High‐throughput sequencing reveals neustonic and planktonic microbial eukaryote diversity in coastal waters

Neustonic organisms inhabit the sea surface microlayer (SML) and have important roles in marine ecosystem functioning. Here, we use high‐throughput 18S rRNA gene sequencing to characterize protist and fungal diversity in the SML at a coastal time‐series station and compare with underlying plankton assemblages. Protist diversity was higher in February (pre‐bloom) compared to April (spring bloom), and was lower in the neuston than in the plankton. Major protist groups, including Stramenopiles and Alveolata, dominated both neuston and plankton assemblages. Chrysophytes and diatoms were enriched in the neuston in April, with diatoms showing distinct changes in community composition between the sampling periods. Pezizomycetes dominated planktonic fungi assemblages, whereas fungal diversity in the neuston was more varied. This is the first study to utilize a molecular‐based approach to characterize neustonic protist and fungal assemblages, and provides the most comprehensive diversity assessment to date of this ecosystem. Variability in the SML microeukaryote assemblage structure has potential implications for biogeochemical and food web processes at the air‐sea interface.     

Evaluation of the Biological Sampling Kit (BiSKit) for large-area surface sampling

Current surface sampling methods for microbial contaminants are designed to sample small areas and utilize culture analysis. The total number of microbes recovered is low because a small area is sampled, making detection of a potential pathogen more difficult. Furthermore, sampling of small areas requires a greater number of samples to be collected, which delays the reporting of results, taxes laboratory resources and staffing, and increases analysis costs. A new biological surface sampling method, the Biological Sampling Kit (BiSKit), designed to sample large areas and to be compatible with testing with a variety of technologies, including PCR and immunoassay, was evaluated and compared to other surface sampling strategies. In experimental room trials, wood laminate and metal surfaces were contaminated by aerosolization of Bacillus atrophaeus spores, a simulant for Bacillus anthracis, into the room, followed by settling of the spores onto the test surfaces. The surfaces were sampled with the BiSKit, a cotton-based swab, and a foam-based swab. Samples were analyzed by culturing, quantitative PCR, and immunological assays. The results showed that the large surface area (1 m2) sampled with the BiSKit resulted in concentrations of B. atrophaeus in samples that were up to 10-fold higher than the concentrations obtained with the other methods tested. A comparison of wet and dry sampling with the BiSKit indicated that dry sampling was more efficient (efficiency, 18.4%) than wet sampling (efficiency, 11.3%). The sensitivities of detection of B. atrophaeus on metal surfaces were 42 +/- 5.8 CFU/m2 for wet sampling and 100.5 +/- 10.2 CFU/m2 for dry sampling. These results demonstrate that the use of a sampling device capable of sampling larger areas results in higher sensitivity than that obtained with currently available methods and has the advantage of sampling larger areas, thus requiring collection of fewer samples per site.     

Evaluation of the Biological Sampling Kit (BiSKit) for Large-Area Surface Sampling

Current surface sampling methods for microbial contaminants are designed to sample small areas and utilize culture analysis. The total number of microbes recovered is low because a small area is sampled, making detection of a potential pathogen more difficult. Furthermore, sampling of small areas requires a greater number of samples to be collected, which delays the reporting of results, taxes laboratory resources and staffing, and increases analysis costs. A new biological surface sampling method, the Biological Sampling Kit (BiSKit), designed to sample large areas and to be compatible with testing with a variety of technologies, including PCR and immunoassay, was evaluated and compared to other surface sampling strategies. In experimental room trials, wood laminate and metal surfaces were contaminated by aerosolization of Bacillus atrophaeus spores, a simulant for Bacillus anthracis, into the room, followed by settling of the spores onto the test surfaces. The surfaces were sampled with the BiSKit, a cotton-based swab, and a foam-based swab. Samples were analyzed by culturing, quantitative PCR, and immunological assays. The results showed that the large surface area (1 m²) sampled with the BiSKit resulted in concentrations of B. atrophaeus in samples that were up to 10-fold higher than the concentrations obtained with the other methods tested. A comparison of wet and dry sampling with the BiSKit indicated that dry sampling was more efficient (efficiency, 18.4%) than wet sampling (efficiency, 11.3%). The sensitivities of detection of B. atrophaeus on metal surfaces were 42 ± 5.8 CFU/m² for wet sampling and 100.5 ± 10.2 CFU/m² for dry sampling. These results demonstrate that the use of a sampling device capable of sampling larger areas results in higher sensitivity than that obtained with currently available methods and has the advantage of sampling larger areas, thus requiring collection of fewer samples per site.     

