Effect of nutrients on the growth of a new alpine strain of Haematococcus (Chlorophyceae) from New Zealand

The microalga Haematococcus lacustris is a source of astaxanthin used widely in aquaculture, pharmaceutical, and cosmetic industries. A new strain of Haematococcus (LCR‐26C‐1f) isolated from the New Zealand alpine zone was evaluated in this study. The influence of vitamins, micronutrients, various carbon and nitrogen sources were investigated to maximize biomass production in batch cultures using shake flasks. Supplementation of vitamins consisting of thiamine, biotin, and cyanocobalamin improved the cell density by 40% over the vitamin‐free medium. Out of the individual vitamins tested, thiamine was shown to be necessary to maintain high cell densities. The best nitrogen source tested was nitrate in the form of sodium nitrate_, at a 40 mM concentration. Heterotrophic growth yielded much lower cell densities compared to autotrophic growth. The micronutrients iron and manganese were essential for growth. However, the best growth was obtained using a micronutrient mix that included iron, copper, cobalt, zinc, manganese and molybdenum.     

Phosphate‐limited growth of the marine diatom Thalassiosira weissflogii (Bacillariophyceae): evidence of non‐monod growth kinetics1

The marine diatom Thalassiosira weissflogii (Grunow) G. A. Fryxell & Hasle was grown in a chemostat over a series of phosphate‐limited growth rates. Ambient substrate concentrations were determined from bioassays involving picomolar spikes of ³³P‐labeled phosphate, and maximum uptake rates were determined from analogous bioassays that included the addition of micromolar concentrations of unlabeled phosphate and tracer concentrations of ³³P. The relationship between cell phosphorus quotas and growth rates was well described by the Droop equation. Maximum uptake rates of phosphate spikes were several orders of magnitude higher than steady state uptake rates. Despite the large size of the T. weissflogii cells, diffusion of phosphate through the boundary layer around the cells had little effect on growth kinetics, in part because the cellular N:P ratios exceeded the Redfield ratio at all growth rates. Fitting the Monod equation to the experimental data produced an estimate of the nutrient‐saturated growth rate that was ~50% greater than the maximum growth rate observed in batch culture. A modified hyperbolic equation with a curvature that is a maximum in magnitude at positive growth rates gave a better fit to the data and an estimate of the maximum growth rate that was consistent with observations. The failure of the Monod equation to describe the data may reflect a transition from substrate to co‐substrate limitation and/or the presence of an inducible uptake system.     

Hyperspectral imaging techniques for the characterization of Haematococcus pluvialis (Chlorophyceae)

A hyperspectral imaging camera was combined with a bright‐field microscope to investigate the intracellular distribution of pigments in cells of the green microalga Haematococcus pluvialis, a synonym for H. lacustris (Chlorophyceae). We applied multivariate curve resolution to the hyperspectral image data to estimate the pigment contents in culture and revealed that the predicted values were consistent with actual measurements obtained from extracted pigments. Because it was possible to estimate pigment contents in every pixel, the intracellular distribution of the pigments was investigated during various life‐cycle stages. Astaxanthin was localized specifically at the eyespot of zoospores in early culture stages. Then, it became widely distributed in cells, but subsequently localized differently than the chl. Integrated with our recently developed image‐processing program “HaematoCalMorph,” the hyperspectral imaging system was useful for monitoring intracellular distributions of pigments during culture as well as for studying cellular responses under various conditions.     

氮、磷营养对雨生血球藻绿色细胞生长的影响

采用BBM培养基,以雨生血球藻(Haematococcus pluvialis CG-11)为研究材料,通过测定细胞生长速率、叶绿素和类胡萝卜素含量、生物量和虾青素含量,探讨氮、磷营养对雨生血球藻营养细胞生长的影响.结果显示:H.pluvialis CG-11生长的适宜氮源形式是NaNO₃和NH₄NO₃;适宜H.pluvialis CG-11生长的氮浓度为41.2mg·L^-1,磷浓度为5.3～53.3mg·L^-1.     

Dwarf and deformed flower 1, encoding an F‐box protein,is critical for vegetative and floral development in rice (Oryza sativa L.)

