High pH Treatment Research Articles

Cryo-EM structures of Banna virus in multiple states reveal stepwise detachment of viral spikes

Banna virus (BAV) is the prototype Seadornavirus, a class of reoviruses for which there has been little structural study. Here, we report atomic cryo-EM structures of three states of BAV virions—surrounded by 120 spikes (full virions), 60 spikes (partial virions), or no spikes (cores). BAV cores are double-layered particles similar to the cores of other non-turreted reoviruses, except for an additional protein component in the outer capsid shell, VP10. VP10 was identified to be a cementing protein that plays a pivotal role in the assembly of BAV virions by directly interacting with VP2 (inner capsid), VP8 (outer capsid), and VP4 (spike). Viral spikes (VP4/VP9 heterohexamers) are situated on top of VP10 molecules in full or partial virions. Asymmetrical electrostatic interactions between VP10 monomers and VP4 trimers are disrupted by high pH treatment, which is thus a simple way to produce BAV cores. Low pH treatment of BAV virions removes only the flexible receptor binding protein VP9 and triggers significant conformational changes in the membrane penetration protein VP4. BAV virions adopt distinct spatial organization of their surface proteins compared with other well-studied reoviruses, suggesting that BAV may have a unique mechanism of penetration of cellular endomembranes.

A Mitochondrial Localized Chaperone Regulator OsBAG6 Functions in Saline-Alkaline Stress Tolerance in Rice

B-cell lymphoma 2 (Bcl-2)-associated athanogene (BAG) family genes play prominent roles in regulating plant growth, development, and stress response. Although the molecular mechanism underlying BAG’s response to abiotic stress has been studied in Arabidopsis, the function of OsBAG underlying saline-alkaline stress tolerance in rice remains unclear. In this study, OsBAG6, a chaperone regulator localized to mitochondria, was identified as a novel negative regulator of saline-alkaline stress tolerance in rice. The expression level of OsBAG6 was induced by high concentration of salt, high pH, heat and abscisic acid treatments. Overexpression of OsBAG6 in rice resulted in significantly reduced plant heights, grain size, grain weight, as well as higher sensitivity to saline-alkaline stress. By contrast, the osbag6 loss-of-function mutants exhibited decreased sensitivity to saline-alkaline stress. The transcriptomic analysis uncovered differentially expressed genes related to the function of “response to oxidative stress”, “defense response”, and “secondary metabolite biosynthetic process” in the shoots and roots of OsBAG6-overexpressing transgenic lines. Furthermore, cytoplasmic levels of Ca2+ increase rapidly in plants exposed to saline-alkaline stress. OsBAG6 bound to calcium sensor OsCaM1-1 under normal conditions, which was identified by comparative interactomics, but not in the presence of elevated Ca2+. Released OsCaM1-1 saturated with Ca2+ is then able to regulate downstream stress-responsive genes as part of the response to saline-alkaline stress. OsBAG6 also interacted with energy biosynthesis and metabolic pathway proteins that are involved in plant growth and saline-alkaline stress response mechanisms. This study reveals a novel function for mitochondrial localized OsBAG6 proteins in the saline-alkaline stress response alongside OsCaM1-1.

Actin-depolymerizing factors 8 and 11 promote root hair elongation at high pH

A root hair is a polarly elongated single-celled structure that derives from a root epidermal cell and functions in uptake of water and nutrients from the surrounding environment. Previous reports have demonstrated that short periods of high pH inhibit root hair extension; but the effects of long-term high-pH treatment on root hair growth are still unclear. Here, we report that the duration of root hair elongation is significantly prolonged with increasing external pH, which counteracts the effect of decreasing root hair elongation rate and ultimately produces longer root hairs, whereas loss of actin-depolymerizing factor 8 and 11 (ADF8/11) function causes shortening of root hair length at high pH (pH 7.4). Accumulation of ADF8/11 at the tips of root hairs is inhibited by high pH, and increasing environmental pH affects the actin filament (F-actin) meshwork at the root hair tip. At high pH, the tip-focused F-actin meshwork is absent in root hairs of the adf8/11 mutant, actin filaments are disordered at the adf8/11 root hair tips, and actin turnover is attenuated. Secretory and recycling vesicles do not aggregate in the apical region of adf8/11 root hairs at high pH. Together, our results suggest that, under long-term exposure to high extracellular pH, ADF8/11 may establish and maintain the tip-focused F-actin meshwork to regulate polar trafficking of secretory/recycling vesicles at the root hair tips, thereby promoting root hair elongation.

