Beta-glucuronidase in the serum and hemolymph cells of Mercenaria mercenaria and Crassostrea virginica (Mollusca: Pelecypoda).

The optimal pH of β-glucuronidase in the serum and cells of the hemolymph of the quahaug clam, Mercenaria mercenaria, and the American oyster, Crassostrea virginica, has been determined to be 4.5 by employing 0.2 M phosphate, 0.2 M glycylglycine, and 0.2 M acetate as the buffers.By comparing the optimal pH of lysozyme from C. virginica (which is either 5.0 or 5.5, depending on the buffer employed) with that of β-glucuronidase, it is concluded that if both of these lysosomal hydrolytic enzymes are to be concurrently operative at maximal efficiency, the hemolymph pH should be maintained between 4.5 and 5.5.The exact physiologic role of β-glucuronidase in M. mercenaria and C. virginica is undetermined; however, since this enzyme can hydrolyze acid mucopolysaccharides, which are constituents of bacterial walls, it may play a role in internal defense by acting on susceptible invading bacteria. Furthermore, the β-glucuronidase in cells may be correlated with cell proliferation.     

Interactive effects of elevated CO2 and temperature on water transport inponderosa pine

Many studies report that water flux through trees declines in response to elevated CO₂, but this response may be modified by exposure to increased temperatures. To determine whether elevated CO₂ and temperature interact to affect hydraulic conductivity, we grew ponderosa pine seedlings for 24 wk in growth chambers with one of four atmospheric CO₂ concentrations (350, 550, 750, and 1100 ppm) and either a low (15°C nights, 25°C days) or high (20°C nights, 30°C days) temperature treatment. Vapor pressure deficits were also higher in the elevated temperature treatment. Seedling biomass increased with CO₂ concentration but was not affected by temperature. Root : shoot ratio was unaffected by CO₂ and temperature. Leaf : sapwood area ratio (A_L/A_S) declined in response to elevated temperature but was not influenced by CO₂. Larger tracheid diameters at elevated temperature caused an increase in xylem-specific hydraulic conductivity (K_S). The increase in K_S and decrease in A_L/A_S led to higher leaf-specific hydraulic conductivity (K_L) at elevated temperature. Stomatal conductance (g_S) was correlated with K_L across all treatments. Neither K_S, K_L, nor g_S were affected by elevated CO₂ concentrations. High K_L in response to elevated temperature may support increased transpiration or reduce the incidence of xylem cavitation in ponderosa pine in future, warmer climates.     

Abundance of Vibrio cholerae,V. vulnificus,and V. parahaemolyticus in Oysters (Crassostrea virginica) and Clams (Mercenaria mercenaria) from Long Island Sound

Vibriosis is a leading cause of seafood-associated morbidity and mortality in the United States. Typically associated with consumption of raw or undercooked oysters, vibriosis associated with clam consumption is increasingly being reported. However, little is known about the prevalence of Vibrio spp. in clams. The objective of this study was to compare the levels of Vibrio cholerae, Vibrio vulnificus, and Vibrio parahaemolyticus in oysters and clams harvested concurrently from Long Island Sound (LIS). Most probable number (MPN)–real-time PCR methods were used for enumeration of total V. cholerae, V. vulnificus, V. parahaemolyticus, and pathogenic (tdh⁺ and/or trh⁺) V. parahaemolyticus. V. cholerae was detected in 8.8% and 3.3% of oyster (n = 68) and clam (n = 30) samples, with levels up to 1.48 and 0.48 log MPN/g in oysters and clams, respectively. V. vulnificus was detected in 97% and 90% of oyster and clam samples, with median levels of 0.97 and −0.08 log MPN/g, respectively. V. parahaemolyticus was detected in all samples, with median levels of 1.88 and 1.07 log MPN/g for oysters and clams, respectively. The differences between V. vulnificus and total and pathogenic V. parahaemolyticus levels in the two shellfish species were statistically significant (P < 0.001). These data indicate that V. vulnificus and total and pathogenic V. parahaemolyticus are more prevalent and are present at higher levels in oysters than in hard clams. Additionally, the data suggest differences in vibrio populations between shellfish harvested from different growing area waters within LIS. These results can be used to evaluate and refine illness mitigation strategies employed by risk managers and shellfish control authorities.     

