Growth, antioxidant capacity and total carotene of Dunaliella salina DCCBC15 in a low cost enriched natural seawater medium

Dunaliella is currently drawing worldwide attention as an alternative source of nutraceuticals. Commercially, β-carotene making up over 10 % of Dunaliella biomass is generating the most interest. These compounds, because of their non-toxic properties, have found applications in the food, drug and cosmetic industry. The β-carotene content of Dunaliella cells, however, depends heavily on the growth conditions and especially on the availability of nutrients, salinity, irradiance and temperature in the growth medium. A chemically well defined medium is usually required, which significantly contributes to the cost of pigment production; hence a desire for low cost marine media. The present study aimed at evaluating the suitability of six different media, especially exploiting local potential resources, for the mass production of Dunaliella salina DCCBC15 as functional food and medicine. The efficacy of a new selected low-cost enriched natural seawater medium (MD4), supplemented with industrial N–P–K fertilizer, was investigated with respect to biomass production, chlorophyll, antioxidant capacity, and total carotene by Dunaliella though culture conditions were not optimized yet. This new medium (MD4) appears extremely promising, since it affords a higher production of Dunaliella biomass and pigments compared with the control, a common artificial medium (MD1), while allowing a substantial reduction in the production costs. The medium is also recommended for culturing other marine algae.     

Life history trade-offs and stress tolerance in green hydra (Hydra viridissima Pallas 1766): the importance of nutritional status and perceived population density

Clonally reproducing animals, such as freshwater hydra, can achieve very quick population growth, potentially resulting in high density when dispersal is limited. The reproductive value of any offspring produced clonally in such a high density population is low because of the strong competition for food. Therefore, animals experiencing such conditions should allocate their resources to self-maintenance, to increase survival chances. Increased allocation to self-maintenance in turn should enable animals to withstand higher levels of genotoxic stress. To test this prediction, we exposed green hydra (Hydra viridissima Pallas 1766) to a perceived high density (by keeping them in crowded culture medium) or low density (fresh culture medium) without altering food availability. We also manipulated nutritional status (by starving animals for different time periods) and previous exposure to mild stress in a full factorial experimental design. At the end of the experiment we exposed animals to a high concentration of hydrogen-peroxide and scored stress tolerance. We found that stress tolerance is greatly elevated in animals perceiving high density, confirming our prediction. Stress tolerance decreased in animals starved for a few days, suggesting that the ability to maintain an elevated stress tolerance function has nutritional costs and is possible only when resource availability is high. On the other hand, previous exposure to mild stress had a small effect on the ability to tolerate subsequent exposure to stress, and only in the low density treatment group. Thus, stress tolerance in hydra is dynamically modulated in response to social, environmental and nutritional cues.     

A Combined Model To Predict the Functionality of the Bacteriocin-Producing Lactobacillus sakei Strain CTC 494

The use of bacteriocin-producing lactic acid bacteria for improved food fermentation processes seems promising. However, lack of fundamental knowledge about the functionality of bacteriocin-producing strains under food fermentation conditions hampers their industrial use. Predictive microbiology or a mathematical estimation of microbial behavior in food ecosystems may help to overcome this problem. In this study, a combined model was developed that was able to estimate, from a given initial situation of temperature, pH, and nutrient availability, the growth and self-inhibition dynamics of a bacteriocin-producing Lactobacillus sakei CTC 494 culture in (modified) MRS broth. Moreover, the drop in pH induced by lactic acid production and the bacteriocin activity toward Listeria as an indicator organism were modeled. Self-inhibition was due to the depletion of nutrients as well as to the production of lactic acid. Lactic acid production resulted in a pH drop, an accumulation of toxic undissociated lactic acid molecules, and a shift in the dissociation degree of the growth-inhibiting buffer components. The model was validated experimentally.     

Foraging behavior and spatial use of a rock specialist: the southern vizcacha (Lagidium viscacia), and the exotic European hare (Lepus europaeus) in rocky outcrops of northwestern Patagonia, Argentina

