Amplification with primer pairs specific for the C245T mutant gyrA and for the wild type gene respectively confirmed all 16 highly resistant ARU 027 isolates, as well as the highly resistant isolates from other ribotypes, had the C245T mutation and that the mutation was absent from all other isolates. Among the five isolates with moderate resistance we found combinations of mutations within gyrA (T128A, Val₄₃ΔAsp and G349T, Ala₁₁₇ΔSer) and gyrB (G1276A, Arg₄₂₆ΔAsn). The G1396A (Glu₄₆₆ΔLys) mutation was not associated with increased resistance.     

The potential role of anthropogenically derived nitrogen in the growth of harmful algae in California,USA

Cultural eutrophication is frequently invoked as one factor in the global increase in harmful algal blooms, but is difficult to definitively prove due to the myriad of factors influencing coastal phytoplankton bloom development. To assess whether eutrophication could be a factor in the development of harmful algal blooms in California (USA), we review the ecophysiological potential for urea uptake by Pseudo-nitzschia australis (Bacillariophyceae), Heterosigma akashiwo (Raphidophyceae), and Lingulodinium polyedrum (Dinophyceae), all of which have been found at bloom concentrations and/or exhibited noxious effects in recent years in California coastal waters. We include new measurements from a large (Chlorophyll a > 500 mg m⁻³) red tide event dominated by Akashiwo sanguinea (Dinophyceae) in Monterey Bay, CA during September 2006. All of these phytoplankton are capable of using nitrate, ammonium, and urea, although their preference for these nitrogenous substrates varies. Using published data and recent coastal time series measurements conducted in Monterey Bay and San Francisco Bay, CA, we show that urea, presumably from coastal eutrophication, was present in California waters at measurable concentrations during past harmful algal bloom events. Based on these observations, we suggest that urea uptake could potentially sustain these harmful algae, and that urea, which is seldom measured as part of coastal monitoring programs, may be associated with these harmful algal events in California.     

PTP1B inhibitors from the seeds of Iris sanguinea and their insulin mimetic activities via AMPK and ACC phosphorylation

To find PTP1B inhibitors from natural products, two new compounds (1 and 2), along with nine known compounds (3–11), were isolated from a methanol-soluble extract of Iris sanguinea seeds. The structures of compounds 1 and 2 were determined based on extensive spectroscopic data analysis including UV, IR, NMR, and MS. The IC₅₀ value of compound 5 on protein tyrosine phosphatase 1B (PTP1B) inhibitory activity is 7.30 ± 0.88 µM with a little activity compared to the IC₅₀ values of the tested positive compound. Compound 5 significantly enhanced glucose uptake and activation of pACC, pAMPK and partially Erk1/2 signaling. These results suggest that compound 5 from Iris sanguinea seeds are utilized as both PTP1B inhibitors and regulators of glucose uptake. These beneficial effects could be applied to treat metabolic diseases such as diabetes and obesity.     

ITS2 sequence variations among members of Diachasmimorpha longicaudata complex (Hymenoptera: Braconidae) in Thailand

The parasitoid Diachasmimorpha longicaudata complex in Thailand contains at least 3 cryptic species informally designated as species D. longicaudata A, B and BB. DNA sequence data of nuclear ITS2 (second internal transcribed spacer) were used to characterize members of this D. longicaudata complex. The polymerase chain reaction (PCR) amplicon of ITS2 region of D. longicaudata B (≈ 650 bp) clearly differentiated this species from A and BB (amplicon of ≈ 590 bp). Sequence alignment of individual parasitoids revealed that low intraspecies differences ranged from 0.457 to 3.991%, but interspecies differences ranged from 7.566 to 12.989%. Phylogenetic trees constructed using Neighbor-Joining (NJ) and Maximum Parsimony (MP) methods, taking the parasitoid Psyttalia concolor complex as an outgroup, revealed that D. longicaudata A, B and BB formed a monophyletic group, with species A and BB being more closely related than species B. ITS2 characterization of D. longicaudata complex has revealed an interesting divergence of the three cryptic sibling species in Thailand.     

