Microbial metabolomic responses to changes in temperature and salinity along the western Antarctic Peninsula.

Seasonal cycles within the marginal ice zones in polar regions include large shifts in temperature and salinity that strongly influence microbial abundance and physiology. However, the combined effects of concurrent temperature and salinity change on microbial community structure and biochemical composition during transitions between seawater and sea ice are not well understood. Coastal marine communities along the western Antarctic Peninsula were sampled and surface seawater was incubated at combinations of temperature and salinity mimicking the formation (cold, salty) and melting (warm, fresh) of sea ice to evaluate how these factors may shape community composition and particulate metabolite pools during seasonal transitions. Bacterial and algal community structures were tightly coupled to each other and distinct across sea-ice, seawater, and sea-ice-meltwater field samples, with unique metabolite profiles in each habitat. During short-term (approximately 10-day) incubations of seawater microbial communities under different temperature and salinity conditions, community compositions changed minimally while metabolite pools shifted greatly, strongly accumulating compatible solutes like proline and glycine betaine under cold and salty conditions. Lower salinities reduced total metabolite concentrations in particulate matter, which may indicate a release of metabolites into the labile dissolved organic matter pool. Low salinity also increased acylcarnitine concentrations in particulate matter, suggesting a potential for fatty acid degradation and reduced nutritional value at the base of the food web during freshening. Our findings have consequences for food web dynamics, microbial interactions, and carbon cycling as polar regions undergo rapid climate change.

An examination of the effects of labor efficiency on the profitability of grass-based, seasonal-calving dairy farms.

The seasonality of grass-based, seasonal-calving dairy systems results in disproportionately higher labor demands during the spring, when cows are calving, than in the remaining seasons. This study aimed to (1) examine the relationship between labor efficiency and profitability; (2) investigate strategies to reduce the hours worked per day by the farmer, family, and farm staff in the spring by having certain tasks outsourced; and (3) quantify the economic implications of those strategies. Data from an existing labor efficiency study on Irish dairy farms were used in conjunction with economic performance data from the farms. Tasks that required the highest level of farm labor per day in the spring were identified and hypothetical strategies to reduce the farm hours worked per day were examined. A stochastic budgetary simulation model was then used to examine the economic implications of employing these strategies and the effects of their use in conjunction with a proportionate increase in cow numbers that would leave the hours worked per day unchanged. The strategies were to use contractors to perform calf rearing, machinery work, or milking. Contracting out milking resulted in the greatest reduction in hours worked per day (5.6 h/d) followed by calf rearing (2.7 h/d) and machinery work (2 h/d). Reducing the hours worked per day by removing those tasks had slight (i.e., <5%) negative effects on profitability; however, maintaining the farm hours worked per day while utilizing the same strategies and increasing herd sizes resulted in profitable options. The most profitable scenario was for farms to increase herd size while contracting out milking.

Measuring labor input on pasture-based dairy farms using a smartphone.

With the cessation of milk quotas in the European Union, dairy herd sizes increased in some countries, including Ireland, with an associated increase in labor requirement. Second to feed costs, labor has been identified as one of the highest costs on pasture-based dairy farms. Compared with other European Union countries, Ireland has historically had low milk production per labor unit; thus, optimization of labor efficiency on farm should be addressed before or concurrently with herd expansion. The objective of this study was to quantify current levels of labor input and labor efficiency on commercial pasture-based dairy farms and to identify the facilities and management practices associated with increased labor efficiency. Thirty-eight dairy farms of varying herd sizes, previously identified as labor-efficient farms, were enrolled on the study and data were collected over 3 consecutive days each month over a 12-mo period, starting in May 2015 and finishing in August of 2016. This was achieved through the use of a smartphone application. For analysis purposes, farms were categorized into 1 of 3 herd size categories (HSC): farms with <150 cows (HSC 1), 150-249 cows (HSC 2), or ≥250 cows (HSC 3). Overall farm labor input increased with HSC with 3,015, 4,499, and 6,023 h worked on HSC 1, 2, and 3, respectively. A higher proportion of work was carried out by hired staff as herd size increased. Labor efficiency was measured as total hours input to the dairy enterprise divided by herd size. Labor efficiency improved as herd size increased above 250 cows with 17.3 h/cow per yr observed for HSC 3; labor efficiency was similar for HSC 1 and 2, at 23.8 and 23.3 h/cow per yr, respectively. A large range of efficiency was observed within HSC. The labor requirements had a distinct seasonal pattern across the 3 HSC with the highest input observed in springtime (February to April) primarily due to calving and calf-care duties, milking, and winter feeding. The lowest input was observed in wintertime (November to January) when cows were dry. Particular facilities and management practices were associated with efficiency within certain tasks, the most notable in regard to milking and winter feeding practices. Additionally, the most efficient farms used contractors to perform a higher proportion of machinery work on farm than the least efficient farms.