Bicarbonate rather than high pH in growth medium induced Fe-deficiency chlorosis in dwarfing rootstock quince A (Cydonia oblonga Mill.) but did not impair Fe nutrition of vigorous rootstock Pyrus betulifolia.

Quince A (Cydonia oblonga Mill.), a typical dwarfing rootstock in pear cultivation, is susceptible to iron (Fe) deficiency in calcareous soils. The aim of this study was to compare the strategies in Fe uptake and utilization in dwarfing rootstock quince A (low Fe efficiency) versus a typical vigorous rootstock Pyrus betulifolia (PB) with high Fe efficiency. Quince A and PB were grown in nutrient solution (pH 6.3) for 4 weeks followed by three pH treatments: pH6.3, pH8.3a (adjusted with hydroxide) and pH8.3b (adjusted with bicarbonate). The Fe uptake and utilization indicators of the rootstocks were assessed at the onset of chlorosis symptoms (after 58 days of treatments). In contrast to PB, quince A exhibited Fe deficiency chlorosis under bicarbonate (pH8.3b). Bicarbonate stimulated the root proton secretion, inhibited root growth and ferric chelate reductase (FCR) activity in both PB and quince A, whereas high pH without bicarbonate (pH8.3a) stimulated only root proton release. Both species accumulated more Fe in roots under high pH treatments than under pH6.3, resulting in Fe sufficiency in leaves. Both high pH treatments increased the activity of leaf FCR in PB and quince A. However, extractable Fe(II) concentration in leaves was increased by high pH treatments in PB only. This study demonstrated that depressed Fe(III) reduction in leaves caused by bicarbonate rather than high pH explained Fe deficiency in quince A grown in bicarbonate-containing medium.

Effects of pH value on stachydrine biosynthesis of hydroponic Leonurus japonicus and its physiological mechanism

The present study explored the physiological mechanism of the effects of different pH treatments on the growth, physiological characteristics, and stachydrine biosynthesis of Leonurus japonicus to provide references for the cultivation and quality control of L. japonicus. Under hydroponic conditions, different pH treatments(pH 5,6,7,8) were set up. The growth, physiology, and the content of stachydrine and total alkaloids of L. japonicus, as well as the content of key intermediate products in stachydrine biosynthesis pathway(i.e., pyruvic acid, α-ketoglutaric acid, glutamic acid, and ornithine) were monitored to explore the physiological mechanism of the effects of pH on the growth and active components of L. japonicus. The results showed that L. japonicus. could grow normally in the pH 5-8 solution. The pH treatment of neutral acidity was more conducive to the accumulation of photosynthetic pigments and the increase in soluble protein in leaves of L. japonicus. to promote its growth and yield. However, since stachydrine is a nitrogen-containing pyrrolidine alkaloid, its synthesis involves the two key rate-limiting steps of nitrogen addition: reductive ammoniation reaction and Schiff base formation reaction. High pH treatments promote the synthesis and accumulation of substrates and products of the above two reactions, indicating that the alkaline environment can promote the nitrogen addition reaction, thereby promoting the biosynthesis and accumulation of stachydrine.

Upwelling-level acidification and pH/pCO2 variability moderate effects of ocean acidification on brain gene expression in the temperate surfperch, Embiotoca jacksoni.