Assessment of the northern distribution range of selected Perkinsus species in eastern oysters (Crassostrea virginica) and hard clams (Mercenaria mercenaria) with the use of PCR-based detection assays

Perkinsus species are protistan parasites of molluscs. In Chesapeake Bay, Perkinsus marinus, Perkinsus chesapeaki, and Perkinsus andrewsi are sympatric, infecting oysters and clams. Although P. marinus is a pathogen for Crassostrea virginica, it remains unknown whether P. andrewsi and P. chesapeaki are equally pathogenic. Perkinsus species have been reported in C. virginica as far north as Maine, sometimes associated with high prevalence, but low mortality. Thus, we hypothesized that, in addition to P. marinus, Perkinsus species with little or no pathogenicity for C. virginica may be present. Accordingly, we investigated the distribution of Perkinsus species in C. virginica and Mercenaria mercenaria, collected from Maine to Virginia, by applying PCR-based assays specific for P. marinus, P. andrewsi, and a Perkinsus sp. isolated from M. mercenaria. DNA samples of M. mercenaria possessed potent PCR inhibitory activity, which was overcome by the addition of 1 mg/ml BSA and 5% (v/v) DMSO to the PCR reaction mixture. All 3 Perkinsus species were found in both host species throughout the study area. Interestingly, the prevalence of P. marinus in M. mercenaria was significantly lower than in C. virginica, suggesting that M. mercenaria is not an optimal host for P. marinus.     

Effects of storage on microbial loads of two commercially important shellfish species, Crassostrea virginica and Mercenaria campechiensis

The effects of storage on the microbial load in two commercially important species of shellfish were examined. Oysters (Crassostrea virginica) were stored as shellstock, shucked meats, and fully processed meats at four temperatures for up to 21 days, and clams (Mercenaria campechiensis) were stored only as shellstock. The concentrations of most microbiological groups of organisms increased with the duration and temperature of storage in both shellfish species, although the increases were significantly lower in claims. Concentrations of Vibrio cholerae rose by approximately 1 log in oysters stored as shellstock after 7 days at 2 degrees C, and Lac+ vibrios increased 2 logs at 8 degrees C. Total counts of bacteria, fungi, coliforms, fecal streptococci, Aeromonas hydrophila, and clostridia were significantly higher in shucked oysters than in those stored as shellstock. Fecal coliforms were statistically the same, but V. cholerae, Vibrio parahaemolyticus, and the Lac+ vibrios were higher in oysters stored as shellstock. The concentrations of all microbial groups were higher in fully processed oysters than in shucked meats, with the exception of the vibrios, which showed no significant difference among the treatments. The results showed that although traditional methods of storing shellfish resulted in an overall increase in the microbial load, vibrio levels increased only in oysters stored as shellstock. Although fecal coliform and total bacterial counts did not correlate with those for vibrios in fresh oysters, strong correlations were observed in oysters stored for 7 days, suggesting that these indicators may be useful in monitoring oyster quality when meats are stored for a limited time as shellstock.     

Interactive effects of elevated CO2 and temperature on the anatomical characteristics of leaves in eleven species

The anatomical features of leaves in 11 species of plants grown in a temperature gradient and a temperature + CO₂ gradient were studied. The palisade parenchyma thickness, the spongy parenchyma thickness and the total leaf thickness were measured and analyzed to investigate the effects of elevated temperature and CO₂ on the anatomical characteristics of the leaves. Our results show that with the increase of temperature, the leaf thickness of C₄ species increased while the leaf thickness of C₃ species showed no constant changes. With increased CO₂, seven out of nine C₃ species exhibited increased total leaf thickness. In C₄ species, leaf thickness decreased. As for the trend on the multi-grades, the plants exhibited linear or non-linear changes. With the increase of temperature or both temperature and CO₂ for the 11 species investigated, leaf thickness varied greatly in different plants (species) and even in different branches on the same plant. These results demonstrated that the effect of increasing CO₂ and temperature on the anatomical features of the leaves were species-specific. Since plant structures are correlated with plant functions, the changes in leaf anatomical characteristics in elevated temperature and CO₂ may lead to functional differences.     