Low-temperature chemotaxis, halotaxis and chemohalotaxis by the psychrophilic marine bacterium Colwellia psychrerythraea 34H.

A variety of ecologically important processes are driven by bacterial motility and taxis, yet these basic bacterial behaviours remain understudied in cold habitats. Here, we present a series of experiments designed to test the chemotactic ability of the model marine psychrophilic bacterium Colwellia psychrerythraea 34H, when grown at optimal temperature and salinity (8°C, 35 ppt) or its original isolation conditions (-1°C, 35 ppt), towards serine and mannose at temperatures from -8°C to 27°C (above its upper growth temperature of 18°C), and at salinities of 15, 35 and 55 ppt (at 8°C and -1°C). Results indicate that C. psychrerythraea 34H is capable of chemotaxis at all temperatures tested, with strongest chemotaxis at the temperature at which it was first grown, whether 8°C or -1°C. This model marine psychrophile also showed significant halotaxis towards 15 and 55 ppt solutions, as well as strong substrate-specific chemohalotaxis. We suggest that such patterns of taxis may enable bacteria to colonize sea ice, position themselves optimally within its extremely cold, hypersaline and temporally fluctuating microenvironments, and respond to various chemical signals therein.

Evidence for marine origin and microbial-viral habitability of sub-zero hypersaline aqueous inclusions within permafrost near Barrow, Alaska.

Cryopegs are sub-surface hypersaline brines at sub-zero temperatures within permafrost; their global extent and distribution are unknown. The permafrost barrier to surface and groundwater advection maintains these brines as semi-isolated systems over geological time. A cryopeg 7 m below ground near Barrow, Alaska, was sampled for geochemical and microbiological analysis. Sub-surface brines (in situtemperature of -6 °C, salinity of 115 ppt), and an associated sediment-infused ice wedge (melt salinity of 0.04 ppt) were sampled using sterile technique. Major ionic concentrations in the brine corresponded more closely to other (Siberian) cryopegs than to Standard seawater or the ice wedge. Ionic ratios and stable isotope analysis of water conformed to a marine or brackish origin with subsequent Rayleigh fractionation. The brine contained ∼1000× more bacteria than surrounding ice, relatively high viral numbers suggestive of infection and reproduction, and an unusually high ratio of particulate to dissolved extracellular polysaccharide substances. A viral metagenome indicated a high frequency of temperate viruses and limited viral diversity compared to surface environments, with closest similarity to low water activity environments. Interpretations of the results underscore the isolation of these underexplored microbial ecosystems from past and present oceans.

Lumen Formation Is an Intrinsic Property of Isolated Human Pluripotent Stem Cells.

We demonstrate that dissociated human pluripotent stem cells (PSCs) are intrinsically programmed to form lumens. PSCs form two-cell cysts with a shared apical domain within 20 hr of plating; these cysts collapse to form monolayers after 5 days. Expression of pluripotency markers is maintained throughout this time. In two-cell cysts, an apical domain, marked by EZRIN and atypical PKCζ, is surrounded by apically targeted organelles (early endosomes and Golgi). Molecularly, actin polymerization, regulated by ARP2/3 and mammalian diaphanous-related formin 1 (MDIA), promotes lumen formation, whereas actin contraction, mediated by MYOSIN-II, inhibits this process. Finally, we show that lumenal shape can be manipulated in bioengineered micro-wells. Since lumen formation is an indispensable step in early mammalian development, this system can provide a powerful model for investigation of this process in a controlled environment. Overall, our data establish that lumenogenesis is a fundamental cell biological property of human PSCs.