An efficient TILLING platform for cultivated tobacco

Targeting-induced local lesions in genomes(TILLING) is a powerful reverse-genetics tool that enables high-throughput screening of genomic variations in plants.Although TILLING has been developed for many diploid plants, the technology has been used in very few polyploid species due to their genomic complexity. Here, we established an efficient capillary electrophoresis-based TILLING platform for allotetraploid cultivated tobacco(Nicotiana tabacum L.) using an ethyl methanesulfonate(EMS)-mutagenized population of 1,536 individuals. We optimized the procedures for endonuclease preparation,leaf tissue sampling, DNA extraction, normalization,pooling, PCR amplification, heteroduplex formation, and capillary electrophoresis. In a test screen using seven target genes with eight PCR fragments, we obtained 118 mutants. The mutation density was estimated to be approximately one mutation per 106kb on average.Phenotypic analyses showed that mutations in two heavy metal transporter genes, HMA2S and HMA4T, led to reduced accumulation of cadmium and zinc, which was confirmed independently using CRISPR/Cas9 to generate knockout mutants. Our results demonstrate that this powerful TILLING platform(available at http://www.croptilling.org)can be used in tobacco to facilitate functional genomics applications.     

A hotspot for cold crenarchaeota in the neuston of high mountain lakes

We have surveyed the first 1 m of 10 oligotrophic high mountain lakes in the Central Pyrenees (Spain) for both abundance and predominant phylotypes richness of the archaeaplankton assemblage, using CARD-FISH and 16S rRNA gene sequencing respectively. Archaea inhabiting the air-water surface microlayer (neuston) ranged between 3% and 37% of total 4,6-diamidino-2-phenylindole (DAPI) counts and were mainly Crenarchaeota of a new freshwater cluster distantly related to the Marine Group 1.1a. Conversely, most of the Archaea from the underlying waters (the remaining first 1 m integrated) were mainly Euryarchaeota of three distantly related branches ranging between 0.4% and 27% of total DAPI counts. Therefore, a consistent qualitative and quantitative spatial segregation was observed for the two main archaeal phyla between neuston and underlying waters at a regional scale. We also observed a consistent pattern along the lakes surveyed between lake area, lake depth and water residence time, and the archaeal enrichment in the neuston: the larger the lake the higher the proportion of archaea in the neuston as compared with abundances from the underlying waters ( n  = 10 lakes; R ² > 0.80; P  < 0.001, in all three cases). This is the first report identifying a widespread non-thermophilic habitat where freshwater planktonic Crenarchaeota can be found naturally enriched. High mountain lakes offer great research opportunities to explore the ecology of one of the most enigmatic and far from being understood group of prokaryotes.     

Der Jahresgang von Neuston im Golf von Neapel

The annual fluctuations in the occurrence of 91 taxa which were identified, made the recognition of 4 main seasonal populations possible. Trends were observed, as well as analogies to other patterns of distribution. On an average, more than half of the fauna was displaced from month to month, but the variation in the density of subtropical neuston appeared to be less over an annual period than it was during the day, and from region to region. The dominance of certain species and occurrence of neuston throughout the year demonstrated, that regionally at least a biocoenosis exists continously at the surface of the sea.     

