Alexandrium catenella and Alexandrium minutum blooms in the Mediterranean Sea: Toward the identification of ecological niches

Annual recurrent blooms of the toxic dinoflagellates Alexandrium catenella and Alexandrium minutum were detected from 2000 to 2003 in harbours along the Catalan coast. The interrelation study between the occurrence of the blooms and specific external conditions at the study sites demonstrated that different factors are required for the bloom of each Alexandrium species. Concentrations higher than 10⁵ cells l⁻¹ of A. catenella were only detected in Tarragona harbour. These blooms were associated with water surface temperature between 21 and 25 °C and salinities of around 34 psu or higher than 37 psu. A. minutum appeared widely spread along the Catalan coast, though the most intensive and recurrent blooms of this species were observed in Arenys de Mar harbour. Concentrations of millions of cells per litre of A. minutum were associated with water temperatures below 14 °C and salinities of around 34–36 psu. A. minutum cell densities showed a positive significant correlation with NO₃ but a negative correlation with NH₄. On the other hand, A. catenella blooms dominated when both NO₃ and NH₄ levels were high. The prevailing inorganic nitrogen form (NO₃ vs. NH₄) could explain why these two species rarely coincide in the same harbours. Accumulation of cysts in the sediment was found to be an important potential factor for the recurrence of these species. The 4.3 × 10³ A. catenella cysts cm⁻³ of wet sediment in Tarragona harbour and the 3.02 × 10³ A. minutum cysts cm⁻³ of wet sediment in Vilanova harbour were the highest concentrations observed from the cyst study. Confined waters such as harbours play an important role as reservoirs for the accumulation of cysts and vegetative cells, which contributes to the expansion of these dinoflagellates in the region. However, the particular environmental conditions are also decisive factors of bloom intensity.     

Profiles of Alexandrium catenella cysts in Puget Sound sediments and the relationship to paralytic shellfish poisoning events

The magnitude of paralytic shellfish poisoning (PSP) toxins in shellfish and the geographical scope of shellfish closures in Puget Sound have increased in recent decades. PSP, monitored by the Washington Department of Health, has spread from Sequim Bay in the 1950s into central Puget Sound in the 1970s and throughout Puget Sound by the 1990s. Alexandrium catenella, the species responsible for PSP toxins, produces a benthic resting cyst that, upon germinating, can seed blooms. This study examined whether there is a relationship between profiles of cysts in the sediment and temporal variation in PSP in shellfish and if the history of the toxin's southward expansion through Puget Sound can be seen in the cyst record. To address this question, sediment cores were collected from three Puget Sound basins, Sequim Bay, Penn Cove, and Carr Inlet, and cyst profiles were determined. Activities of ²¹⁰Pb were fitted to a depth-dependent diagenetic model to date the sediment cores and determine mixing and sediment-accumulation rates. In order to compare historical variation in PSP with cyst profiles that have been altered by bioturbation, a depth and time-dependent diagenetic model was then used to predict vertical profiles of cysts that would occur under the assumption that cyst deposition rates are proportional to PSP concentration in shellfish measured over several decades at each site. The cyst profiles predicted by the model were compared to measured cyst profiles. These comparisons suggested that Alexandrium blooms and resulting PSP concentration in shellfish are more closely linked to cyst germination and deposition at some stations than at others. Sequim Bay had relatively large numbers of cysts and it is likely that the persistent toxicity here is the result of recurrent seeding from the cyst bed. Penn Cove and Carr Inlet had few cysts despite occasional blooms, suggesting that blooms are transported into those areas, perhaps from other sites of cyst germination. Sequim Bay and Penn Cove had cysts from top to bottom of the cores so it was not possible to determine the date when cysts were first introduced into these bays, but it is likely that A. catenella has been in Penn Cove since at least 1955 or for about two decades before the WDOH PSP toxicity data would indicate. The cyst profile in Carr Inlet suggested a first appearance date of 1985 that is consistent with the first appearance of PSP in shellfish in 1988.     