Acidification‐induced changes in neurological function have been documented in several tropical marine fishes. Here, we investigate whether similar patterns of neurological impacts are observed in a temperate Pacific fish that naturally experiences regular and often large shifts in environmental pH/pCO2. In two laboratory experiments, we tested the effect of acidification, as well as pH/pCO2 variability, on gene expression in the brain tissue of a common temperate kelp forest/estuarine fish, Embiotoca jacksoni. Experiment 1 employed static pH treatments (target pH = 7.85/7.30), while Experiment 2 incorporated two variable treatments that oscillated around corresponding static treatments with the same mean (target pH = 7.85/7.70) in an eight‐day cycle (amplitude ± 0.15). We found that patterns of global gene expression differed across pH level treatments. Additionally, we identified differential expression of specific genes and enrichment of specific gene sets (GSEA) in comparisons of static pH treatments and in comparisons of static and variable pH treatments of the same mean pH. Importantly, we found that pH/pCO2 variability decreased the number of differentially expressed genes detected between high and low pH treatments, and that interindividual variability in gene expression was greater in variable treatments than static treatments. These results provide important confirmation of neurological impacts of acidification in a temperate fish species and, critically, that natural environmental variability may mediate the impacts of ocean acidification.

Growth and physiological characteristics of four blueberry cultivars under different high soil pH treatments

Blueberry is economically important small pulp shrub that prefers to grow in acidic soil, and soil pH is an important factor affecting blueberry growth and development. In this study, one-year-old seedlings of four blueberry cultivars, namely, ‘Primadonna’, ‘Anna’, ‘Baldwin’ and ‘Bluegold’, were used as experimental materials to study the changes in physiological and biochemical indexes, such as growth potential, osmoregulatory substance levels, antioxidant enzyme activities and antioxidant capacity, of blueberry cultivars under soil pH values of 5.5 (CK), 6.0 (T1), 6.5 (T2), 7.0 (T3) and 7.5 (T4), and the relationships between various indexes were analyzed. The overall results showed that soil pH stress led to deficiency in green color development in blueberry leaves and inhibited plant growth in terms of the height, main basal diameter and biomass (dry weight). Through determination of the blueberry growth phenotype and growth indexes, it was found that the ‘Primadonna’, ‘Anna’, ‘Baldwin’ and ‘Bluegold’ cultivars had a certain tolerance to high soil pH, and the tolerance threshold was pH 6.5. Correlation and principal component analyses showed that the MDA content, SOD and CAT antioxidant enzyme activities, DPPH free radical scavenging ability, and SPAD values of blueberry leaves were closely related to the growth and dry matter accumulation of blueberry under high soil pH environments; these values were important reference indexes for assessing the adaptation of blueberry to a high soil pH environment. Our study provides a reference for soil management for blueberry cultivation and for research on the mechanism underlying blueberry resistance to high soil pH. • Soil pH 6.5 was the threshold for growth of the four blueberry cultivars. • High soil pH destroys blueberry photosynthesis and antioxidant system. • SPAD, DPPH, CAT, SOD were positively correlated with blueberry growth. • MDA was negatively correlated with blueberry growth and dry matter accumulation.

Similar zooplankton responses to low pH and calcium may impair long-term recovery from acidification.

Throughout much of the 20th century, unprecedented industrial emissions have led to widespread acidification of regions in North America and Europe and, as lake water pH dropped, aquatic ecosystems have experienced dramatic declines in biodiversity. International emission-control agreements have led to sweeping increases in lake pH, however acid-structured zooplankton communities still persist in many lakes. Concomitantly, calcium concentrations have been declining as a legacy of acidification and are approaching or have reached concentrations that could represent a barrier to the re-establishment of zooplankton communities similar to those in non-acidified or circumneutral reference lakes. To understand how declining calcium may influence the re-establishment of zooplankton in acid-damaged lakes we manipulated calcium and pH using a factorial in-lake mesocosm experiment and assessed their individual and combined effects on a regionally diverse zooplankton assemblage. We found that the impacts of low calcium on zooplankton species were similar to those of acidification and, consequently, may prevent the recovery of acid-structured communities. Abundance of the larger bodied and acid-sensitive Daphnia pulex/pulicaria increased in high pH treatments, albeit nonsignificantly yet, by the end of our experiment, only two individuals were sampled among our 10 low calcium enclosures. In contrast, small acid-tolerant cladocerans, such as Daphnia catawba, Daphnia ambigua, and eubosminids maintained significantly higher abundances in low calcium treatments relative to all other treatment combinations. Although we did not detect an effect of calcium on mean body size, the disproportionately high abundance of small cladocerans in low calcium treatments resulted in low calcium communities with higher overall abundance and lower cladoceran evenness. Our results, along with a landscape comparison demonstrating parallel changes in zooplankton relative abundance from 34 historically acidified lakes, suggests that declining calcium will be an important, on-going factor that may limit the recovery of zooplankton, despite regional improvements in lake pH.