沉水植物在治理滇池草海污染中的作用

本文论证了在滇池草海中恢复沉水植物并建立以优化的沉水植物为基础的湖泊生态系统,是利用水生植物治理草海的最佳生物防治措施。这种以优化的沉水植物为基础的湖泊生态系统优于以浮游植物或以漂浮植物为基础的湖泊生态系统。结合水生植物的综合利用,它是一种一举多得、经济而又实效的治理手段。     

The mysterious ecosystem at the ocean’s surface

Life on the ocean’s surface connects worlds. From shallow waters to the deep sea, the open ocean to rivers and lakes, numerous terrestrial and marine species depend on the surface ecosystem and the organisms found therein. Organisms that live freely at the surface, termed “neuston,” include keystone organisms like the golden seaweed Sargassum that makes up the Sargasso Sea, floating barnacles, snails, nudibranchs, and cnidarians. Many ecologically and economically important fish species live as or rely upon neuston. Species at the surface are not distributed uniformly; the ocean’s surface harbors unique neustonic communities and ecoregions found at only certain latitudes and only in specific ocean basins. But the surface is also on the front line of climate change and pollution. Despite the diversity and importance of the ocean’s surface in connecting disparate habitats, and the risks it faces, we know very little about neustonic life. This Essay will introduce you to the neuston, their connections to diverse habitats, the threats they face, and new opportunities for research and discovery at the air-sea interface.

The mysterious ’neuston’ ecosystem at the ocean’s surface includes keystone organisms like the golden seaweed Sargassum that makes up the Sargasso Sea, floating barnacles, snails, nudibranchs, and cnidarians; this Essay explores threats to its wellbeing and the importance of further research.     

Temporal changes in essential fatty acid availability in different food sources in the littoral macrophyte zone

In littoral environments, different food resources are available for zooplankters. In addition to seston, species may feed on biofilms growing on sediments, plants (epiphyton), and at the air–water interface (neuston). However, despite a growing interest in these different biofilms, little is known about their food quality for microcrustaceans. In a field study, we measured changes in the food quality over time in terms of the essential fatty acid (EFA) content of different potential food sources for littoral consumers. The food quality of seston, neuston, and epiphyton growing on three different aquatic macrophytes were assessed. Our results showed that there is an important seasonal variability within each food source. However, in the system studied, epiphytic biofilms, especially those of Ludwigia and Callitriche offered the highest food quality, in terms of EFA content, throughout the year. As the highest EFA concentrations in each food source were found consecutively, high concentrations of these physiologically important compounds are maintained in the system throughout the year. Therefore, greater diversity of food resources could affect ecosystem productivity.     

A simple strategy for mitigating the effect of data variability on the identification of active chemotypes from high-throughput screening data

Among the several goals of a high-throughput screening campaign is the identification of as many active chemotypes as possible for further evaluation. Often, however, the number of concentration response curves (e.g., IC(50)s or K(i)s) that can be collected following a primary screen is limited by practical constraints such as protein supply, screening workload, and so forth. One possible approach to this dilemma is to cluster the hits from the primary screen and sample only a few compounds from each cluster. This introduces the question as to how many compounds must be selected from a cluster to ensure that an active compound is identified, if it exists at all. This article seeks to address this question using a Monte Carlo simulation in which the dependence of the success of sampling is directly linked to screening data variability. Furthermore, the authors demonstrate that the use of replicated compounds in the screening collection can easily assess this variability and provide a priori guidance to the screener and chemist as to the extent of sampling required to maximize chemotype identification during the triage process. The individual steps of the Monte Carlo simulation provide insight into the correspondence between the percentage inhibition and eventual IC(50) curves.     

Vertical distribution of the European sardine (Sardina pilchardus) larvae and its implications for their survival

This study presents results of the vertical behaviour of theEuropean sardine (Sardina pilchardus) larvae as observed atsea off the NW Iberian coast during an oceanographic cruiseconducted in May 2002. Samples were taken in a grid of 38 stations(conductivity-temperature-depth [CTD] measurements and LonghurstHardy Plankton Recorder [LHPR] plankton hauls); a 69-h fixedstation study was also performed (hourly CTD measurements andLHPR/neuston hauls every 2 h). The horizontal distribution oflarvae is closely related to the circulation patterns measuredby a current metre-mooring array deployed during the cruise.Larvae were mainly distributed in the upper 20–25 m ofthe water column, in evident association with the waters ofthe Western Iberia Buoyant Plume (WIBP). Large (older) larvaeare found mainly in the surface layers, and larval size decreaseswith depth. A diel rhythm of migration to the neuston layerwas observed, correlated with the inflation/deflation activityof the swim bladder. Larvae with lengths greater than 12.5 mmand inflated swim bladders were only found in this layer. Consideringthe near surface stratification conditions for food availabilityand Ekman transport in the upper few metres, even small verticalmigrations of larvae can be very important for their survivaland subsequent recruitment success.     