Selective occurrence of Rhizobiales in frost flowers on the surface of young sea ice near Barrow, Alaska and distribution in the polar marine rare biosphere.

Frost flowers are highly saline ice structures that grow on the surface of young sea ice, a spatially extensive environment of increasing importance in the Arctic Ocean. In a previous study, we reported organic components of frost flowers in the form of elevated levels of bacteria and exopolymers relative to underlying ice. Here, DNA was extracted from frost flowers and young sea ice, collected in springtime from a frozen lead offshore of Barrow, Alaska, to identify bacteria in these understudied environments. Evaluation of the distribution of 16S rRNA genes via four methods (microarray analysis, T-RFLP, clone library and shotgun metagenomic sequencing) indicated distinctive bacterial assemblages between the two environments, with frost flowers appearing to select for Rhizobiales. A phylogenetic placement approach, used to evaluate the distribution of similar Rhizobiales sequences in other polar marine studies, indicated that some of the observed strains represent widely distributed members of the marine rare biosphere in both the Arctic and Antarctic.

Associations of housing, management, milking activity, and standing and lying behavior of dairy cows milked in automatic systems.

The objective of this cross-sectional study was to describe the housing, feeding management, and characteristics (parity and stage of lactation) of cows on commercial automatic milking system (AMS) dairies and their associations with the standing and lying behavior patterns and milking activity (frequency and yield) of lactating dairy cows. Thirteen AMS herds were enrolled in the study, with an average herd size of 71±30 (mean ± SD; range: 34 to 131) lactating cows. All of the herds used freestall barns, each set up for free cow traffic to the AMS. On-farm measurements were taken to determine stocking density at the freestalls (0.9±0.1 cows/stall; mean ± SD), feed bunk (0.66±0.17 m of feed bunk space/cow; mean ± SD), and AMS units (55±11 cows/AMS; mean ± SD). A random sample of 30 cows/herd was selected to monitor standing and lying behavior for 4d using electronic data loggers. Times of feed delivery and feed push-up were recorded daily by the herd managers. Milking times, frequency, and yield were automatically recorded by the AMS units. Data were analyzed in a multivariable mixed regression model to determine which herd-level (housing and feeding management) and cow-level (parity, DIM, and milk yield) factors were associated with behavior and milking activity measures. Lying bout lengths were found to be negatively associated with milk yield and tended to be positively associated with more space at the feed bunk. Increased lying duration was associated with cows of lower milk production, increased space at the feed bunk, and increased frequency of feed push-up. Longer postmilking standing durations were associated with cows of higher parity. An association existed between cows milking less frequently when they were further in lactation, were of higher parity, and as stocking density at the AMS (cows/AMS) increased. Milk yield was positively associated with increased space at the feed bunk and higher parity and negatively associated with DIM. From this study, it can be concluded that increased milking frequency may be achieved in AMS herds by reducing stocking density at the AMS unit. Further, in AMS systems, greater milk yield and lying duration may be achieved by ensuring that cows have ample feed bunk space and have their feed readily available to them in the bunk.

Short communication: Kelp taste preferences by dairy calves.

Kelp is a common feed additive used on many dairy farms in the United States. However, few data are available supporting the efficacy of its addition to cattle feed. The purpose of this study was to evaluate the taste preferences of calves provided with 0, 30, or 60 g of kelp daily in a sequential elimination experiment. Calves in this study always ranked the control treatment first when given a choice and consumed 34.5% more dry matter from the control treatment in the first 3-d segment of the experiment. During the second feeding segment (d 4 and 5), when the control treatment was removed, daily dry matter consumption was reduced in 4 out of 6 calves compared with control calves when this treatment was available (first feeding segment). However, calves did not differentiate between the 2 amounts of kelp. Results indicated that calves preferred calf starter grains without kelp.