Towards an optimal sampling strategy for Alexandrium catenella (Dinophyceae) benthic resting cysts

The study proposes methodological developments to optimize sampling strategy of resting cysts of Alexandrium catenella to estimate their abundance with a predefined error. This work also aims to provide information on spatial distribution of resting cysts in sediments. The distribution mode of A. catenella resting cysts related to the abundance variability was studied through sediment cores sampling on four different spatial scales and using Ludox CLX gradient density method. The quantification method underestimates by a factor of 2 the resting cysts abundance in one gram of sediment. Application of Taylor's power law allowed us to define a compromise between sampling effort and abundance estimation error. In the case of A. catenella resting cysts from Thau lagoon, the optimal sampling strategy consists of sampling 10 stations on a surface of 2 km² for a given coefficient of variability (C) of 15%, sampling 3 sediment cores at each station (C = 30%) and counting only one replicate by core (C = 18%). Results related to the application of Taylor's power law are closely dependent on resting cyst density and aggregation in a given sediment. In our area, A. catenella resting cysts are mainly observed in the upper 3 cm of sediment. Horizontally, their heterogeneity is lower on 10 cm² surface and tends to stabilize itself beyond a surface of 10 m². Each author has to carry out this pre-sampling effort for his own resting cysts-forming species, in his own area, in order to increase accuracy of resting cyst mapping.     

Contribution of several nitrogen sources to growth of Alexandrium catenella during blooms in Thau lagoon, southern France

A monitoring program with a weekly sampling frequency over a 15-month period indicates that urea concentrations above a certain threshold level may trigger the blooms of Alexandrium catenella in Thau lagoon. However, urea concentrations were also sometimes related to ammonium and dissolved organic nitrogen concentrations, indicating that the role of urea may not be a direct one. An original approach is used to assess the relative contribution of several nitrogen sources (nitrate, nitrite, ammonium, urea) to growth of A. catenella by comparing nitrogen uptake rates to nitrogen-based growth rates estimated from dilution experiments during four blooms over a 4-year period (2001–2004) in Thau lagoon. Nitrate and nitrite contributed 0.1–14% and 0.1–5% respectively of growth requirements. Ammonium and urea were the main N sources fueling growth of A. catenella (30–100% and 2–59%, respectively). Indirect estimates indicated that an unidentified N source could also contribute significantly to growth at specific times. Concerning ammonium and urea uptake kinetics, half-saturation constants varied between 0.2 and 20 μgat N L⁻¹ for ammonium and between 0.1 and 44 μgat N L⁻¹ over the 4-year period, indicating that A. catenella can have a competitive advantage over other members of the phytoplankton even under low concentrations of ammonium and urea. However, the observed large changes in ammonium and urea uptake kinetics on a short time scale (days) during blooms preclude more precise estimates of those contributions to growth and require further investigation.     

Temperature as a factor regulating growth and toxin content in the dinoflagellate Alexandrium catenella

Controlled laboratory culture of Alexandrium catenella was used to determine the effects of a range of temperatures between 10 and 16 °C on the growth and saxitoxin content of this dinoflagellate, using strain ACC02 isolated from seawater at Aysen, XI Region, Southern Chile. Cell cultures were made using L1 culture medium at 30‰ salinity, and a photon flux density of 59.53 μmol m² s⁻¹. The results showed that the duration of the exponential growth phase was determined by the experimental temperature, with maximum cell concentrations obtained at 12 °C; significantly lower cell concentrations and growth rates were obtained at 16 °C. Cell dry weight and chlorophyll a values followed cell growth trends. The toxicity of A. catenella was variable at all the experimental temperatures, with a tendency towards having an inverse relation to temperature, with the highest values occurring at 10 °C and the lowest at 16 °C. The optimal range of temperature for the growth of the Chilean strain of A. catenella differed from rates reported for this species isolated at other latitudes, and was correlated with natural temperature conditions predominant in the environment from which it was isolated. The inverse relation of toxicity with temperature in the laboratory was broadly reflected in observations on the toxicity of this dinoflagellate in the field, and coincided with results from the literature.     