Recent studies have shown that F‐box proteins constitute a large family in eukaryotes, and play pivotal roles in regulating various developmental processes in plants. However, their functions in monocots are still obscure. In this study, we characterized a recessive mutant dwarf and deformed flower 1‐1 (ddf1‐1) in Oryza sativa (rice). The mutant is abnormal in both vegetative and reproductive development, with significant size reduction in all organs except the spikelet. DDF1 controls organ size by regulating both cell division and cell expansion. In the ddf1‐1 spikelet, the specification of floral organs in whorls 2 and 3 is altered, with most lodicules and stamens being transformed into glume‐like organs and pistil‐like organs, respectively, but the specification of lemma/palea and pistil in whorls 1 and 4 is not affected. DDF1 encodes an F‐box protein anchored in the nucleolus, and is expressed in almost all vegetative and reproductive tissues. Consistent with the mutant floral phenotype, DDF1 positively regulates B‐class genes OsMADS4 and OsMADS16, and negatively regulates pistil specification gene DL. In addition, DDF1 also negatively regulates the Arabidopsis LFY ortholog APO2, implying a functional connection between DDF1 and APO2. Collectively, these results revealed that DDF1, as a newly identified F‐box gene, is a crucial genetic factor with pleiotropic functions for both vegetative growth and floral organ specification in rice. These findings provide additional insights into the molecular mechanism controlling monocot vegetative and reproductive development.     

Effect of long‐term injection of dopamine on the ovarian growth of Cherax quadricarinatus juvenile females (Parastacidae,Decapoda)

The neurotransmitter dopamine (DA) modulates many physiological processes in decapod crustaceans, including reproduction. This study was aimed at evaluating whether the long‐term injection of DA affects ovarian growth in the freshwater crayfish Cherax quadricarinatus. Three experiments were performed with females of different mean initial size: 4.15 g (Group A); 9.65 g (Group B); and 13.98 g (Group C). Treated females were injected with DA and control females with physiological saline twice a week for 90 (Group B), 105 (Group A) and 120 (Group C) days. At the end of the experiments, the animals were killed, and the stage of ovarian development, gonadosomatic index, and mean oocyte diameter were determined. DA had a differential effect according to female size: it negatively affected ovarian growth of females in a weight range of 4–14 g and had no effect on ovarian maturation when injected to females with an initial weight of 14 g. The results are compared with previous reports in freshwater decapod crustaceans.     

An AGAMOUS intron‐driven cytotoxin leads to flowerless tobacco and produces no detrimental effects on vegetative growth of either tobacco or poplar

Flowerless trait is highly desirable for poplar because it can prevent pollen‐ and seed‐mediated transgene flow. We have isolated the second intron of PTAG2, an AGAMOUS (AG) orthologue from Populus trichocarpa. By fusing this intron sequence to a minimal 35S promoter sequence, we created two artificial promoters, fPTAG2I (forward orientation of the PTAG2 intron sequence) and rPTAG2I (reverse orientation of the PTAG2 intron sequence). In tobacco, expression of the β‐glucuronidase gene (uidA) demonstrates that the fPTAG2I promoter is non‐floral‐specific, while the rPTAG2I promoter is active in floral buds but with no detectable vegetative activity. Under glasshouse conditions, transgenic tobacco plants expressing the Diphtheria toxin A (DT‐A) gene driven by the rPTAG2I promoter produced three floral ablation phenotypes: flowerless, neuter (stamenless and carpel‐less) and carpel‐less. Further, the vegetative growth of these transgenic lines was similar to that of the wild‐type plants. In field trials during 2014 and 2015, the flowerless transgenic tobacco stably maintained its flowerless phenotype, and also produced more shoot and root biomass when compared to wild‐type plants. In poplar, the rPTAG2I::GUS gene exhibited no detectable activity in vegetative organs. Under field conditions over two growing seasons (2014 to the end of 2015), vegetative growth of the rPTAG2I::DT‐A transgenic poplar plants was similar to that of the wild‐type plants. Our results demonstrate that the rPTAG2I artificial promoter has no detectable activities in vegetative tissues and organs, and the rPTAG2I::DT‐A gene may be useful for producing flowerless poplar that retains normal vegetative growth.     

Insights from ecological niche modeling on the taxonomic distinction and niche differentiation between the black‐spotted and red‐spotted tokay geckoes (Gekko gecko)