Effects of Episodic Exposure to High-pH Water on Survival of Atlantic Salmon Eggs and Juveniles: Results from Laboratory and Field Studies.

Although effects of acidification on salmonid fish are well studied and documented, effects of episodic high pH have rarely received attention. In the present study, we investigated effects of high-pH events on Atlantic salmon (Salmo salar) using both field and laboratory data. Effects of an episodic high-pH event on juvenile densities in a Norwegian river were studied using data from several electrofishing surveys conducted both before and after the event. Effects of high pH on survival of eggs were studied by exposing eggs to a range of high-pH treatments for different durations. Juvenile densities from the field study showed that the high-pH event had little or no effect on the cohort that had been exposed to pH 9.7-10.3 during the egg stage. This finding was in accordance with the laboratory experiment that showed no excess mortality on eggs until pH was >12. The high-pH event occurred in March during low winter flows, and densities of older juveniles in May were significantly lower in the affected area compared to controls upstream. In June and September the difference was not significant, but there was a clear spatial trend indicating that the event had a negative effect on densities of older juvenile salmon. Environ Toxicol Chem 2022;41:771-780. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Disentangling the Effects of Ocean Carbonation and Acidification on Elemental Contents and Macromolecules of the Coccolithophore Emiliania huxleyi.

Elemental contents change with shifts in macromolecular composition of marine phytoplankton. Recent studies focus on the responses of elemental contents of coccolithophores, a major calcifying phytoplankton group, to changing carbonate chemistry, caused by the dissolution of anthropogenically derived CO2 into the surface ocean. However, the effects of changing carbonate chemistry on biomacromolecules, such as protein and carbohydrate of coccolithophores, are less documented. Here, we disentangled the effects of elevated dissolved inorganic carbon (DIC) concentration (900 to 4,930μmolkg−1) and reduced pH value (8.04 to 7.70) on physiological rates, elemental contents, and macromolecules of the coccolithophore Emiliania huxleyi. Compared to present DIC concentration and pH value, combinations of high DIC concentration and low pH value (ocean acidification) significantly increased pigments content, particulate organic carbon (POC), and carbohydrate content and had less impact on growth rate, maximal relative electron transport rate (rETRmax), particulate organic nitrogen (PON), and protein content. In high pH treatments, elevated DIC concentration significantly increased growth rate, pigments content, rETRmax, POC, particulate inorganic carbon (PIC), protein, and carbohydrate contents. In low pH treatments, the extents of the increase in growth rate, pigments and carbohydrate content were reduced. Compared to high pH value, under low DIC concentration, low pH value significantly increased POC and PON contents and showed less impact on protein and carbohydrate contents; however, under high DIC concentration, low pH value significantly reduced POC, PON, protein, and carbohydrate contents. These results showed that reduced pH counteracted the positive effects of elevated DIC concentration on growth rate, rETRmax, POC, PON, carbohydrate, and protein contents. Elevated DIC concentration and reduced pH acted synergistically to increase the contribution of carbohydrate–carbon to POC, and antagonistically to affect the contribution of protein–nitrogen to PON, which further shifted the carbon/nitrogen ratio of E. huxleyi.

Synthetic bacterial vesicles combined with tumour extracellular vesicles as cancer immunotherapy.