The dinoflagellate Alexandrium minutum in Cape Town harbour (South Africa): Bloom characteristics, phylogenetic analysis and toxin composition

A novel bloom of Alexandrium minutum occurred in an inner basin of the Cape Town harbour from November 2003 to February 2004. Cellular concentrations reached a maximum of 1.4 × 10⁸ cells l⁻¹ during the mid-December period with corresponding chlorophyll a concentrations of 243 mg m⁻³. Primary productivity measurements conducted during the latter part of the bloom revealed a maximum assimilation number of 11.17 mg C mg Chl a⁻¹ h⁻¹ during the middle of the day. Productivity during this post-peak period was sustained largely by the reduced nitrogen species NH₄ and urea (96%) as measured using ¹⁵N tracer techniques. The large subunit ribosomal DNA sequence of A. minutum isolates from Cape Town harbour was identical to conspecifics collected in Western Europe and in Australia. The composition of tetrahydropurine neurotoxins associated with paralytic shellfish poisoning (PSP) was limited to gonyautoxins (GTX1-GTX4). This profile combined with evidence of a low toxin cell quota (1.5 fmol GTX cell⁻¹) supports a close association of this taxon with other members of the A. minutum species complex, particularly from Europe. Toxin analysis from black mussels collected during this bloom indicated that the accumulated PSP toxins originated from A. minutum and not from Alexandrium catenella as is most often the case along the South African coast.     

Allelopathic effects of Ulva lactuca on selected species of harmful bloom-forming microalgae in laboratory cultures

Allelopathic effects of the green macroalgae Ulva lactuca on the growth of three species of red tide microalgae, Heterosigma akashiwo, Alexandrium tamarense, and Skeletonema costatum were tested in laboratory co-cultures precluding the nutrient and light limitation and the effect of high pH. The growth of all three species of microalgae was significantly (p < 0.01) inhibited by fresh U. lactuca. In nutrient replete semicontinuous co-cultures with U. lactuca, H. akashiwo was completely dead in 12 days, and the growth of A. tamarense and S. costatum was reduced by 48 and 46%, respectively by U. lactuca within 12 days. The U. lactuca culture filtrate exhibited a significant (p < 0.05) inhibitory effect on the microalgae in the first 1 or 2 days, but growth resumed in the following days, and S. costatum growth was slightly (p > 0.05) promoted from day 3. The results suggested that the allelopathic compounds are quickly degradable and a long-term inhibition might need the continuous addition of compounds originated from macroalgae. Dried U. lactuca also exhibited inhibitory effects on the microalgae, and the normalized mean growth rates of microalgae decreased with the biomass of dried U. lactuca. The dependent relationships were y = −2.1208x² + 1.0159x + 0.9752 for H. akashiwo, y = 0.7133x² − 3.5813x + 1.1665 for A. tamarense, and y = −0.2114x² − 1.063x + 1.0873 for S. costatum, respectively. The potential feasibility of utilization of dried U. lactuca against red tide microalgae was 2.0 g dry wt L⁻¹. The present study shows that U. lactuca exhibits negative allelopathic effects on harmful bloom-forming microalgae.     

Grazing on toxic Alexandrium fundyense resting cysts and vegetative cells by the eastern oyster (Crassostrea virginica)

In laboratory experiments, oysters (Crassostrea virginica) were fed Alexandrium fundyense (strain CB501) vegetative cells or resting cysts (from strains CB501 and GMT25) produced from laboratory cultures. The toxicity per cyst was 1.7 pg STXequiv/cyst and for vegetative cells 3.9 pg STXequiv/cell. The toxic, resting cysts and vegetative cells were removed from suspension in the experimental containers within about 4 h. Oysters fed toxic vegetative cells digested 72% of cells ingested, and 28% survived gut passage by forming temporary cysts. Toxin levels of oysters fed vegetative cells averaged 27 μg STXequiv/100 g meat. Resting cysts added to the experimental containers adhered to the walls so that only 40% of the cysts added were available to the oysters during the experiment. Of the cysts that were ingested, approximately 59% were digested, and oysters accumulated toxins (an average of 1.2 μg STXequiv/100 g meat), showing that consumption of resting cysts can cause toxicity in oysters. Direct consumption of resting cysts, thus, may explain shellfish toxicity in areas without known blooms, but with toxic resting cysts in the sediment. These results suggest a possible role of toxic cysts in mediating time-lags between surface blooms and appearance of toxicity in benthic grazers, and the possible role of benthic grazers in controlling seed populations, except in anoxic areas, which can serve as cyst “refuges” from grazing mortality.     