The southern vizcacha (Lagidium viscacia) and the exotic European hare (Lepus europaeus) are two medium-sized herbivores that inhabit rocky outcrops in Patagonian steppe. These species overlap in diet and spatial use at medium distances from rocky outcrops in summer. We evaluated the spatial use through feces distribution in winter and determined seasonal foraging intensity in relation to the distances from rocky outcrops in order to elucidate how these herbivores use food and spatial resources in food scarcity periods. The vizcacha utilized the habitat close to rocky outcrops (<40 m) independent of season, while the hare exploited the space more widely, especially distances >40 m. However, in winter, at medium distances from rocky outcrops, there was partial spatial overlap because hares' activities were closer to rocky outcrops. Foraging intensity increased significantly in areas used by the vizcacha closer to rocky outcrops when food availability decreased, and the grasses Stipa speciosa, Poa sp., and Festuca pallescens were strongly foraged. In contrast, foraging intensity showed no changes in further distances to rocky outcrops and more use by the hare. The spatial and feeding behavior of the vizcacha, restricted to vicinity of rocky outcrops, showed high vulnerability to food availability changes. In resource scarcity situations, the spatial opportunistic behavior of the hare and the overlap in diet with the vizcacha constitutes a threat to this native herbivore. It is necessary to monitor populations of hare, since high densities could lead to food competition, impacting the small colonies of the southern vizcacha.     

A Combined Enrichment and Aptamer Pulldown Assay for Francisella tularensis Detection in Food and Environmental Matrices

Francisella tularensis, a Gram-negative bacterium and causative agent of tularemia, is categorized as a Class A select agent by the Centers for Disease Control and Prevention due to its ease of dissemination and ability to cause disease. Oropharyngeal and gastrointestinal tularemia may occur due to ingestion of contaminated food and water. Despite the concern to public health, little research is focused on F. tularensis detection in food and environmental matrices. Current diagnostics rely on host responses and amplification of F. tularensis genetic elements via Polymerase Chain Reaction; however, both tools are limited by development of an antibody response and limit of detection, respectively. During our investigation to develop an improved culture medium to aid F. tularensis diagnostics, we found enhanced F. tularensis growth using the spent culture filtrate. Addition of the spent culture filtrate allowed for increased detection of F. tularensis in mixed cultures of food and environmental matrices. Ultraperformance liquid chromatography (UPLC)/MS analysis identified several unique chemicals within the spent culture supernatant of which carnosine had a matching m/z ratio. Addition of 0.625 mg/mL of carnosine to conventional F. tularensis medium increased the growth of F. tularensis at low inoculums. In order to further enrich F. tularensis cells, we developed a DNA aptamer cocktail to physically separate F. tularensis from other bacteria present in food and environmental matrices. The combined enrichment steps resulted in a detection range of 1–10⁶ CFU/mL (starting inoculums) in both soil and lettuce backgrounds. We propose that the two-step enrichment process may be utilized for easy field diagnostics and subtyping of suspected F. tularensis contamination as well as a tool to aid in basic research of F. tularensis ecology.     

Intracellular Changes in Ions and Organic Solutes in Halotolerant Brevibacterium sp. Strain JCM 6894 after Exposure to Hyperosmotic Shock

In the present study we aimed to observe the intracellular responses when there was a hyperosmotic shock with a large shift in ionic strength in nutrient-rich and nutrient-poor external environments in order to clarify the availability of substrates. To do this, we used the halotolerant organism Brevibacterium sp. strain JCM 6894, which is able to grow in the presence of a wide range of salt concentrations. Hyperosmotic shock was induced by transferring cells in the late exponential phase of growth in a complex medium containing 0.5 M NaCl into either old or fresh culture medium containing 2 M NaCl. Changes in the growth rate, in the pH of the medium, and in the internal cation or organic solute concentrations in the cytosol after an upshock were analyzed as a function of incubation time. The cells exhibited very different responses to upshocks in fresh culture medium and in old culture medium; in fresh culture medium, growth was stimulated and the medium became more acidic, whereas the old culture medium repressed growth and the medium became more alkaline. The intracellular free Na⁺ concentrations remained low (80 nmol mg of protein⁻¹) after an upshock in fresh culture medium, although they quickly increased twofold in the old culture medium. In contrast, K⁺ ions immediately accumulated in the cells in fresh culture medium, whereas K⁺ ions were taken up quite slowly in old culture medium. Furthermore, the cells placed in fresh culture medium transiently accumulated alanine and glutamine in response to the upshock, but the cells placed in old culture medium did not. Growth of the Brevibacterium strain at higher levels of salinity was supported by ectoine synthesis but was not observed after the shift to high-osmolarity conditions in the old culture. In the fresh culture, however, ectoine was vigorously synthesized in cells for more than 5 h after the upshock; the concentration of ectoine in cells was more than 3,500 nmol mg of protein⁻¹ at 10 h, which corresponded to a ninefold increase compared to the concentration before the shock. These findings are consistent with the results of an analysis of the extracellular medium composition before and after the upshock.     