Bacterial outer membrane vesicles (OMV) have gained attention as a promising new cancer vaccine platform for efficiently provoking immune responses. However, OMV induce severe toxicity by activating the innate immune system. In this study, we applied a simple isolation approach to produce artificial OMV that we have named Synthetic Bacterial Vesicles (SyBV) that do not induce a severe toxic response. We also explored the potential of SyBV as an immunotherapy combined with tumour extracellular vesicles to induce anti‐tumour immunity. Bacterial SyBV were produced with high yield by a protocol including lysozyme and high pH treatment, resulting in pure vesicles with very few cytosolic components and no RNA or DNA. These SyBV did not cause systemic pro‐inflammatory cytokine responses in mice compared to naturally released OMV. However, SyBV and OMV were similarly effective in activation of mouse bone marrow‐derived dendritic cells. Co‐immunization with SyBV and melanoma extracellular vesicles elicited tumour regression in melanoma‐bearing mice through Th‐1 type T cell immunity and balanced antibody production. Also, the immunotherapeutic effect of SyBV was synergistically enhanced by anti‐PD‐1 inhibitor. Moreover, SyBV displayed significantly greater adjuvant activity than other classical adjuvants. Taken together, these results demonstrate a safe and efficient strategy for eliciting specific anti‐tumour responses using immunotherapeutic bacterial SyBV.

Effect of Growing Media pH on Performance of Huanglongbing-Affected Young Citrus Trees

Since the advent of Huanglongbing (HLB) disease, Florida’s citrus production has dropped by 70%. HLB-affected trees decline rapidly under high pH growing conditions. Limited nutrient availability at high soil pH has been speculated to be the cause of such rapid decline in HLB-affected trees; however, such decline is not observed in healthy citrus trees. Therefore, the objective of this study was to understand the nutrient uptake, physiological, and molecular responses of healthy (HLY) and HLB-affected (HLB) sweet orange (Citrus sinensis (L.) Osbeck) trees when grown in media maintained at different pH levels 6.0, 7.0, or 8.0. Overall, the performance of HLY and HLB trees decreased with the increase in pH conditions. HLB trees showed a significant inverse correlation between growth parameters (leaf number, shoot growth, SPAD value, stem water potential) and growing media pH whereas no such correlation was observed for HLY trees. Interesting, superior performance of HLB trees at pH 6.0 coincided with higher accumulation of Fe, Mn, and N in whole plant body as compared to high pH treatments. In contrast, HLY tree performance was significantly better at pH 7.0 compared to other pH conditions. At pH 7.0, stress related genes were upregulated in HLB leaves as compared to HLY leaves, indicating the stress response in terms of leaf abscission, reduced growth, and natural aging process was exacerbated in HLB trees at higher pH treatments. Altogether, all the physiological and molecular observations supports an interaction between HLB trees and pH, where HLB trees at pH 6.0 perform better than at higher pH.

Inactivation of Piscine orthoreovirus.

Piscine orthoreovirus infects various salmonid fish species, and the infection is associated with diseases such as heart and skeletal muscle inflammation (HSMI) in farmed Atlantic salmon (Salmo salar). There are no vaccines available or genetically selected resistant hosts that can efficiently control piscine orthoreovirus (PRV) infection. Currently, the only prophylactic measure against PRV is general biosecurity measures aiming to break the transmission cycle. Methods to eradicate infectious virus from contaminated facilities are desirable, but the knowledge on how to inactivate PRV is lacking. A major bottleneck for inactivation studies is the lack of ability to propagate PRV in cell culture. Therefore, in this study we developed an in vivo model for detection of infectious PRV particles after treatment of the virus with inactivation tools such as heat, pH, iodine, UV and commercially available disinfectants. The results show that standard iodine treatment is efficient in inactivation of the virus, and similarly are high and low pH extremes and treatment with Virocid, a commercially available disinfectant. A UV dose of at least 50mJ/cm2 is required for inactivation, and the virus has high resistance against heat treatment.