Growth of eight Gambierdiscus (Dinophyceae) species: Effects of temperature,salinity and irradiance

Little is known about how the growth of individual Gambierdiscus species responds to environmental factors. This study examined the effects of temperature (15–34 °C), salinity (15–41) and irradiance (2–664 μmol photons m⁻² s⁻¹) on growth of Gambierdiscus: G. australes, G. belizeanus, G. caribaeus, G. carolinianus, G. carpenteri, G. pacificus and G. ruetzleri and one putative new species, Gambierdiscus ribotype 2. Depending on species, temperatures where maximum growth occurred varied between 26.5 and 31.1 °C. The upper and lower thermal limits for all species were between 31–34 °C and 15–21 °C, respectively. The shapes of the temperature vs. growth curves indicated that even small differences of 1–2 °C notably affected growth potentials. Salinities where maximum growth occurred varied between 24.7 and 35, while the lowest salinities supporting growth ranged from <14 to 20.9. These data indicated that Gambierdiscus species are more tolerant of lower salinities than is generally appreciated. Growth of all species began to decline markedly as salinities exceed 35.1–39.4. The highest salinity tested in this study (41), however, was lethal to only one species, Gambierdiscus ribotype 2. The combined salinity data indicated that differences in salinity regimes may affect relative species abundances and distributions, particularly when salinities are <20 and >35. All eight Gambierdiscus species were adapted to relatively low light conditions, exhibiting growth maxima at 50–230 μmol photons m⁻² s⁻¹ and requiring only 6–17 μmol photons m⁻² s⁻¹ to maintain growth. These low light requirements indicate that Gambierdiscus growth can occur up to 150 m depth in tropical waters, with optimal light regimes often extending to 75 m. The combined temperature, salinity and light requirements of Gambierdiscus can be used to define latitudinal ranges and species-specific habitats, as well as to inform predictive models.     

Bacterial community dynamics during a bloom caused by Akashiwo sanguinea in the Xiamen sea area,China

Phytoplankton blooms are a worldwide ecological problem and one of the major algae that cause phytoplankton blooms is Akashiwo sanguinea. Though much research has addressed the abiotic causes (e.g. growth condition) of A. sanguinea blooms, few studies have examined the dynamics of microbial communities associated with these blooms. In this study, polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) analysis of 16S rDNA genes was used to document changes in the phylogenetic diversity of microbial communities associated with an A. sanguinea bloom that occurred in the Xiamen sea in May 2010. Surface sea water was sampled once a day within five consecutive days at four sites, and the microbial community composition was determined using DGGE. Sea water concentrations of chlorophyll a, nitrate and phosphate were also measured. The results indicated that the A. sanguinea bloom was probably stimulated by low salinity (26–30‰) and ended probably because inorganic nutrients were consumed and resulted in a N/P ratio unfavorable for this alga. Gammaproteobacteria populations increased significantly during bloom declines and then decreased post-bloom. Divergences in the microbial community composition during different bloom periods were the result of changes in Candidatus, Pelagibacter, Alteromonas, Rhodobacteraceae, Vibrio and Pseudoalteromonas populations. Sediminimonas qiaohouensis was the first bacterium shown to be significantly negatively correlated with A. sanguinea concentration. This study indicated that bacteria may play an important role in A. sanguinea–bloom regulation and provides a deeper insight into bacterial community succession during and after an A. sanguinea–bloom.     