Effects of storage on microbial loads of two commercially important shellfish species, Crassostrea virginica and Mercenaria campechiensis.

The effects of storage on the microbial load in two commercially important species of shellfish were examined. Oysters (Crassostrea virginica) were stored as shellstock, shucked meats, and fully processed meats at four temperatures for up to 21 days, and clams (Mercenaria campechiensis) were stored only as shellstock. The concentrations of most microbiological groups of organisms increased with the duration and temperature of storage in both shellfish species, although the increases were significantly lower in claims. Concentrations of Vibrio cholerae rose by approximately 1 log in oysters stored as shellstock after 7 days at 2 degrees C, and Lac+ vibrios increased 2 logs at 8 degrees C. Total counts of bacteria, fungi, coliforms, fecal streptococci, Aeromonas hydrophila, and clostridia were significantly higher in shucked oysters than in those stored as shellstock. Fecal coliforms were statistically the same, but V. cholerae, Vibrio parahaemolyticus, and the Lac+ vibrios were higher in oysters stored as shellstock. The concentrations of all microbial groups were higher in fully processed oysters than in shucked meats, with the exception of the vibrios, which showed no significant difference among the treatments. The results showed that although traditional methods of storing shellfish resulted in an overall increase in the microbial load, vibrio levels increased only in oysters stored as shellstock. Although fecal coliform and total bacterial counts did not correlate with those for vibrios in fresh oysters, strong correlations were observed in oysters stored for 7 days, suggesting that these indicators may be useful in monitoring oyster quality when meats are stored for a limited time as shellstock.     

Interactive effects of elevated CO2 and drought stress on leaf water potential and growth in Caragana intermedia

We studied the responses of leaf water potential (Ψ_w), morphology, biomass accumulation and allocation, and canopy productivity index (CPI) to the combined effects of elevated CO₂ and drought stress in Caragana intermedia seedlings. Seedlings were grown at two CO₂ concentrations (350 and 700 μmol mol⁻¹) interacted with three water regimes (60–70%, 45–55%, and 30–40% of field capacity of soil). Elevated CO₂ significantly increased Ψ_w, decreased specific leaf area (SLA) and leaf area ratio (LAR) of drought-stressed seedlings, and increased tree height, basal diameter, shoot biomass, root biomass as well as total biomass under the all the three water regimes. Growth responses to elevated CO₂ were greater in well-watered seedlings than in drought-stressed seedlings. CPI was significantly increased by elevated CO₂, and the increase in CPI became stronger as the level of drought stress increased. There were significant interactions between elevated CO₂ and drought stress on leaf water potential, basal diameter, leaf area, and biomass accumulation. Our results suggest that elevated CO₂ may enhance drought avoidance and improved water relations, thus weakening the effect of drought stress on growth of C. intermedia seedings.     

Interactive effects of elevated temperature and drought on plant carbon metabolism: A meta-analysis

Elevated temperature (T_e) and drought often co-occur and interactively affect plant carbon (C) metabolism and thus the ecosystem C cycling; however, the magnitude of their interaction is unclear, making the projection of global change impacts challenging. Here, we compiled 107 journal articles in which temperature and water availability were jointly manipulated, and we performed a meta-analysis of interactive effects of T_e and drought on leaf photosynthesis (A_growth) and respiration (R_growth) at growth temperature, nonstructural carbohydrates and biomass of plants, and their dependencies on experimental and biological moderators (e.g., treatment intensity, plant functional type). Our results showed that, overall, there was no significant interaction of T_e and drought on A_growth. T_e accelerated R_growth under well-watered conditions rather than under drought conditions. The T_e × drought interaction on leaf soluble sugar and starch concentrations were neutral and negative, respectively. The effect of T_e and drought on plant biomass displayed a negative interaction, with T_e deteriorating the drought impacts. Drought induced an increase in root to shoot ratio at ambient temperature but not at T_e. The magnitudes of T_e and drought negatively modulated the T_e × drought interactions on A_growth. Root biomass of woody plants was more vulnerable to drought than that of herbaceous plants at ambient temperature, but this difference diminished at T_e. Perennial herbs exhibited a stronger amplifying effect of T_e on plant biomass in response to drought than did annual herbs. T_e exacerbated the responses of A_growth and stomatal conductance to drought for evergreen broadleaf trees rather than for deciduous broadleaf and evergreen coniferous trees. A negative T_e × drought interaction on plant biomass was observed on species-level rather than on community-level. Collectively, our findings provide a mechanistic understanding of the interactive effects of T_e and drought on plant C metabolism, which would improve the prediction of climate change impacts.     