Effects of low and high pH on sea urchin settlement, implications for the use of alkali to counter the impacts of acidification

Respiration, photosynthesis, and calcification of cultured organisms and biological substrata can substantially alter the pH and other carbonate parameters of water in aquaculture systems. One such example is the diel cycle of photosynthesis and respiration by diatoms and seaweeds growing on ‘settlement plates’ used to induce metamorphosis of invertebrate larvae and as food for post-larvae. We documented low pH and high pH conditions in nursery raceways and simulated settlement tanks that were as much as 0.26 pH units lower and 0.52 pH units higher than the pH of the source seawater supplied to the systems. To better understand whether the low pH and high pH conditions commonly found in aquaculture culture systems affected the success of the settlement stage of the sea urchin Centrostephanus rodgersii, we induced larvae to settle at pH 7.6, 7.8 (created by injecting CO2), 8.1 (ambient), 8.2, and 8.3 (created by raising total alkalinity), and followed post-settlement growth, development, and survival for 16 d. At metamorphosis, low pH significantly increased the occurrence of abnormalities and reduced the number and length of the sea urchins' spines and pedicellaria, but did not affect settlement rate or size compared to ambient pH. In contrast, high pH generally had little effect on morphological traits, but settlement was significantly reduced by 14–26% compared to ambient and low pH treatments. After 16 d, juveniles in the low pH treatments were as much as 7% smaller, had 2–4 fewer and 9–13% shorter spines, and had less-developed digestive systems compared to juveniles in ambient or high pH treatments, and there was a non-significant trend towards lower survival in low pH treatments. Our results highlight that the low pH and high pH conditions in invertebrate settlement and nursery culture systems have the potential to hamper production through reduced settlement or growth rates. We need to understand the impacts of fluctuating pH in culture systems, especially day-night oscillations. Treating seawater with alkali chemicals to stabilise pH and counter acidification should be done with caution. Due to the potential for deleterious effects on settlement, dosage regimens will need to be optimised.

Effects of pH and Mineral Nutrition on Growth and Physiological Responses of Trembling Aspen (Populus tremuloides), Jack Pine (Pinus banksiana), and White Spruce (Picea glauca) Seedlings in Sand Culture.

Responses of trembling aspen (Populus tremuloides), jack pine (Pinus banksiana), and white spruce (Picea glauca) seedlings to root zone pH ranging from 5 to 9 were studied in sand culture in the presence of two mineral nutrition levels. After eight weeks of treatments, effects of pH on plant dry weights varied between the plant species and were relatively minor in white spruce. Higher nutrient supply significantly increased dry weights only in trembling aspen subjected to pH 5 treatment. There was little effect of pH and nutrition level on net photosynthesis and transpiration rates in white spruce and jack pine, but net photosynthesis markedly declined in aspen at high pH. Chlorophyll concentrations in young foliage decreased the most in trembling aspen and jack pine. The effects of high pH treatments on the concentrations of Mg, P, Ca, Mn, Zn, and Fe in young foliage varied between the plant species with no significant decreases of Fe and Zn recorded in trembling aspen and white spruce, respectively. This was in contrast to earlier reports from the studies carried out in hydroponic culture. The sand culture system that we developed could be a more suitable alternative to hydroponics to study plant responses to pH in the root zone. Plant responses to high pH appear to involve complex events with a likely contribution of nutritional effects and altered water transport processes.

Stress response of Asian redtail catfish (Hemibagrus nemurus) fingerlings subjected to various pH exposure

Asian redtail catfish (Hemibagrus nemurus) is one of the economic freshwater fish species in Indonesia. There is still a lack of information regarding water quality tolerance of Asian redtail catfish. Therefore, this study was conducted to obtain information on the stress response of Asian redtail catfish under various pH exposures. The experimental fish was the second generation of domesticated Asian redtail catfish from the Cirata population (Length: 11.7 ± 0.6 cm; Weight: 13.2 ± 1.6 g). The pH treatment in this experiment was low pH (4-5), optimal pH (6-9), and high pH (10-11) with three replicates in each treatment. Stress response was observed by measuring blood glucose and plasma cortisol. Moreover, fish behavior and survival were also observed. Results showed that glucose level of Asian redtail catfish fingerlings was higher at low pH treatment (258.8 ± 75.1 mg/dL) than optimal (58.7 ± 7.5 mg/dL) and high pH treatment (151.0 ± 21.5 mg/dL) (P<0.05). Cortisol level at low (34.02 ± 3.16 IU) and high pH (32.88 ± 13.85 IU) was significantly higher than optimal pH treatment (12.56 ± 5.18 IU) (P<0.05). Low and high pH exposure to Asian redtail catfish increased their cortisol and glucose levels, which will lead them to mortality.