The black‐spotted tokay and the red‐spotted tokay are morphologically distinct and have largely allopatric distributions. The black‐spotted tokay is characterized by a small body size and dark skin with sundry spots, while the red‐spotted tokay has a relatively large body size and red spots. Based on morphological, karyotypic, genetic, and distribution differences, recent studies suggested their species status; however, their classifications remain controversial, and additional data such as ecological niches are necessary to establish firm hypotheses regarding their taxonomic status. We reconstructed their ecological niches models using climatic and geographic data. We then performed niche similarity tests (niche identity and background tests) and point‐based analyses to explore whether ecological differentiation has occurred, and whether such differences are sufficient to explain the maintenance of their separate segments of environmental ranges. We found that both niche models of the black‐ and the red‐spotted tokay had a good fit and a robust performance, as indicated by the high area under the curve (AUC) values (“black” = 0.982, SD = ± 0.002, “red” = 0.966 ± 0.02). Significant ecological differentiation across the entire geographic range was found, indicating that the involvement of ecological differentiation is important for species differentiation. Divergence along the environmental axes is highly associated with climatic conditions, with isothermality being important for the “black” form, while temperature seasonality, precipitation of warmest quarter, and annual temperature range together being important for the “red” form. These factors are likely important factors in niche differentiation between the two forms, which result in morphological replacement. Overall, beside morphological and genetic differentiation information, our results contribute to additional insights into taxonomic distinction and niche differentiation between the black‐ and the red‐spotted tokay.     

A quantitative real‐time PCR assay for identification and enumeration of the occasionally co‐occurring ichthyotoxic Pseudochattonella farcimen and P. verruculosa (Dictyochophyceae) and analysis of variation in gene copy numbers during the growth phase of single and mixed cultures

The ichthyotoxic genus Pseudochattonella forms recurrent extensive blooms in coastal waters in Japan, New Zealand and Northern Europe. It comprises of two morphologically similar species, P. verruculosa and P. farcimen, which complicates visual species identification and enumeration of live and fixed material. Primers designed previously could not quantitatively distinguish species in mixed assemblages. To address this issue we developed two primer sets: one revealed itself to be genus specific for Pseudochattonella and the other species‐specific for P. verruculosa. By subtracting cell estimates for P. verruculosa from combined results we could calculate cell numbers for P. farcimen. This approach has overcome the challenges posed by the very limited sequence availability and low gene variability between the two species. The qPCR assay was extensively tested for specificity, efficiency and sensitivity over an entire growth cycle in both single and mixed assemblages. Comparison of cell abundance estimates obtained by qPCR assay and microscopy showed no statistically significant difference until stationary and death phases. The assay was also tested on environmental samples collected during a small Pseudochattonella bloom in Denmark in March–April 2015. It was impossible to distinguish P. farcimen and P. verruculosa by light microscopy but qPCR showed both species were present. The two methods provided nearly identical cell numbers but the assay provided discrimination and enumeration of both species.     

Logistic growth of the host‐specific obligate insect pathogenic fungus Entomophthora muscae in house flies (Musca domestica)

Insect pathogenic fungi (IPF) need to overcome the host immune system in order to sporulate and ensure transmission to new hosts. Some IPF produce immunosuppressive toxins, whereas others rely on rapid fungal proliferation to kill the host by sheer fungal mass, resulting in a trade‐off between allocating resources to toxin production and fungal proliferation. The obligate entomopathogenic fungus, Entomophthora muscae sensu stricto, is host specific to the common house fly, Musca domestica. E. muscae grows as protoplast cells without cell walls and is not known to produce toxins. Here, we assessed the growth of E. muscae, in vivo, using real‐time PCR to measure the amount of a single‐copy actin gene. We find that E. muscae exhibits S‐shaped logistic growth between time post‐exposure and the number of fungal nuclei. The results show that E. muscae initially grows exponentially inside the host until depletion of available nutrient sources signifies the ‘limiting capacity’ where after the host is killed. This growth pattern differs markedly from toxin‐producing IPF species of Metarhizium and Beauveria in which maximal (plateau) growth and sporulation do not occur until well after the death of the host.     

The 12th International Workshops on Opportunistic Protists (IWOP‐12)

The 12th International Workshops on Opportunistic Protists (IWOP‐12) was held in August 2012 in Tarrytown, New York. The objectives of the IWOP meetings are to: (1) serve as a forum for exchange of new information among active researchers concerning the basic biology, molecular genetics, immunology, biochemistry, pathogenesis, drug development, therapy, and epidemiology of these immunodeficiency‐associated pathogenic eukaryotic microorganisms that are seen in patients with AIDS and (2) foster the entry of new and young investigators into these underserved research areas. The IWOP meeting focuses on opportunistic protists, e.g. the free‐living amoebae, Pneumocystis, Cryptosporidium, Toxoplasma, the Microsporidia, and kinetoplastid flagellates. This conference represents the major conference that brings together research groups working on these opportunistic pathogens. Slow but steady progress is being achieved on understanding the biology of these pathogenic organisms, their involvement in disease causation in both immune‐deficient and immune‐competent hosts, and is providing critical insights into these emerging and reemerging pathogens. This IWOP meeting demonstrated the importance of newly developed genomic level information for many of these pathogens and how analysis of such large data sets is providing key insights into the basic biology of these organisms. A great concern is the loss of scientific expertise and diversity in the research community due to the ongoing decline in research funding. This loss of researchers is due to the small size of many of these research communities and a lack of appreciation by the larger scientific community concerning the state of art and challenges faced by researchers working on these organisms.     