The physiological and molecular effects of elevated CO2 levels

Carbon dioxide (CO₂) is an end product of cellular respiration, a process by which organisms including all plants, animals, many fungi and some bacteria obtain energy¹. CO₂ has several physiologic roles in respiration, pH buffering, autoregulation of the blood supply and others². Here we review recent findings from studies in mammalian lung cells, Caenorhabditis elegans and Drosophila melanogaster that help shed light on the molecular sensing and response to hypercapnia.     

Population structures of two genotypes of Vibrio vulnificus in oysters (Crassostrea virginica) and seawater

Vibrio vulnificus biotype 1 strains can be classified into two genotypes based on the PCR analysis of variations in the virulence-correlated gene (vcg). Genotype has been correlated with human infection for 90% of isolates from human cases having the vcgC sequence type and 87% of environmental strains having the vcgE variant. In this study we examined the dynamics of V. vulnificus populations and the distribution of the two genotypes recovered from oysters and surrounding estuarine wasters. Analysis of 880 isolates recovered from oysters showed a disparity in the ratio of the two genotypes, with those of the vcgE (E) genotype accounting for 84.4% of the population. In contrast, 292 isolates recovered from the waters surrounding the oyster sites revealed an almost equal distribution of the two genotypes. The levels of vcgC (C genotype) strains from both sources increased as a percentage of the population as water temperatures increased, while no culturable V. vulnificus cells were recovered from December through February. Our results suggest that there is a selective advantage for strains of the E genotype within oysters while survival of the C genotype strains may be favored by increased water column temperatures. These data suggest that the low incidence of infections may be due to the comparatively rare consumption of an oyster that contains a greater number of V. vulnificus vcgC genotype strains than of vcgE genotype strains. Levels of the two genotypes as well as seasonal dynamics within both oyster tissue and the surrounding waters may aid in identifying risk factors associated with human infection.     

The effects of elevated temperature and dissolved ρCO2 on a marine foundation species

Understanding how climate change and other environmental stressors will affect species is a fundamental concern of modern ecology. Indeed, numerous studies have documented how climate stressors affect species distributions and population persistence. However, relatively few studies have investigated how multiple climate stressors might affect species. In this study, we investigate the impacts of how two climate change factors affect an important foundation species. Specifically, we tested how ocean acidification from dissolution of CO₂ and increased sea surface temperatures affect multiple characteristics of juvenile eastern oysters (Crassostrea virginica). We found strong impacts of each stressor, but no interaction between the two. Simulated warming to mimic heat stressed summers reduced oyster growth, survival, and filtration rates. Additionally, we found that CO₂‐induced acidification reduced strength of oyster shells, which could potentially facilitate crab predation. As past studies have detected few impacts of these stressors on adult oysters, these results indicate that early life stages of calcareous marine organisms may be more susceptible to effects of ocean acidification and global warming. Overall, these data show that predicted changes in temperature and CO₂ can differentially influence direct effects on individual species, which could have important implications for the nature of their trophic interactions.     