Bacterivory in the common foraminifer Ammonia tepida: Isotope tracer experiment and the controlling factors

The majority of sediment dweller foraminifera are deposit feeders. They use their pseudopodia to gather sediment with associated algae, organic detritus and bacteria. Uptake of bacteria by foraminifera have been observed but rarely quantified. We measured uptake of bacteria by the common foraminifera Ammonia tepida using ¹⁵N pre-enriched bacteria as tracers. In intertidal flats, seasonal, tidal and circadian cycles induce strong variations in environmental parameters. Grazing experiments were performed in order to measure effects of abiotic (temperature, salinity and irradiance) and biotic (bacterial and algal abundances) factors on uptake rates of bacteria. In mean conditions, A. tepida grazed 78 pgC ind^− 1 h^− 1 during the first eight hours of incubation, after which this uptake rate decreased. Uptake of bacteria was optimal at 30 °C, decreased with salinity and was unaffected by light. Above 7 × 10⁸ bacteria ml wt sed^− 1, uptake of bacteria remained unchanged when bacterial abundance increased. Algal abundance strongly affected algal uptake but did not affect uptake of bacteria. As uptake of bacteria represented 8 to 19% of microbes (algae plus bacteria) uptake, Ammonia seemed to be mainly dependant on algal resource.     

Effects of Acacia nilotica, A. polyacantha and Leucaena leucocephala leaf meal supplementation on performance of Small East African goats fed native pasture hay basal forages

Optimal utilisation of tannin-rich browse tree fodders including Acacia spp. foliages as crude protein (CP) supplements to ruminants in the tropics is limited by less available information on their feed nutritive potential. Two studies were conducted to: (1) determine rate and extent of ruminal dry matter (DM) degradability (DMD) and (2) investigate effect of sun-dried Acacia nilotica (NLM), A. polyacantha (PLM) and Leucaena leucocephala leaf meal (LLM) supplementation on growth performance of 20 growing (7–9 months old) Small East African male goats (14.6 ± 0.68 kg) fed on native pasture hay (NPH) basal diet for 84 days in a completely randomised design experiment in north-western Tanzania. The goats were randomised into four treatment groups consisting of five animals each. Three supplement diets: 115.3 g NLM (T₂), 125.9 g PLM (T₃) and 124.1 g LLM (T₄), which was used as a positive control, were supplemented at 20% of the expected DM intake (DMI; i.e., 3% body weight) to the three animal groups fed on NPH (basal diet) compared to the animals in a control group that were fed on NPH without browse supplementation (T₁).

NPH had significantly the lowest (P < 0.05) CP of 45.5 g kg⁻¹ DM compared to NLM, PLM and LLM (159, 195 and 187 g kg⁻¹ DM, respectively). NPH had higher (P < 0.05) fibre fractions; lower ruminal DM degradability characteristics and ME than NLM, PLM and LLM. Supplementation of the animals with browse resulted to (P < 0.05) higher average daily weight gains (ADG) of 157.1 g day⁻¹ in T₄ than the animals fed on T₂ (114.3 g day⁻¹) and T₃ (42.9 g day⁻¹), and even to those fed on T₁ (control), which lost weight (−71.4 g day⁻¹). Improved weight gains were mainly due to corrected feed nitrogen (N) or CP due to supplementation of the animals with browse fodder. Too low CP of the NPH would not meet the normal requirements of CP (80 g CP kg⁻¹ DM) for optimal rumen microbial function in ruminants. Higher ADG due to LLM (T₄) and NLM (T₂) supplementation suggest optimised weight gains due to browse supplementation (20% of expected DMI); while lower weight gains from supplementation with PLM (T₃) indicate the possible utilisation of A. polyacantha leaves to overcome weight losses especially during dry seasons.     

Effect of tree leaf as supplementation on nutrient digestion and rumen fermentation pattern in sheep grazing semi-arid range of India – II