Phenotypic plasticity of reproductive traits in response to food availability in invasive and native species of nematode

Invasive species cause severe ecological and economic damage; however, the mechanisms underlying their successful invasion often remain elusive. In the case of Bursaphelenchus xylophilus, a global quarantine pest which invaded Asia and Europe, it has been suggested that this species possesses highly competitive abilities, which promotes its establishment and rapid spread. To explore biological traits that may explain its highly competitive abilities, we focused on expression of phenotypic plasticity in response to the food conditions experienced by the females during their development as juveniles in the invasive species B. xylophilus and native species Bursaphelenchus mucronatus. We report an unexpected significant difference of phenotypic trade-off between egg number and egg size in the invasive species B. xylophilus and native species B. mucronatus. This leads to superior propagation ability of invasive species, under high and low food conditions in culture. These effects reflect adaptive optimal resource allocation where more eggs are produced in favorable environments to enhance population viability. Furthermore, we show that B. xylophilus eggs hatched earlier than B. mucronatus when their parents experienced high food availability. Thus, this study revealed, for the first time, phenotypic plasticity of reproductive traits in B. xylophilus which empowers the species a competitive advantage relative to their native counterpart B. mucronatus when they are under different range of food availability. These results are a step towards answering the vital question of how an exotic invasive species exclude a native species from its original niche.     

Observations on variation in skull size of three mammals in Israel during the 20th century

Among mammals, food availability, especially during the growth period, is a key predictor in determining final body size, and improved nutrition may lead to an increase in their body size. In Israel during the last century food availability for animals commensal with humans increased greatly, due to a 16-fold increase in the human population and the accompanying changes, such as a 135-fold increase in the area of irrigated agriculture and the availability of large quantities of organic garbage.Using museum material, we studied temporal changes in skull size of a sample of 89 red foxes (Vulpes vulpes Linnaeus, 1758), 108 golden jackals (Canis aureus Linnaeus, 1758) and 117 Cape hares (Lepus capensis Linnaeus, 1758) collected during the 20th century. Four measurements (condylobasal length, zygomatic breadth, the length of the upper cheek teeth row and the length of the mandible) were taken for each skull, and principal component analysis was used to combine the measurements into principal components.We found that skull size of the red fox increased significantly during the 20th century, possibly due to improved food availability from man-made resources such as agricultural produce and garbage. No temporal trend in body size was detected for the jackal and hare. These differences are discussed.     

From breeding pairs to fox towns: the social organisation of arctic fox populations with stable and fluctuating availability of food

Food availability can impact group formation in Carnivora. Specifically, it has been suggested that temporal variation in food availability may allow a breeding pair to tolerate additional adults in their territory at times when food abundance is high. We investigate group occurrence and intraspecific tolerance during breeding in a socially flexible canid, the arctic fox (Vulpes lagopus). We compare Iceland and Sweden where resource conditions differ considerably. A breeding pair was the most common social unit in both populations, but as predicted, groups were more frequent where food abundance varied substantially between years (Sweden: 6 %) than where food availability was stable (Iceland: ≤2 %). Within Sweden, supplemental feeding increased group occurrence from 6 to 21 %, but there was no effect of natural variation in lemming (Lemmus lemmus) availability since group formation was rare also at lemming highs. Thus, additional factors appeared to influence the trade-off between intraspecific territoriality and tolerance. We report two cases where related females showed enduring social relationships with good-neighbour strategies. Related females also engaged in alloparental behaviour in a ‘fox town’ with 31 foxes (4 adults, 3 litters). In contrast, when unrelated foxes bred close to each other, they moved or split their litters during summer, presumably because of territorial conflict. We suggest that fluctuating food availability is linked to group formation in this Arctic carnivore, but also when food availability increases, additional factors such as relatedness, alloparental benefits, competition and predator defence appear necessary to explain group formation.     

Characterization of Amylolytic Enzyme Activities Associated with the Hyperthermophilic Archaebacterium Pyrococcus furiosus

The hyperthermophilic archaebacterium Pyrococcus furiosus produces several amylolytic enzymes in response to the presence of complex carbohydrates in the growth medium. These enzyme activities, α-glucosidase, pullulanase, and α-amylase, were detected in both cell extracts and culture supernatants. All activities were characterized by temperature optima of at least 100°C as well as a high degree of thermostability. The existence of this collection of activities in P. furiosus suggests that polysaccharide availability in its growth environment is a significant aspect of the niche from which it was isolated.     