Interactive effects of elevated CO2 and drought on photosynthetic capacity and PSII performance in maize

Elevated atmospheric CO₂ concentration [CO₂] and the change of water distribution in arid and semiarid areas affect plant physiology and ecosystem processes. The interaction of elevated [CO₂] and drought results in the complex response such as changes in the energy flux of photosynthesis. The performance of photosystem (PS) II and the electron transport were evaluated by using OJIP induction curves of chlorophyll a fluorescence and the P _N-C _i curves in the two-factor controlled experiment with [CO₂] of 380 (AC) or 750 (EC) [μmol mol^?1] and water stress by 10% polyethylene glycol 6000. Compared to water-stressed maize (Zea mays L.) under AC, the EC treatment combined with water stress decreased the number of active reaction centers but it increased the antenna size and the energy flux (absorbed photon flux, trapping flux, and electron transport flux) of each reaction center in PSII. Thus, the electron transport rate was enhanced, despite the indistinctively changed quantum yield of the electron transport and energy dissipation. The combination of EC and the water-stress treatment resulted in the robust carboxylation rate without elevating the saturated photosynthetic rate (P _max). This study demonstrated that maize was capable of transporting more electrons into the carboxylation reaction, but this could not be used to increase P _max under EC.     

Survival of Vibrio vulnificus in shellstock and shucked oysters (Crassostrea gigas and Crassostrea virginica) and effects of isolation medium on recovery

When two species of shellstock oysters were artificially contaminated with Vibrio vulnificus, the bacterium survived when the oysters were stored at 10 degrees C and below. Large numbers of endogenous V. vulnificus cells were found after 7 days at both 0.5 and 10 degrees C in uninoculated control oysters (Crassostrea virginica). Oysters allowed to take up V. vulnificus from seawater retained the bacterium for 14 days at 2 degrees C. The presence of V. vulnificus in the drip exuded from the shellstock presented a possibility of contamination of other shellstock in storage. V. vulnificus injected into shucked Pacific (Crassostrea gigas) and Eastern (C. virginica) oysters survived at 4 degrees C for at least 6 days. An 18-h most-probable-number enrichment step in alkaline peptone water gave higher recovery levels of V. vulnificus than did direct plating to selective agars. The survival of this pathogen in both shellstock and shucked oysters suggests a potential for human illness, even though the product is refrigerated.     

Modulatory effects of hard clam (Mercenaria mercenaria) tissue extracts on the in vitro growth of its pathogen QPX

Quahog parasite unknown (QPX) is a fatal protistan parasite affecting cultured and wild hard clams Mercenaria mercenaria along the northeastern coasts of the USA and maritime Canada. Field investigations and laboratory transmission studies revealed some variations in the susceptibility of different hard clam stocks to QPX infection. In this study, we used in vitro QPX cultures to investigate the effect of plasma and tissue extracts from two different clam stocks on parasite survival and growth. Results demonstrated the presence of factors in clams that significantly modulate QPX growth. Extracts from gills and mantle tissues as well as plasma inhibited in vitro QPX growth, whereas foot and adductor muscle extracts enhanced parasite growth. Investigations of anti-QPX activities in plasma from two clam stocks displaying different susceptibility toward QPX disease in vivo demonstrated higher inhibition of QPX growth by plasma from New York (resistant) clams compared to Florida (susceptible) clams. Some clams appeared to be deficient in inhibitory factors, suggesting that such animals may become more easily infected by the parasite.     