A study was conducted to evaluate the effects of supplementing with different tree leaves on nutrient digestion, rumen fermentation and blood parameters of sheep. Thirty adult Malpura rams (39.0 ± 0.56 kg) were divided into five groups of six each. They were grazed as a single flock on a semi-arid rangeland and after the end of routine grazing period (08:00–17:00 h), first group (G1), which was not provided with any supplementation, served as control group. Second group (G2) was supplemented with 200 g of a conventional concentrate mixture per head per day, whereas third, fourth and fifth groups (G3, G4 and G5) were supplemented with approximately 200 g dry matter (DM) per day freshly cut foliage from Ailanthes excelsa, Azardirachta indica and Bauhinia racemosa, respectively. Protein content (g kg⁻¹ DM) in A. excelsa, A. indica and B. racemosa foliage was 197, 128 and 132, respectively. A. indica and B. racemosa foliages also contained 123.2 and 211.2 g kg⁻¹ DM condensed tannin (CT) with protein precipitating capacity (PPC) of 16.5 and 46.5 g kg⁻¹ DM. None of the tree leaves contained hydrolysable tannin (HT). Dry matter intake (DMI, g day⁻¹) was 591, 766, 865, 974 and 939 in G1, G2, G3, G4 and G5, respectively. Digestible crude protein (DCP) and metabolisable energy (ME) intakes in supplemented groups G2–G5 were higher (P < 0.05) compared to control (G1). Supplementation improved digestibility of all nutrients in all groups. Rumen fermentation study indicated lower (P < 0.05) ammonia and total N in the rumen liquor collected from G5 sheep compared to the other supplemented groups. Although haemoglobin (Hb, g dl⁻¹) levels showed small changes among groups, blood urea nitrogen (BUN, mg dl⁻¹) was lowest in G5 compared to the other groups. Initial BW were similar among the groups. After 60 days of experimental feeding, all animals maintained their BW, except sheep in the control group (G1), which lost BW. Results indicate that for adult sheep grazing on a semi-arid range, supplementation with a concentrate mixture could be replaced by tree leaves like A. excelsa, A. indica and B. racemosa, during the lean season to maintain their BW. In addition, supplementing with tree leaves containing condensed tannin has advantages in terms of N utilization.     

Temporal changes in the cyst densities of Pyrodinium bahamense var. compressum and other dinoflagellates in Manila Bay, Philippines

Temporal variation in the type and abundance of dinoflagellate cysts in Manila Bay, Philippines, is established using ²¹⁰Pb-dated sediment cores. At least 17 dinoflagellate cyst species, including those of the toxic species, Pyrodinium bahamense var. compressum, were identified. P. bahamense may have been present in the area since at least the 1920s. Total cyst density has increased beginning about 1988 to 1998 coinciding with records of P. bahamense blooms in the area. Heterotrophs have always dominated the cysts assemblage. These changes in the dinoflagellate record and the P. bahamense blooms in recent years may have been induced by the interplay of warmer temperatures, high rainfall leading to higher river discharge and less turbulent waters due to passage of few tropical cyclones.     

Protein-based complex medium design for recombinant serine alkaline protease production

This work reports on the design of a complex medium based on simple and complex carbon sources, i.e. glucose, sucrose, molasses, and defatted-soybean, and simple and complex nitrogen sources, i.e. (NH₄)₂HPO₄, casein, and defatted-soybean, for serine alkaline protease (SAP) production by recombinant Bacillus subtilis carrying pHV1431::subC gene. SAP activity was obtained as 3050 U cm⁻³ with the initial defatted-soybean concentration C_soybean^o=20 kg m⁻³ and initial glucose concentration C_G^o=8 kg m⁻³; whereas, addition of the inorganic nitrogen source (NH₄)₂HPO₄ decreased SAP production considerably. Further increase in SAP production (3850 U cm⁻³) was obtained when sucrose was replaced with glucose at C_sucrose^o=15 kg m⁻³ and C_soybean^o=20 kg m⁻³. Nevertheless, when molasses was replaced with sucrose, the maximum activity was obtained with molasses having 10 kg m⁻³ initial sucrose concentration and C_soybean^o=15 kg m⁻³as 2130 U cm⁻³; moreover, when casein was replaced with defatted-soybean SAP production decreased considerably (ca. 250 U cm⁻³). Thereafter, the effects of inorganic ionic compounds were investigated; and except phosphate, inorganic compounds supplied from defatted-soybean were found to be sufficient for the bioprocess. The highest SAP activity was obtained as 5350 U cm⁻³ in the medium that contained (kg m⁻³): C_soybean^o=20, C_sucrose^o=15, C_Na2HPO4^o=0.021, and C_NaH2PO4^o=2.82, that was 6.5-fold higher than that of the SAP produced in the defined medium. By using the designed complex medium, oxygen transfer characteristics of the bioprocess were investigated; and, Damköhler number that is the oxygen transfer limitation increases with the cultivation time until t=14 h; and, at t>20 h both mass transfer and biochemical reaction resistances were effective. Overall oxygen transfer coefficient varied between 0.010 and 0.044 s⁻¹; volumetric oxygen uptake rate varied between 0.001 and 0.006 mol m⁻³ s⁻¹; and specific oxygen uptake rate varied between 0.0001 and 0.0022 mol kg⁻¹ DW s⁻¹ throughout the bioprocess.     