Association of standing and lying behavior patterns and incidence of intramammary infection in dairy cows milked with an automatic milking system.

The standing and lying behavior patterns of dairy cows, particularly the length of time cows spend standing after milking, have the potential to influence the incidence of intramammary infection (IMI). The objectives were to describe the standing and lying behavior patterns of cows milked with an automatic milking system (AMS) and to determine how these patterns relate to the incidence of IMI. One hundred and eleven lactating Holstein dairy cows were monitored over a 4-mo period. These cows were kept in a sand-bedded freestall barn with 2 pens, each with a free cow traffic AMS. Feed was delivered once daily, and pushed up 2 to 3 times daily. Quarter milk samples were collected for bacteriological culture from each cow once every 4 wk. A new IMI was defined as a positive culture sample following a negative culture. For 7 d before each of the last 3 milk samplings, standing and lying behavior, and times of milking and feed manipulation (feed delivery and push up) were recorded. Daily lying time and lying bout length were negatively related with milk yield (r=-0.23 and -0.20, respectively) and milking frequency (r=-0.32 and -0.20, respectively); milk yield was positively related to milking frequency (r=0.58). Feed manipulation near the time cows were milked (1h before 2h after) resulted in the longest post-milking standing times (mean=86 min; 95% confidence interval=78, 94 min), whereas feed manipulation occurring outside that time frame resulted in shorter post-milking standing times. Over the study period, 171 new IMI were detected. Of these new IMI detected, those caused by coagulase-negative staphylococci were the only ones associated with post-milking standing time; as post-milking standing time increased past 2.5h after milking, the odds of acquiring a new IMI tended to also increase. In summary, standing and lying behavior patterns of cows milked with an AMS were affected by both feed manipulation and their milking activity. Further, the post-milking standing time of cows milked with an AMS can be managed by providing fresh feed, as well as by pushing up feed, frequently throughout the day. Finally, cows that spend long periods of time (>2.5h) standing following milking may be at higher risk of acquiring a new CNS IMI.

Phylogenetic diversity of numerically important Arctic sea-ice bacteria cultured at subzero temperature.

Heterotrophic bacteria in sea ice play a key role in carbon cycling, but little is known about the predominant players at the phylogenetic level. In a study of both algal bands and clear ice habitats within summertime Arctic pack ice from the Chukchi Sea, we determined the abundance of total bacteria and actively respiring cells in melted ice samples using epifluorescence microscopy and the stains 4', 6'-diamidino-2-phenylindole 2HCl (DAPI) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), respectively. Organic-rich and -poor culturing media were used to determine culturable members by plating (at 0 degrees C and 5 degrees C) and most-probable-number (MPN) analyses (at -1 degrees C). Total bacterial counts ranged from 5.44 x 10(4) ml(-1) in clear ice to 2.41 x 10(6) ml(-1) in algal-band ice samples, with 2-27% metabolically active by CTC stain. Plating and MPN results revealed a high degree of culturability in both types of media, but greater success in oligotrophic media (to 62% of total abundance) and from clear ice samples. The bacterial enumeration anomaly, commonly held to mean

Remarkably low temperature optima for extracellular enzyme activity from Arctic bacteria and sea ice.

Extracellular degradative enzymes released by psychrophilic marine bacteria (growing optimally at or below 15 degrees C and maximally at 20 degrees C) typically express activity optima at temperatures well above the upper growth limit of the producing strain. In the present study, we investigated whether or not near-zero Arctic environments contain extracellular enzymes with activity optimized to temperatures lower than previously reported. By applying fluorescently tagged substrate analogues to measure leucine-aminopeptidase and chitobiase activity, the occurrence of extracellular enzymatic activity (EEA) with remarkably low temperature optima (15 degrees C) was documented in sea-ice samples. An extremely psychrophilic bacterial isolate, strain 34H, yielded an extract of cell-free protease with activity optimized at 20 degrees C, the lowest optimum yet reported for cell-free EEA from a pure culture. The use of zymogram gels revealed the presence of three proteolytic bands (between 37 and 45 kDa) in the extract and the release of the greatest quantities of the proteases when the strain was grown at -1 degrees C, suggesting a bacterial strategy for counteracting the effects of very cold temperatures on the catalytic efficiency of released enzymes. The detection of unusually cold-adapted EEA in environmental samples has ramifications not only to polar ecosystems and carbon cycling but also to protein evolution, biotechnology and bioremediation.