Regional‐scale dominance of non‐framework building corals on Caribbean reefs affects carbonate production and future reef growth

Coral cover on Caribbean reefs has declined rapidly since the early 1980's. Diseases have been a major driver, decimating communities of framework building Acropora and Orbicella coral species, and reportedly leading to the emergence of novel coral assemblages often dominated by domed and plating species of the genera Agaricia, Porites and Siderastrea. These corals were not historically important Caribbean framework builders, and typically have much smaller stature and lower calcification rates, fuelling concerns over reef carbonate production and growth potential. Using data from 75 reefs from across the Caribbean we quantify: (i) the magnitude of non‐framework building coral dominance throughout the region and (ii) the contribution of these corals to contemporary carbonate production. Our data show that live coral cover averages 18.2% across our sites and coral carbonate production 4.1 kg CaCO₃ m^?2 yr^?1. However, non‐framework building coral species dominate and are major carbonate producers at a high proportion of sites; they are more abundant than Acropora and Orbicella at 73% of sites; contribute an average 68% of the carbonate produced; and produce more than half the carbonate at 79% of sites. Coral cover and carbonate production rate are strongly correlated but, as relative abundance of non‐framework building corals increases, average carbonate production rates decline. Consequently, the use of coral cover as a predictor of carbonate budget status, without species level production rate data, needs to be treated with caution. Our findings provide compelling evidence for the Caribbean‐wide dominance of non‐framework building coral taxa, and that these species are now major regional carbonate producers. However, because these species typically have lower calcification rates, continued transitions to states dominated by non‐framework building coral species will further reduce carbonate production rates below ‘predecline’ levels, resulting in shifts towards negative carbonate budget states and reducing reef growth potential.     

High‐resolution time‐resolved imaging of in vitro Arabidopsis rosette growth

Although quantitative characterization of growth phenotypes is of key importance for the understanding of essential networks driving plant growth, the majority of growth‐related genes are still being identified based on qualitative visual observations and/or single‐endpoint quantitative measurements. We developed an in vitro growth imaging system (IGIS) to perform time‐resolved analysis of rosette growth. In this system, Arabidopsis plants are grown in Petri dishes mounted on a rotating disk, and images of each plate are taken on an hourly basis. Automated image analysis was developed in order to obtain several growth‐related parameters, such as projected rosette area, rosette relative growth rate, compactness and stockiness, over time. To illustrate the use of the platform and the resulting data, we present the results for the growth response of Col–0 plants subjected to three mild stress conditions. Although the reduction in rosette area was relatively similar at 19 days after stratification, the time‐lapse analysis demonstrated that plants react differently to salt, osmotic and oxidative stress. The rosette area was altered at various time points during development, and leaf movement and shape parameters were also affected differently. We also used the IGIS to analyze in detail the growth behavior of mutants with enhanced leaf size. Analysis of several growth‐related parameters over time in these mutants revealed several specificities in growth behavior, underlining the high complexity of leaf growth coordination. These results demonstrate that time‐resolved imaging of in vitro rosette growth generates a better understanding of growth phenotypes than endpoint measurements.     

Global distribution,diversity hot spots and niche transitions of an astaxanthin‐producing eukaryotic microbe

Microbes establish very diverse but still poorly understood associations with other microscopic or macroscopic organisms that do not follow the more conventional modes of competition or mutualism. Phaffia rhodozyma, an orange‐coloured yeast that produces the biotechnologically relevant carotenoid astaxanthin, exhibits a Holarctic association with birch trees in temperate forests that contrasts with the more recent finding of a South American population associated with Nothofagus (southern beech) and with stromata of its biotrophic fungal parasite Cyttaria spp. We investigated whether the association of Phaffia with Nothofagus–Cyttaria could be expanded to Australasia, the other region of the world where Nothofagus are endemic, studied the genetic structure of populations representing the known worldwide distribution of Phaffia and analysed the evolution of the association with tree hosts. The phylogenetic analysis revealed that Phaffia diversity in Australasia is much higher than in other regions of the globe and that two endemic and markedly divergent lineages seem to represent new species. The observed genetic diversity correlates with host tree genera rather than with geography, which suggests that adaptation to the different niches is driving population structure in this yeast. The high genetic diversity and endemism in Australasia indicate that the genus evolved in this region and that the association with Nothofagus is the ancestral tree association. Estimates of the divergence times of Phaffia lineages point to splits that are much more recent than the break‐up of Gondwana, supporting that long‐distance dispersal rather than vicariance is responsible for observed distribution of P. rhodozyma.     