Brown tide alga, Aureococcus anophagefferens, can affect growth but not survivorship of Mercenaria mercenaria larvae

Since the collapse of populations of northern quahogs (hard clam), Mercenaria mercenaria, in Long Island bays, brown tide blooms have been proposed to pose a barrier to recovery. We tested whether the brown tide alga, Aureococcus anophagefferens, affects survivorship, development or growth in the larvae of M. mercenaria. There was no effect of A. anophagefferens (clone CCMP1708) on survivorship of hard clam larvae, even at bloom concentrations. Under most experimental conditions, larvae fed a mixed diet of Isochrysis galbana (T-Iso) and A. anophagefferens or a single species diet of A. anophagefferens, developed faster than those fed a single species diet of Isochrysis. A mixed diet of I. galbana and A. anophagefferens either had no effect on larval growth, or produced enhanced growth at moderate cell densities (8 × 10⁴ cells ml⁻¹ of A. anophagefferens). Similarly, moderate cell densities of a single food diet of A. anophagefferens (1.6 × 10⁵ cells ml⁻¹) generally had no effect on the growth of larvae. When fed bloom concentrations (10⁶ cells ml⁻¹) of A. anophagefferens, larvae developed faster, but growth was reduced, compared to those fed an equal biovolume of Isochrysis. Larvae fed slow growing or near stationary phase cultures of A. anophagefferens experienced reduced growth and slowed development. These data suggest a qualitative difference between slow or stationary phase and fast growing cultures of the brown tide alga. They also suggest that impacts of A. anophagefferens, when present, are likely to be due to the nutritional quality of this alga as a food source for hard clam larvae, which could have a lasting legacy through ontogeny. Additional studies are needed to test whether our findings apply to more recently isolated strains of A. anophagefferens.     

Effect of feeding tree leaves as supplements on the nutrient digestion and rumen fermentation pattern in sheep grazing on semi-arid range of India – I

A study was carried out to determine the effect of feeding different tree leaves as supplements on nutrient digestion, rumen fermentation and blood parameters of sheep grazing on a semi-arid rangeland. Thirty adult Malpura rams of uniform body weight (39.0 ± 0.75) were divided into five groups of six each. They were grazed as a single flock from 08.00 to 17.00 h on a semi-arid rangeland. After the end of the grazing period, the first group (G1), which was not provided with any supplementation, served as the control. The second group (G2) was supplemented with 200 g of a concentrate mixture per head per day, whereas the third, fourth and fifth groups (G3–G5) were provided with approximately 200 g DM d⁻¹ of freshly cut foliage from Prosopis cineraria, Acacia nilotica and Albezia lebbek. The foliage from P. cineraria contained 133.4 g kg⁻¹ DM condensed tannin (CT) with protein precipitating capacity (PPC) of 66 g kg⁻¹ DM, whereas A. nilotica contained 18.9 g kg⁻¹ DM hydrolysable tannin (HT) with PPC of 11.5 g kg⁻¹ DM. However, A. lebbek did not contain any tannin. The protein contents were 119, 139 and 194 g kg⁻¹ DM, respectively. The DMI (g d⁻¹) was 688, 916, 1024, 1003, 999 in G1, G2, G3, G4 and G5, respectively. Digestible crude protein (DCP) and metabolizable energy (ME) intakes in supplemented groups G2–G5 were higher (P < 0.05) than in the control (G1). Supplementation improved the DM digestibility in all groups, whereas CP digestibility was lower (P < 0.05) in G3 compared to G2, G4 and G5. Rumen fermentation study conducted 6 h after supplementation revealed that total N, ammonia N, and total VFA levels were lower (P < 0.05) in G3 compared to the other supplemented groups. Although the haemoglobin (Hb) levels were similar among groups, blood urea N (BUN) was lowest in G3 compared to the other groups. The initial body weights were similar among groups (mean 39 kg). After 60 days of experimental feeding, all groups maintained their body weight, except the control group (G1), which lost body weight. It was observed, that supplementation with tree leaves containing CT like P. cineraria helps in better rumen fermentation pattern by preventing excessive loss of nitrogen. It was concluded that maximum nutritional benefits of tree leaves could be harvested, if used as supplement rather than as a sole feed.     