Diversity of anaerobic fungal populations in cattle revealed by selective enrichment culture using different carbon sources

We employed most probable numbers (MPNs) enumeration of enrichment cultures, combined with the use of a range of carbon sources (glucose, cellobiose, cellulose, xylan and wheat straw), to recover and identify morphologically different groups of anaerobic fungi (monocentric rhizoidal [Neocallimastix, Piromyces spp.], polycentric rhizoidal [Anaeromyces, Orpinomyces spp.], bulbous non-rhizoidal [Caecomyces, Cyllamyces spp.]) from rumen digesta, and fresh or frozen–thawed faeces of silage-fed cattle. Highest MPN counts (>10⁶ thallus forming units [TFU] g^?1 dry matter (DM)) were obtained using wheat straw but use of other carbon sources revealed large variation in the relative abundance of the morphotypes recovered in culture. Polycentric morphotypes were overall the most abundant fungi, comprising ca. 60 % of observations and recovered most frequently with xylan and wheat straw. Bulbous morphotypes showed a reciprocal pattern of occurrence, being most frequently observed on glucose, cellobiose and cellulose. Monocentric morphotypes were surprisingly the least abundant (<10 % overall), occurring mostly on glucose and wheat straw. Freezing of faeces (?20 °C/5 weeks) and thawing prior to enrichment culture reduced MPN counts by ca. 40 % from a mean of 1.8 × 10⁵ TFU g^?1 DM, but greater relative abundance of polycentric morphotypes in frozen–thawed faeces suggested differential survival in response to environmental stresses. PCR–RFLP demonstrated the simultaneous presence of seven ribotypes in one animal, but not all ribotypes could be associated with a particular genus.     

Gambierdiscus (Dinophyceae) species diversity in the Flower Garden Banks National Marine Sanctuary,Northern Gulf of Mexico,USA

Globally, ciguatera fish poisoning (CFP) is the principal cause of non-bacterial illness associated with seafood consumption. The toxins (ciguatoxins) responsible for CFP are produced by dinoflagellates in the genus Gambierdiscus, which are endemic to tropical and sub-tropical areas. Ciguatoxins are lipophilic and bioaccumulate in marine food webs, typically reaching their highest concentrations in fish. Following a CFP event in 2008, the U.S. Food and Drug Administration (USFDA) issued a ciguatera toxin alert that included fish harvested in the northern Gulf of Mexico in and near the Flower Garden Banks National Marine Sanctuary (FGBNMS). The East Flower Garden Bank (EFGB) and West Flower Garden Bank (WFGB) are characterized by thriving coral communities that support Gambierdiscus growth. This study was undertaken specifically to document the diversity of Gambierdiscus species present in the sanctuary that may be sources of ciguatoxins entering the food web. Samples collected from the FGBNMS over a three year period were screened using species-specific polymerase chain reaction assays. A diverse assemblage of Gambierdiscus species was distributed to depths of >45 m, a new depth record for Gambierdiscus. Gambierdiscus belizeanus, Gambierdiscus caribaeus, Gambierdiscus carolinianus, Gambierdiscus carpenteri and Gambierdiscus ribotype 2 were all found on both East and West FGB with Gambierdiscus ruetzleri also recorded from the WFGB. The most common species was G. carolinianus, originally identified from samples collected between 35 and 40 m off the coast of NC, USA. Our findings are consistent with recent physiological studies showing that some Gambierdiscus species can grow year round at the temperatures and salinities at the FGBNMS and at light levels as low as 10 μmol photons m⁻² s⁻¹. Such irradiances are estimated to occur in the FGBNMS at depths of ∼70–80 m. The consistent recovery of Gambierdiscus species from deep sampling sites in areas known to produce ciguatoxic fish signals a substantial change in our concept of suitable habitats for Gambierdiscus to include depths greater than 50 m.     

Mixotrophy in the phototrophic dinoflagellate Takayama helix (family Kareniaceae): Predator of diverse toxic and harmful dinoflagellates