Engineering of a Bacillus subtilis Strain with Adjustable Levels of Intracellular Biotin for Secretory Production of Functional Streptavidin

Streptavidin is a biotin-binding protein which has been widely used in many in vitro and in vivo applications. Because of the ease of protein recovery and availability of protease-deficient strains, the Bacillus subtilis expression-secretion system is an attractive system for streptavidin production. However, attempts to produce streptavidin using B. subtilis face the problem that cells overproducing large amounts of streptavidin suffer poor growth, presumably because of biotin deficiency. This problem cannot be solved by supplementing biotin to the culture medium, as this will saturate the biotin binding sites in streptavidin. We addressed this dilemma by engineering a B. subtilis strain (WB800BIO) which overproduces intracellular biotin. The strategy involves replacing the natural regulatory region of the B. subtilis chromosomal biotin biosynthetic operon (bioWAFDBIorf2) with an engineered one consisting of the B. subtilis groE promoter and gluconate operator. Biotin production in WB800BIO is induced by gluconate, and the level of biotin produced can be adjusted by varying the gluconate dosage. A level of gluconate was selected to allow enhanced intracellular production of biotin without getting it released into the culture medium. WB800BIO, when used as a host for streptavidin production, grows healthily in a biotin-limited medium and produces large amounts (35 to 50 mg/liter) of streptavidin, with over 80% of its biotin binding sites available for future applications.     

Protein utilization by larvae of the black carpet beetle,Attagenus megatoma

An increase in dietary protein for larvae of the black carpet beetle, Attagenus megatoma, resulted in a decreased digestibility that was partly compensated for by substantial increases in both the gross and net efficiency of conversion of digested food into body substance. When the dietary amino acids were in a free form (not protein bound), the rate of food consumption was significantly lower; however, the greater ease of digestion and more efficient utilization of the amino acid diet resulted in a growth rate comparable to that observed on an all-casein diet. Maximum larval weight gain occurred on the stock culture medium, probably due to a combination of a faster rate of food consumption, ease of digestion, and more efficient utilization. Larvae preferentially selected protein from some of the diets, but when no selection was possible, the efficiency of N utilization was inversely proportional to the concentration of N in the diet. The concentrations of faecal uric acid decreased with an increase in dietary protein concentration, especially when expressed as a percentage of the soluble faecal nitrogen.     

Optimization of Pseudoalteromonas sp. JYBCL 1 culture conditions, medium composition and extracellular β-agarase activity

Extracellular agarase produced by the Pseudoalteromonas strain JYBCL 1 is used in a variety of applications in the biotechnology, pharmaceutical, cosmetic, and food industries. The optimization of culture conditions for agarase-producing microbes and agarase activity is thus an important consideration in many industrial applications. In this study, the optimum medium composition and culture conditions for the JYBCL 1 strain were determined using the ??one factor at a time?? (OFAT) method and a Plackett-Burman design. Optimal cell growth was obtained at a temperature of 25°C and when 10 g/L tryptone was present in the culture medium. Optimal agarase activity occurred at a temperature of 40°C and at pH 6. The presence of carbonyl groups in the extracellular agarase hydrolysis products was verified using FT-IR. LC-MS identified the hydrolyzates as neoagarohexaose, neoagarotetraose, and neoagarobiose. The extracellular agarase produced by the JYBCL 1 strain used in this study was identified as ??-agarase by ¹³C-NMR spectroscopy.     

Effects of Juvenile Host Density and Food Availability on Adult Immune Response,Parasite Resistance and Virulence in a Daphnia-Parasite System

Host density can increase infection rates and reduce host fitness as increasing population density enhances the risk of becoming infected either through increased encounter rate or because host condition may decline. Conceivably, potential hosts could take high host density as a cue to up-regulate their defence systems. However, as host density usually covaries with food availability, it is difficult to examine the importance of host density in isolation. Thus, we performed two full-factorial experiments that varied juvenile densities of Daphnia magna (a freshwater crustacean) and food availability independently. We also included a simulated high-density treatment, where juvenile experimental animals were kept in filtered media that previously maintained Daphnia at high-density. Upon reaching adulthood, we exposed the Daphnia to their sterilizing bacterial parasite, Pasteuria ramosa, and examined how the juvenile treatments influenced the likelihood and severity of infection (Experiment I) and host immune investment (Experiment II). Neither juvenile density nor food treatments affected the likelihood of infection; however, well-fed hosts that were well-fed as juveniles produced more offspring prior to sterilization than their less well-fed counterparts. By contrast, parasite growth was independent of host juvenile resources or host density. Parasite-exposed hosts had a greater number of circulating haemocytes than controls (i.e., there was a cellular immune response), but the magnitude of immune response was not mediated by food availability or host density. These results suggest that density dependent effects on disease arise primarily through correlated changes in food availability: low food could limit parasitism and potentially curtail epidemics by reducing both the host’s and parasite’s reproduction as both depend on the same food.     