Early biomarker indicators of health in two commercially produced microalgal species important for aquaculture

In bivalve hatcheries, microalgae production accounts for a large proportion of hatchery operation costs. A reliable supply of good quality algae is essential for optimal output. Two commercially important algal species, Tisochrysis lutea (T-Iso) and Chaetoceros calcitrans were grown in batch culture under optimal and high-pH conditions for 7 and 5 days respectively. A suite of parameters were compared to identify those that could be considered as early markers of algal stress. One day after inoculation of both algal species, carbon dioxide (CO2) addition was removed to cause the high pH treatment condition and cultures were monitored to determine any changes in algal health. A variety of indicators of algal function including photosynthetic parameters by Pulse Amplitude Modulation (PAM) fluorometry and morphological and functional changes by flow cytometry were determined in parallel during the pH stress to correlate them and identify parameters that could be used as an early indicator of decreasing algal health. The pH in the sub-optimal treatments was significantly higher compared with controls from 1 day after the removal of CO2 addition. At the same sampling time (Day 2), in the high-pH treatment, the PSII photosynthetic efficiency in the light measured by PAM fluorometry and, chlorophyll fluorescence and reactive oxygen species production measured by flow cytometry were significantly lower for T-Iso, compared with controls. In sub-optimal cultures of C. calcitrans, relative algal size, complexity, chlorophyll and neutral lipid content (as measured by flow cytometry) were higher as early as Day 2, compared with controls. Additionally, the bacterial abundance associated with T-Iso and C. calcitrans cells was increased in the high pH treatment compared with controls, from Day 6 and Day 5 respectively. These responses to a subtle negative change in the culture environment were detected 24 h prior to a change in cell density and indicate the promise of PAM fluorometry and flow cytometry as tools to provide a rapid, sensitive and reliable assessment of microalgal health in a hatchery setting. Such advances in algal health monitoring will improve bivalve production systems.

Molecular cloning, expression and characterization of secreted ferritin in the silkworm, Bombyx mori.

Ferritin is a ubiquitous iron storage protein which plays key role in regulating iron homeostasis and metabolism. In this paper, the ferritin heavy chain homologs (HCH) and light chain homologs (LCH) from Bombyx mori (BmFerHCH and BmFerLCH) were amplified through PCR and cloned into the expression vector pET-30a(+). The recombinant BmFerHCH and BmFerLCH expressed in Escherichia coli were in the form of insoluble inclusion bodies, indicating that the two proteins were not in their natural structural conformation. In order to obtain refolded ferritin in vitro, the inclusion bodies (BmFerHCH and/or BmFerLCH) were dissolved in denaturing buffer (100mM Tris, 50mM Glycine, 8M urea, 5mM DTT, pH 8.0) and then refolded in refolding buffer (100mM Tris, 400mML-arginine, 0.2mM PMSF, 0.5mM DTT). The result showed that it was only when both BmFerHCH and BmFerLCH were present together in the denaturing buffer that refolding was successful and resulted in the formation of heteropolymers (H-L chain dimers) over homopolymers (H-H chain or L-L chain dimers). Moreover, the molecules (NaCl, Triton and glycerol) were found to enhance protein refolding. The optimum temperature, pH and ratios of BmFerHCH/BmFerLCH required for refolding were found to be 10°C, pH 7, 1:1 or 1:2, respectively. Finally, the refolded ferritin had the ability to store iron, exhibited ferroxidase activity, and could withstand high temperatures and pH treatment, which is consistent with ferritin in other species.