Growth response of major USA cowpea cultivars: II. Effect of salinity on leaf gas exchange

In an effort to elucidate the physiological processes involved in cowpea differential growth response of four major USA cowpea cultivars (CB5, CB27, 8517 and 7964) to increasing salinity, we investigated the effect of salinity on leaf gas exchange of net photosynthetic rate per unit leaf mass (Pnm) and per unit leaf area (Pna), and stomatal conductance (gs) of the four cowpea cultivars. The experiment was set up as a standard split-plot design in which cowpea plants were grown in greenhouse sand tanks irrigated with nutrient solutions. Seven salinities ranging from 2.6 to 20.5 dS m⁻¹ were constructed based on Colorado River water salt composition with NaCl, CaCl₂ and MgSO₄ as the salinization salts. Light-saturated Pnm, Pna and gs of fully expanded trifoliage were examined at the vegetative growth and flowering stages, and the data were analyzed using a split-plot analysis of variance (ANOVA) model. We found a highly significant (P ≤ 0.0001) reduction of Pnm, Pna and gs due to salinity. The responses of Pnm, Pna and gs to salinity could be further described by a general model of log(y) = a₁ + a₂x + a₃x², where y represents either Pnm, Pna, or gs; a₁, a₂ and a₃, empirical constants; x, salinity. We found that Pnm was more sensitive to salinity than Pna. Additionally, we found that increasing stomatal closure with increasing salinity might limit Pnm or Pna. While we did not find any significant difference (P > 0.05) of Pnm and Pna among the four cultivars, we did find a significant difference (P ≤ 0.05) in gs. No significant salt × cultivar interaction effect (P > 0.05) was found with Pnm, Pna and gs indicating that the four cowpea cultivars have the same response pattern of their leaf gas exchange to salinity.     

Organ-specific distribution of dietary alkaloids in the marine opisthobranch Tylodina perversa

Specimens of the opisthobranch Tylodina perversa that were observed while feeding on the sponge Aplysina aerophoba were transferred to seawater tanks along with their prey and kept under controlled conditions. After one week the opisthobranchs were anaesthetized, dissected and studied for sequestered sponge-derived brominated alkaloids. All parts of T. perversa analyzed including feces, mucus and egg masses that had been produced during captivity contained alkaloids derived from A. aerophoba. The highest total alkaloid concentration (24.6 mg g⁻¹ dry wt) was found in mantles of T. perversa (compared to 51.2 mg g⁻¹ dry wt of total alkaloids in A. aerophoba). Hepatopancreas, egg masses and mucus (respective total alkaloid concentrations ranging from 20.4 to 12.5 mg g⁻¹ dry wt) were also rich in alkaloids. Whereas in A. aerophoba the isoxazoline alkaloids aerophobin-2 and isofistularin-3 were present in almost equal concentrations, aerophobin-2 constituted by far the major alkaloid (amounting to approximately 70% of all identified alkaloids) in mantles, mucus and egg masses of T. perversa, indicating selective sequestration by the opisthobranchs. Mantles as well as mucus also contained appreciable concentrations (approximately 20% of all identified compounds) of the brominated alkaloid aerothionin; this is not detected in A. aerophoba. It is possible that aerothionin originates from a previous encounter of T. perversa with the sponge A. cavernicola, the latter being closely related to A. aerophoba. The enrichment of aerophobin-2 (and of aerothionin) in mantles, mucus and egg masses that are vulnerable and exposed (mantles and egg masses) to predators and/or pathogens argues for defensive functions of the respective alkaloids even though this hypothesis still needs to be experimentally corroborated.     