Next‐generation sequencing (NGS)‐based identification of induced mutations in a doubly mutagenized tomato (Solanum lycopersicum) population

The identification of mutations in targeted genes has been significantly simplified by the advent of TILLING (Targeting Induced Local Lesions In Genomes), speeding up the functional genomic analysis of animals and plants. Next‐generation sequencing (NGS) is gradually replacing classical TILLING for mutation detection, as it allows the analysis of a large number of amplicons in short durations. The NGS approach was used to identify mutations in a population of Solanum lycopersicum (tomato) that was doubly mutagenized by ethylmethane sulphonate (EMS). Twenty‐five genes belonging to carotenoids and folate metabolism were PCR‐amplified and screened to identify potentially beneficial alleles. To augment efficiency, the 600‐bp amplicons were directly sequenced in a non‐overlapping manner in Illumina MiSeq, obviating the need for a fragmentation step before library preparation. A comparison of the different pooling depths revealed that heterozygous mutations could be identified up to 128‐fold pooling. An evaluation of six different software programs (camba , crisp , gatk unified genotyper , lofreq , snver and vipr ) revealed that no software program was robust enough to predict mutations with high fidelity. Among these, crisp and camba predicted mutations with lower false discovery rates. The false positives were largely eliminated by considering only mutations commonly predicted by two different software programs. The screening of 23.47 Mb of tomato genome yielded 75 predicted mutations, 64 of which were confirmed by Sanger sequencing with an average mutation density of 1/367 Kb. Our results indicate that NGS combined with multiple variant detection tools can reduce false positives and significantly speed up the mutation discovery rate.     

The dependence of leaf senescence on the balance between 1‐aminocyclopropane‐1‐carboxylate acid synthase 1 (ACS1)‐catalysed ACC generation and nitric oxide‐associated 1 (NOS1)‐dependent NO accumulation in Arabidopsis

• Ethylene and nitric oxide (NO) act as endogenous regulators during leaf senescence. Levels of ethylene or its precursor 1‐aminocyclopropane‐1‐carboxylate acid (ACC) depend on the activity of ACC synthases (ACS), and NO production is controlled by NO‐associated 1 (NOA1). However, the integration mechanisms of ACS and NOA1 activity still need to be explored during leaf senescence.
• Here, using experimental techniques, such as physiological and molecular detection, liquid chromatography‐tandem mass spectrometry and fluorescence measurement, we investigated the relevant mechanisms.
• Our observations showed that the loss‐of‐function acs1‐1 mutant ameliorated age‐ or dark‐induced leaf senescence syndrome, such as yellowing and loss of chlorophyll, that acs1‐1 reduced ACC accumulation mainly in mature leaves and that acs1‐1‐promoted NOA1 expression and NO accumulation mainly in juvenile leaves, when compared with the wild type (WT). But the leaf senescence promoted by the NO‐deficient noa1 mutant was not involved in ACS1 expression. There was a similar sharp reduction of ACS1 and NOA1 expression with the increase in WT leaf age, and this inflection point appeared in mature leaves and coincided with the onset of leaf senescence.
• These findings suggest that NOA1‐dependent NO accumulation blocked the ACS1‐induced onset of leaf senescence, and that ACS1 activity corresponds to the onset of leaf senescence in Arabidopsis.

     

绞股蓝总皂甙含量与其营养器官和生长期相关性的研究

应用组织化学和植物化学方法,研究绞股蓝总皂甙含量与其营养器官和生长期的相关性。结果表明,不同营养器官中绞股蓝总皂甙含量叶最高,茎次之,根茎和根最低。绞股蓝地上部分总皂甙含量从营养生长→开花→结果不同生长期呈现由低→高→低的变化趋势,其中7、8、9月的开花期绞股蓝总皂甙含量达到顶峰,为药材最佳采收时间。研究还得出,绞股蓝叶的形态结构特征与其总皂甙含量相关,可作为选育绞股蓝优良品种的指标。