The retrieval of material from the surface micro layer with screen and plate samplers and its implications for partitioning of material within the micro layer

SUMMARY.

• 1 The surface micro layers of two small ponds in Wisconsin were sampled over several years using screen and plate samplers. These devices performed identically for dissolved substances but for particulate material the glass plate sampler produced values 4 times those obtained with the screen.
• 2 These differences in sampling efficiency are probably the result of the failure of the screen sampler to release all of the particulate material it has collected and also to differences in the partitioning of dissolved and particulate material within the micro layer.
• 3 Results suggest that particulates are concentrated at the air-water interface and that there is a four-fold dilution with subsurface water in the screen samples when compared to the plate samples. Dissolved material, in contrast, is concentrated in a thicker layer below the surface mono layer, resulting in equal sampling efficiency with the plate and screen samplers.

     

MDL1 is a high copy suppressor of ATM1: evidence for a role in resistance to oxidative stress

The yeast ATM1 gene is essential for normal cellular iron homeostasis. Deletion of ATM1 results in mitochondrial iron accumulation and increased sensitivity to oxidative stress and transition metal toxicity. Atm1p is an ATP-binding cassette (ABC) transporter localized to the mitochondrial inner membrane. The specific function of Atm1p has not been determined, though roles in both mitochondrial iron export and cytosolic Fe-S cluster assembly have been proposed. We undertook a screen for yeast genes capable of suppressing the abnormalities of cellular iron metabolism demonstrated by Deltaatm1 cells. One of the genes we identified was MDL1, which like ATM1, encodes a mitochondrial inner membrane ABC transporter. Mdl1p has previously been shown to function in the export of peptides from the mitochondrial matrix. We demonstrate that over-expression of MDL1 in Deltaatm1 cells results in a reduction of mitochondrial iron content, and decreased sensitivity to H(2)O(2) and transition metal toxicity. Additionally, in studies of the effect of over-expression and deletion of MDL1, we have identified a novel role for Mdl1p in the regulation of cellular resistance to oxidative stress.     

Histidine protects against zinc and nickel toxicity in Caenorhabditis elegans

Zinc is an essential trace element involved in a wide range of biological processes and human diseases. Zinc excess is deleterious, and animals require mechanisms to protect against zinc toxicity. To identify genes that modulate zinc tolerance, we performed a forward genetic screen for Caenorhabditis elegans mutants that were resistant to zinc toxicity. Here we demonstrate that mutations of the C. elegans histidine ammonia lyase (haly-1) gene promote zinc tolerance. C. elegans haly-1 encodes a protein that is homologous to vertebrate HAL, an enzyme that converts histidine to urocanic acid. haly-1 mutant animals displayed elevated levels of histidine, indicating that C. elegans HALY-1 protein is an enzyme involved in histidine catabolism. These results suggest the model that elevated histidine chelates zinc and thereby reduces zinc toxicity. Supporting this hypothesis, we demonstrated that dietary histidine promotes zinc tolerance. Nickel is another metal that binds histidine with high affinity. We demonstrated that haly-1 mutant animals are resistant to nickel toxicity and dietary histidine promotes nickel tolerance in wild-type animals. These studies identify a novel role for haly-1 and histidine in zinc metabolism and may be relevant for other animals.     