Evidence for the microbial basis of a chemoautotrophic invertebrate community at a whale fall on the deep seafloor: bone-colonizing bacteria and invertebrate endosymbionts.

To explore the microbial basis for a remarkable macrofaunal community at the site of a whale skeleton on the seafloor of the Santa Catalina Basin, we obtained samples of whale bone, bone-colonizing invertebrates, microbial mats, and the dominant fauna in the adjacent sulfide-rich sediments during Alvin expeditions in 1988 and 1991. Invertebrate tissues were examined by transmission electron microscopy (TEM) and mats and bone-penetrating bacteria by epifluorescence microscopy (EM). Tissues from the dominant bivalve Vesicomya c.f. gigas, the mytilid mussel Idasola washingtonia, and selected gastropods and limpets were also assayed chemically for enzymes diagnostic of sulfur- and methane-based chemoautotrophy and for stable carbon isotopic composition. Results of all analyses were consistent with dominant sulfur-based endosymbioses in the clam and mussel (the first record of endosymbiosis in the genus Idasola) and the general absence of methane symbioses at the site, strengthening the analogy of the whale-skeleton faunal community to those known from distant Pacific hydrothermal vent sites. Examples of minor endosymbionts, either nitrifying or methanotrophic cells according to internal membrane structures by TEM, raised the possibility of a supplemental mode of nutrition to the clam, or means to remove ammonia in the gill tissue, in the event of significant changes in the chemical environment.

Enumeration and phylogenetic analysis of polycyclic aromatic hydrocarbon-degrading marine bacteria from Puget sound sediments.

Naphthalene- and phenanthrene-degrading bacteria in Puget Sound sediments were enumerated by most-probable-number enumeration procedures. Sediments from a creosote-contaminated Environmental Protection Agency Superfund Site (Eagle Harbor) contained from 10(4) to 10(7) polycyclic aromatic hydrocarbon (PAH)-degrading bacteria g (dry weight) of sediment-1, whereas the concentration at an uncontaminated site ranged from 10(3) to 10(4) g of sediment(-1). Isolates of PAH-degrading bacteria were obtained from these most-probable-number tubes as well as from sediment samples from noncontaminated sites and from bioreactors enriched with PAHs. The 18 resulting strains were grouped by whole-cell fatty acid analysis into two subgroups. The larger group of strains belonged to the newly described genus Cycloclasticus, whereas the other group contained members of the genus Vibrio. The Cycloclasticus group seems to be widespread in noncontaminated sediments. PAH degradation was confirmed in selected strains on the basis of removal of phenanthrene from growing cultures.

Deep-sea smokers: windows to a subsurface biosphere?

Since the discovery of hyperthermophilic microbial activity in hydrothermal fluids recovered from "smoker" vents on the East Pacific Rise, the widely accepted upper temperature limit for life (based on pure culture data) has risen from below the boiling point of water at atmospheric pressure to approximately 115 degrees C. Many microbiologists seem willing to speculate that the maximum may be closer to 150 degrees C. We have postulated not only higher temperatures than these (under deep-sea hydrostatic pressures), but also the existence of a biosphere subsurface to accessible seafloor vents. New geochemical information from the Endeavour Segment of the Juan de Fuca Ridge indicative of subsurface organic material caused us to re-examine both the literature on hyperthermophilic microorganisms cultured from deep-sea smoker environments and recent results of microbial sampling efforts at actively discharging smokers on the Endeavour Segment. Here we offer the case for a subsurface biosphere based on an interdisciplinary view of microbial and geochemical analyses of Endeavour smoker fluids, a case in keeping with rapidly evolving geophysical understanding of organic stability under deep-sea hydrothermal conditions.

Kinetics of peptide hydrolysis and amino acid decomposition at high temperature.