Arctic Small Rodents Have Diverse Diets and Flexible Food Selection

The ecology of small rodent food selection is poorly understood, as mammalian herbivore food selection theory has mainly been developed by studying ungulates. Especially, the effect of food availability on food selection in natural habitats where a range of food items are available is unknown. We studied diets and selectivity of grey-sided voles (Myodes rufocanus) and tundra voles (Microtus oeconomus), key herbivores in European tundra ecosystems, using DNA metabarcoding, a novel method enabling taxonomically detailed diet studies. In order to cover the range of food availabilities present in the wild, we employed a large-scale study design for sampling data on food availability and vole diets. Both vole species had ingested a range of plant species and selected particularly forbs and grasses. Grey-sided voles also selected ericoid shrubs and tundra voles willows. Availability of a food item rarely affected its utilization directly, although seasonal changes of diets and selection suggest that these are positively correlated with availability. Moreover, diets and selectivity were affected by availability of alternative food items. These results show that the focal sub-arctic voles have diverse diets and flexible food preferences and rarely compensate low availability of a food item with increased searching effort. Diet diversity itself is likely to be an important trait and has previously been underrated owing to methodological constraints. We suggest that the roles of alternative food item availability and search time limitations for small rodent feeding ecology should be investigated.

Nomenclature

Annotated Checklist of the Panarctic Flora (PAF), Vascular plants. Available at: http://nhm2.uio.no/paf/, accessed 15.6.2012.     

中华绒螯蟹血细胞原代培养条件的优化

比较了几种常见血细胞培养基(L-15、2×L-15、3×L-15、M199和RMPI-1640)对中华绒螯蟹(Eriocheir sinensis)血细胞原代培养中细胞形态以及存活率的影响,以及在筛选获得的最佳培养基中添加不同比例胎牛血清(FBS)(0%、5%、10%和15%),进一步观察了血清对中华绒螯蟹血细胞培养效果的比较。结果表明,3×L-15培养基培养效果较好,所培养的细胞形态相对完整,数量较多,培养至96 h时血细胞存活率仍大于60%;而其他4种培养基效果较差,培养12 h存活率均低于50%,且细胞形态结构变化明显。以3×L-15培养基为基础,添加不同比例胎牛血清后发现,对细胞存活有显著影响,存活率明显降低。因此,不添加血清的3×L-15培养基对中华绒螯蟹血细胞的生长较为适宜。     

Field Assessment of the Predation Risk - Food Availability Trade-Off in Crab Megalopae Settlement

Settlement is a key process for meroplanktonic organisms as it determines distribution of adult populations. Starvation and predation are two of the main mortality causes during this period; therefore, settlement tends to be optimized in microhabitats with high food availability and low predator density. Furthermore, brachyuran megalopae actively select favorable habitats for settlement, via chemical, visual and/or tactile cues. The main objective in this study was to assess the settlement of Metacarcinus edwardsii and Cancer plebejus under different combinations of food availability levels and predator presence. We determined, in the field, which factor is of greater relative importance when choosing a suitable microhabitat for settling. Passive larval collectors were deployed, crossing different scenarios of food availability and predator presence. We also explore if megalopae actively choose predator-free substrates in response to visual and/or chemical cues. We tested the response to combined visual and chemical cues and to each individually. Data was tested using a two-way factorial design ANOVA. In both species, food did not cause significant effect on settlement success, but predator presence did, therefore there was not trade-off in this case and megalopae respond strongly to predation risk by active aversion. Larvae of M. edwardsii responded to chemical and visual cues simultaneously, but there was no response to either cue by itself. Statistically, C. plebejus did not exhibit a differential response to cues, but reacted with a strong similar tendency as M. edwardsii. We concluded that crab megalopae actively select predator-free microhabitat, independently of food availability, using chemical and visual cues combined. The findings in this study highlight the great relevance of predation on the settlement process and recruitment of marine invertebrates with complex life cycles.