Plankton reach new heights in effort to avoid predators

The marine environment associated with the air-water interface (neuston) provides an important food source to pelagic organisms where subsurface prey is limited. However, studies on predator-prey interactions within this environment are lacking. Copepods are known to produce strong escape jumps in response to predators, but must contend with a low-Reynolds-number environment where viscous forces limit escape distance. All previous work on copepod interaction with predators has focused on a liquid environment. Here, we describe a novel anti-predator behaviour in two neustonic copepod species, where individuals frequently exit the water surface and travel many times their own body length through air to avoid predators. Using both field recordings with natural predators and high-speed laboratory recordings, we obtain detailed kinematics of this behaviour, and estimate energetic cost associated with this behaviour. We demonstrate that despite losing up to 88 per cent of their initial kinetic energy, copepods that break the water surface travel significantly further than those escaping underwater and successfully exit the perceptive field of the predator. This behaviour provides an effective defence mechanism against subsurface-feeding visual predators and the results provide insight into trophic interactions within the neustonic environment.     

Evaluation of Two Surface Sampling Methods for Detection of Erwinia herbicola on a Variety of Materials by Culture and Quantitative PCR

This research was designed to evaluate surface sampling protocols for use with culture and quantitative PCR (QPCR) amplification assay for detection of the gram-negative bacterial biothreat simulant Erwinia herbicola on a variety of surface materials. Surfaces selected for evaluation were wood laminate, glass and computer monitor screens, metal file cabinets, plastic arena seats, nylon seat cushions, finished concrete flooring, and vinyl tile flooring. Laboratory and test chamber studies were performed to evaluate two sampling methods, a sponge and a macrofoam swab, for detection of E. herbicola on surface materials. In laboratory trials, seven materials were inoculated with a known concentration of E. herbicola cells and samples were collected from the surfaces of the materials to determine sampling efficiencies. Culture analysis was ineffective for assessing E. herbicola collection efficiency because very few culturable cells were obtained from surface samples. QPCR demonstrated that E. herbicola DNA was present in high concentrations on all of the surface samples, and sampling efficiencies ranged from 0.7 to 52.2%, depending on the sampling method and the surface material. The swab was generally more efficient than the sponge for collection of E. herbicola from surfaces. Test chamber trials were also performed in which E. herbicola was aerosolized into the chamber and allowed to settle onto test materials. Surface sampling results supported those obtained in laboratory trials. The results of this study demonstrate the capabilities of QPCR to enhance the detection and enumeration of biocontaminants on surface materials and provide information on the comparability of sampling methods.     

A Metal Detector Study to Locate Inactive Small Arms Range Impact Areas

Historical records suggested the existence of .45 caliber Thompson sub-machine gun ranges near the Fort Lewis Evergreen Range; however, the precise location of the bullet impact area was not known. The site covered 20 acres, too large to cost-effectively characterize using convention grid sampling techniques. As an alternative, a study was conducted using a hand-held metal detector to locate the bullet impact areas.

The metal detector chosen, a Garrett Graphic Target Imaging? (GTI) 2500, has an LCD screen, which provides the user information on the potential size and depth of the object causing the signal. A two-man team performed the study and bullet detection was confirmed by excavation.

The metal detector proved effective at finding .45 caliber bullets. Three impact areas were successfully identified and marked using a global positioning system (GPS). The metal detecting strategy saved substantial sampling time and over $35,000.00 by decreasing the sampling area by 93 percent.     

Adaptive responses to static conditions in nutrient-rich cultures of luminous Ralstonia eutropha

The lux-gene fused Ralstonia eutropha, when adapting to static conditions, causes stratification of air-exposed and nutrient-rich cultures at above 0.15 mg biomass ml(-1). The O2 respiring biofilm (luminous neuston) phase, along with the dark sub-neustonic suspension phase, develops within 5-60 min. The instability of the biphasic static culture was identified as a reason for occasionally observable oscillatory bioluminescence.     

A membrane adsorption-SEM technique for observing neuston organisms

A technique to observe neuston organisms, combining membrane adsorption and scanning electron microscopy, is described. Bacteria, algae, and protozoa collected from the surface of 2 freshwater ponds using this methodology appeared well preserved by the fixation and dehydration procedures used.