CO2浓度升高和降水增加协同作用对玉米产量及生长发育的影响

关于[CO₂]升高和降水变化等多因子共同作用对植物的影响报道较少, 制约着人们对植物对全球气候变化响应的认识和预测。玉米(Zea mays)作为重要的C₄植物, 受[CO₂]和降水影响显著, 但鲜有[CO₂]升高和降水增加协同作用对其产量及生长发育影响的报道。该研究利用开顶式生长箱模拟[CO₂]升高(390 (环境)、450和550 μmol·mol^-1), 降水增加量设置为增加自然降水量的15% (以试验地锦州1981-2010年6至8月月平均降水量为基准), 从而形成6个处理: C₅₅₀W_+15%、C₅₅₀W₀、C₄₅₀W_+15%、C₄₅₀W₀、C₃₉₀W_+15%和C₃₉₀W₀。试验材料选用玉米品种‘丹玉39’。结果表明: [CO₂]升高和降水增加的协同作用在玉米的籽粒产量和生物产量上均达到了显著水平(p< 0.05), 二因子均起正作用, 使籽粒产量和生物产量均升高。籽粒产量在[CO₂] 390、450和550 μmol·mol^-1水平下的降水增加处理较自然降水处理分别增加15.94%、9.95%和9.45%, 而生物产量分别增加13.06%、8.13%和6.49%。因为籽粒产量的增幅略大于生物产量的增幅, 所以促进了经济系数的升高。穗部性状变化显著, 其中, 穗粒数、穗粒重、穗长和穗粗等性状值均随[CO₂]升高而升高, 且各[CO₂]水平下均表现为降水增加处理>自然降水处理, 而瘪粒数相反。但是, [CO₂]升高和降水增加的协同作用也促进了轴粗的升高, 对玉米产量的增加起着限制作用。二因子协同作用在净光合速率(P_n)和叶面积上达到了极显著水平(p< 0.01), 而在株高和干物质积累量上达到了显著水平(p< 0.05)。二因子协同作用使玉米叶片的P_n升高, 植株高度升高, 穗位高升高, 茎粗增加, 叶面积变大, 从而促进了干物质积累量的升高, 为玉米增产打下了良好的基础。这表明: 在未来[CO₂]升高条件下, 一定程度的降水增加对玉米的产量具有正向促进作用。     

Survival of Vibrio vulnificus in shellstock and shucked oysters (Crassostrea gigas and Crassostrea virginica) and effects of isolation medium on recovery.

When two species of shellstock oysters were artificially contaminated with Vibrio vulnificus, the bacterium survived when the oysters were stored at 10 degrees C and below. Large numbers of endogenous V. vulnificus cells were found after 7 days at both 0.5 and 10 degrees C in uninoculated control oysters (Crassostrea virginica). Oysters allowed to take up V. vulnificus from seawater retained the bacterium for 14 days at 2 degrees C. The presence of V. vulnificus in the drip exuded from the shellstock presented a possibility of contamination of other shellstock in storage. V. vulnificus injected into shucked Pacific (Crassostrea gigas) and Eastern (C. virginica) oysters survived at 4 degrees C for at least 6 days. An 18-h most-probable-number enrichment step in alkaline peptone water gave higher recovery levels of V. vulnificus than did direct plating to selective agars. The survival of this pathogen in both shellstock and shucked oysters suggests a potential for human illness, even though the product is refrigerated.     

Effects of salinity on hard clam (Mercenaria mercenaria) defense parameters and QPX disease dynamics

QPX (Quahog Parasite Unknown) is a protistan parasite affecting hard clams (Mercenaria mercenaria) along the Northeast coast of the United States. The fact that QPX disease epizootics are usually observed in field sites with high salinities led to the general assumption that salinity represents an important factor for disease distribution. This study was designed to investigate the effect of salinity on QPX disease development as well as constitutive and QPX-induced defense factors in M. mercenaria. Naïve and QPX-infected (both experimentally and naturally) clams were submitted to 17 and 30 psu for 4 months. Standard and QPX-specific cellular and humoral defense parameters were assessed after 2 and 4 months. These included total and differential hemocyte counts, reactive oxygen species production, phagocytic activity of hemocytes, lysozyme concentration in plasma, anti-QPX activity in plasma and resistance of hemocytes to cytotoxic QPX extracellular products. Results demonstrated higher QPX-associated mortality in naturally infected clams maintained at high salinity compared to those held at 17 psu. Our findings also showed an increase in mortality following experimental challenge with QPX in clams submitted to 30 psu but not in those held at 17 psu. Constitutive clam defense factors and the response to QPX challenge were also affected by salinity. QPX challenge caused significant but transitory changes in hemolymph parameters that were obvious at 2 months but disappeared at 4 months. Overall, our results show that salinity modulates clam immunity and the progress of QPX disease although its impact appears secondary as compared to findings we reported earlier for temperature.