Takayama spp. are phototrophic dinoflagellates belonging to the family Kareniaceae and have caused fish kills in several countries. Understanding their trophic mode and interactions with co-occurring phytoplankton species are critical steps in comprehending their ecological roles in marine ecosystems, bloom dynamics, and dinoflagellate evolution. To investigate the trophic mode and interactions of Takayama spp., the ability of Takayama helix to feed on diverse algal species was examined, and the mechanisms of prey ingestion were determined. Furthermore, growth and ingestion rates of T. helix feeding on the dinoflagellates Alexandrium lusitanicum and Alexandrium tamarense, which are two optimal prey items, were determined as a function of prey concentration. T. helix ingested large dinoflagellates ≥15 μm in size, except for the dinoflagellates Karenia mikimotoi, Akashiwo sanguinea, and Prorocentrum micans (i.e., it fed on Alexandrium minutum, A. lusitanicum, A. tamarense, A. pacificum, A. insuetum, Cochlodinium polykrikoides, Coolia canariensis, Coolia malayensis, Gambierdiscus caribaeus, Gymnodinium aureolum, Gymnodinium catenatum, Gymnodinium instriatum, Heterocapsa triquetra, Lingulodinium polyedrum, and Scrippsiella trochoidea). All these edible prey items are dinoflagellates that have diverse eco-physiology such as toxic and non-toxic, single and chain forming, and planktonic and benthic forms. However, T. helix did not feed on small flagellates and dinoflagellates <13 μm in size (i.e., the prymnesiophyte Isochrysis galbana; the cryptophytes Teleaulax sp., Storeatula major, and Rhodomonas salina; the raphidophyte Heterosigma akashiwo; the dinoflagellates Heterocapsa rotundata, Amphidinium carterae, Prorocentrum minimum; or the small diatom Skeletonema costatum). T. helix ingested Heterocapsa triquetra by direct engulfment, but sucked materials from the rest of the edible prey species through the intercingular region of the sulcus. With increasing mean prey concentration, the specific growth rates of T. helix on A. lusitanicum and A. tamarense increased continuously before saturating at prey concentrations of 336–620 ng C mL⁻¹. The maximum specific growth rates (mixotrophic growth) of T. helix on A. lusitanicum and A. tamarense were 0.272 and 0.268 d⁻¹, respectively, at 20 °C under a 14:10 h light/dark cycle of 20 μE m⁻² s⁻¹ illumination, while its growth rates (phototrophic growth) under the same light conditions without added prey were 0.152 and 0.094 d⁻¹, respectively. The maximum ingestion rates of T. helix on A. lusitanicum and A. tamarense were 1.23 and 0.48 ng C predator⁻¹d⁻¹, respectively. The results of the present study suggest that T. helix is a mixotrophic dinoflagellate that is able to feed on a diverse range of toxic species and, thus, its mixotrophic ability should be considered when studying red tide dynamics, food webs, and dinoflagellate evolution.     

Cool gleaners: Thermoregulation in sympatric bat species

Thermoregulatory behavior in temperate bats is influenced by gender, food availability, ambient temperature and reproduction. Ecologically and morphologically similar bat species (Myotis bechsteinii, M. nattereri, and Plecotus auritus; Vespertilionidae) facing similar diurnal conditions should therefore not differ in their thermoregulatory behavior. Identified day roosts (n = 23) of radio-tagged bats (n = 30) were spread over an area of 33.1 ha, but ambient temperature did not differ between roosting sites. Furthermore, there was no significant difference in cardinal direction, roost height, canopy coverage, and breast height diameter between day roosts used by the three species. Minimum roost temperatures and isolation values, however, differed significantly between our species with lowest values in P. auritus. The range of skin temperatures (min–max) recorded by temperature-sensitive transmitters was not species-specific with the lowest ranges in late pregnancy (mean ± SD: 7.1 ± 1.1 °C) and highest in post-lactation (mean ± SD: 13.1 ± 1.1 °C). The minimum skin temperature, however, was species-specific with the lowest values in P. auritus (mean ± SD: 20.2 ± 1.1 °C), intermediate in M. nattereri (mean ± SD: 23.4 ± 1.0 °C), and the highest in M. bechsteinii (mean ± SD: 26.8 ± 1.0 °C). Species-specific usage of energy-saving mechanisms might represent an important niche differentiation of species. Different mechanisms might allow, e.g. one species to occupy colder roosts with higher temperature variations or to shorten foraging times due to distinct thermoregulatory behavior.