Growth, Fruit Yield, Photosynthetic Characteristics, and Leaf Microelement Concentration of Two Blueberry Cultivars under Different Long-Term Soil pH Treatments

Soil pH is a key factor affecting the growth of blueberries. Understanding the response mechanism of blueberries to different pH values and selecting suitable evaluation indexes are the basis of breeding new blueberry cultivars with high pH tolerances. The effects of different soil pH treatments for 17 months on the plant growth, fruit yield, photosynthetic characteristics, and leaf microelement concentration of Vaccinium ashei Reade ‘Climax’ and V. corymbosum hybrid ‘Chaoyue No. 1′ were studied. Plant height, main stem diameter, branch number per plant, leaf dry weight, stem dry weight, root dry weight, and total dry weight decreased with increasing soil pH. With an increase in soil pH, the first flowering date, 50% flowering date, first ripening date, and 50% ripening date of the two cultivars were postponed, and the flower bud numbers per plant, the floret numbers per bud, and yield per plant showed a downward trend. Moreover, the fruit quality decreased, which was reflected in the increase in the titratable acid content (TA) and the decrease in the total soluble solids content (TSS) and the TSS:TA ratio in the high pH treatment. With increasing soil pH, the chlorophyll content index (CCI), maximal photochemical efficiency of the PSII (Fv/Fm), quantum photosynthetic yield of the PSII (Y(II)) and net photosynthetic rate (Pn) of the two cultivars showed a downward trend, and some microelement concentrations in the leaves were imbalanced. Under high pH treatment, ‘Chaoyue No. 1′ had a relatively higher plant biomass and fruit yield, so it had a stronger tolerance to high pH than ‘Climax’ did. More strongly acidified rhizosphere soil capacity, as well as higher CCI, Fv/Fm, Y(II), and Pn values were the main reasons for the high pH tolerance of ‘Chaoyue No. 1′. Compared with destructive biomass indicators such as plant weight, nondestructive indicators such as CCI, Fv/Fm, and Y(II) can be more valuable indicators for fast and accurate evaluation of blueberry tolerance to high pH at early stages of treatment.

Metabolic and NH4 excretion rate of fresh water species, Chondrostoma regium in response to environmental stressors, different scenarios for temperature and pH

Changes in water temperature and pH levels have substantial adverse effects on aquatic organisms, hence causing physiological constraint on their well-being. To understand the physiological responses of Chondrostoma regium to temperature or pH changes, standard metabolic rate (SMR), maximum metabolic rate (MMR), absolute and factorial aerobic scope (AS&FAS) as well as the specific rate of ammonia excretion (Jamm) were measured at following temperatures: acute low (3.5–4.5 °C), 24 h low (5.5–6.5 °C), 7 d low (5.5–6.5 °C), acute high (30–31 °C), 24 h high (29–30 °C), 7 d high (28–29 °C), and different pH treatments: acute low (4.3–4.4), 24 h low (4.3–4.4), 7 d low (6.3–6.4), acute high (9.8–9.9), 24 h high (9.8–9.9), 7 d high (8.8–8.9). A control group was also assigned to optimum temperature = 22–23 °C and pH = 7.8–7.9. These experimental ranges for each treatment were obtained based on critical thermal and pH thresholds, i.e., 1.9 to 31.7 °C and 2.7 to 11.1, respectively. SMR was enhanced significantly (P < 0.05) following pH treatments, except for 24 h low pH treatment. Results showed significant (P < 0.05) changes in both SMR and MMR at low and high temperature treatments. The AS was elevated following pH treatments except for acute low pH treatment in which AS significantly was declined (P < 0.05). Low temperature treatments resulted in lower AS while no significant changes in AS were observed in high temperatures. In all treatments, FAS value did not differ significantly from control, except for acute and 24 h low treatments of both temperature and pH. All high pH and temperature treatments showed a significant increase (P < 0.05) in Jamm. Histopathological results of gills indicated hyperplasia and fusion of secondary lamella and kidneys histopathology revealed necrosis and loss of tubular lumen in the most treatments. Results indicated that increases in water temperature or pH are more stressful than the lower ranges of them, suggesting higher capability of fish to adjust to the low levels of temperature or pH.