Dipeptide hydrolysis and amino acid decomposition appear to follow a first-order rate law. The hydrolysis rate increases exponentially with increasing temperature in aqueous solution at both 265 atm and water steam pressures over the temperature range of 100 to 220 degrees C. Dipeptide hydrolysis has a lower apparent activation energy at 265 atm (44.1 KJ/mol) than at water steam pressure (98.9 KJ/mol). At lower temperatures (<200-220 degrees C), the rate of peptide bond hydrolysis is faster at 265 atm than at water steam pressure. At higher temperatures (>200-220 degrees C), however, peptide bond hydrolysis is slower at 265 atm than at water steam pressure. In aqueous solution, amino acid decomposition rates also increase exponentially with increasing temperature. Amino acid decomposition rates are much higher at 265 atm than at water steam pressure over the entire temperature range investigated.

Effects of hydrostatic pressure on growth of hyperthermophilic archaebacteria from the juan de fuca ridge.

Two new strains (AL1 and AL2) of hyperthermophilic, sulfur-reducing, heterotrophic archaebacteria from high-temperature (350 degrees C) vents on the Juan de Fuca Ridge were highly barotolerant at their optimal growth temperatures (90 and 100 degrees C, respectively). A trend towards barophily at pressures greater than those encountered in situ at the sea floor was demonstrated for the more extremely thermophilic strain (AL2), implying an ability to thrive in (unexplored) habitats well below accessible vent formations.

Particulate DNA in smoker fluids: evidence for existence of microbial populations in hot hydrothermal systems.

As part of an interdisciplinary study of hydrothermal vents on the Endeavour Segment of the Juan de Fuca Ridge, we used the submersible ALVIN to collect 57 fluid samples in titanium syringes and Go Flo Niskin bottles from 17 different hot vents (smokers and flanges) and their environs for the purpose of extracting particulate DNA. The relative purity of the vent fluids collected was determined by Mg content as an indicator of seawater entrainment. Particulate material concentrated from these samples was lysed enzymatically (enz) and by a combination of enzyme and French press treatment (fp). Concentrations of partially purified DNA recovered from these lysates were determined spectrofluorometrically by using the dye Hoechst 33258. Ambient seawater surrounding the vents was found to contain low DNA concentrations, 0.18 to 0.32 ng of DNA per ml (n = 4; mean(enz) = 0.23 +/- 0.05; mean(fp) = 0.26 +/- 0.05), while low-temperature vent samples yielded significantly higher concentrations of 0.37 to 2.12 ng of DNA per ml (n = 4; mean(enz) = 0.97 +/- 0.68; mean(fp) = 1.05 +/- 0.54). Although DNA recovery values from superheated (210 to 345 degrees C) flange samples (mean(enz) = 0.14 +/- 0.10; mean(fp) = 0.12 +/- 0.14) were not significantly different from ambient seawater values, most of the superheated (174 to 357 degrees C) smoker fluid samples contained particulate DNA in concentrations too high to be attributable to entrained seawater. Detailed sampling at one smoker site demonstrated not only the existence of significant levels of particulate DNA in the superheated smoker fluids but also the presence of an elevated microbial population in the buoyant plume 20 to 100 m above the smoker. These results underscore the heterogeneity of smoker environments within a given hydrothermal vent field and indicate that microorganisms exist in some superheated fluids.

Evaluation of practical dry dog foods suitable for all life stages.

We evaluated the nutritional adequacy of 2 dry-type commercially prepared dog diets by use of a gestation/lactation/growth protocol outlined by the Association of American Feed Control Officials. Both diets were formulated to contain minimal concentrations of nutrients, compared with canine nutrient requirements listed in the 1974 or 1985 National Research Council publications. Although there were no significant differences in the performance of dogs fed either diet, only one of the diets (diet 2) passed the complete life-stage protocol test. The nutritional profile of diet 1 was only sufficient to support a claim of complete and balanced nutrition for adult maintenance. Any commercial dry dog food meeting the nutrient levels in diet 2, and of similar nutrient bioavailability, should also pass an Association of American Feed Control Officials complete life-stage protocol test.