Seasonal patterns of viruses, bacteria and dissolved organic carbon in a riverine wetland

SUMMARY 1. Viral and bacterial abundances were studied in relation to environmental attributes over an annual period, for both planktonic and attached (sediment, aquatic macrophyte and submerged wood) habitats, in a riverine wetland.
2. Annual mean abundance of planktonic viruses ranged from 2.3 × 10⁵−3.8 × 10⁵ particles mL⁻¹ and varied according to sampling site. Significant seasonal patterns in viral abundance were evident and appeared to be linked to variations in bacterial abundance, dissolved organic carbon and inorganic nutrients.
3. Annual mean abundance of viruses associated with surfaces ranged from 1.3 × 10⁶ particles cm⁻² on aquatic macrophytes to 1.1 × 10⁷ particles cm⁻² on wood and also showed seasonal patterns. The difference in viral dynamics among the different sites emphasizes the importance of considering habitat diversity within wetlands when examining microbial communities.     

Antimicrobial activity of steady-state cultures of Bacillus sp. CCMI 1051 against wood contaminant fungi

The effect of changing dilution rate (D) on Bacillus sp. CCMI 1051 at dilution rates between 0.1 and 0.55 h⁻¹ in a glucose-limited medium was studied. Biomass values varied between 0.88 and 1.1 g L⁻¹ at D values of 0.15–0.35 h⁻¹. Maximal biomass productivity was found to be 0.39 g L⁻¹ h⁻¹, obtained at D = 0.35 h⁻¹ and corresponding to a 54.4% conversion of the carbon into cell mass. The highest rate of glucose consumption was 4.45 mmol g⁻¹ h⁻¹ occurring at D = 0.4 h⁻¹. The glucose concentration inside the chemostat was below the detection level starting to accumulate around 0.4 h⁻¹. Growth inhibition of fifteen strains of fungi by the broth of the steady-state cell-free supernatants was assessed. Results showed that the relative inhibition differ among the target species but was not influenced by the dilution rate changing.     

PAH-DNA adducts in environmentally exposed population in relation to metabolic and DNA repair gene polymorphisms

Epidemiologic studies indicate that prolonged exposure to particulate air pollution may be associated with increased risk of cardiovascular diseases and cancer in general population. These effects may be attributable to polycyclic aromatic hydrocarbons (PAHs) adsorbed to respirable air particles. It is expected that metabolic and DNA repair gene polymorphisms may modulate individual susceptibility to PAH exposure. This study investigates relationships between exposure to PAHs, polymorphisms of these genes and DNA adducts in group of occupationally exposed policemen (EXP, N = 53, males, aged 22–50 years) working outdoors in the downtown area of Prague and in matched “unexposed” controls (CON, N = 52). Personal exposure to eight carcinogenic PAHs (c-PAHs) was evaluated by personal samplers during working shift prior to collection of biological samples. Bulky-aromatic DNA adducts were analyzed in lymphocytes by ³²P-postlabeling assay. Polymorphisms of metabolizing (GSTM1, GSTP1, GSTT1, EPHX1, CYP1A1-MspI) and DNA repair (XRCC1, XPD) genes were determined by PCR-based RFLP assays. As potential modifiers and/or cofounders, urinary cotinine levels were analyzed by radioimmunoassay, plasma levels of vitamins A, C, E and folates by HPLC, cholesterol and triglycerides using commercial kits. During the sampling period ambient particulate air pollution was as follows: PM10 32–55 μg/m³, PM2.5 27–38 μg/m³, c-PAHs 18–22 ng/m³; personal exposure to c-PAHs: 9.7 ng/m³ versus 5.8 ng/m³ (P < 0.01) for EXP and CON groups, respectively. The total DNA adduct levels did not significantly differ between EXP and CON groups (0.92 ± 0.28 adducts/10⁸ nucleotides versus 0.82 ± 0.23 adducts/10⁸ nucleotides, P = 0.065), whereas the level of the B[a]P-“like” adduct was significantly higher in exposed group (0.122 ± 0.036 adducts/10⁸ nucleotides versus 0.099 ± 0.035 adducts/10⁸ nucleotides, P = 0.003). A significant difference in both the total (P < 0.05) and the B[a]P-“like” DNA adducts (P < 0.01) between smokers and nonsmokers within both groups was observed. A significant positive association between DNA adduct and cotinine levels (r = 0.368, P < 0.001) and negative association between DNA adduct and vitamin C levels (r = −0.290, P = 0.004) was found. The results of multivariate regression analysis showed smoking, vitamin C, polymorphisms of XPD repair gene in exon 23 and GSTM1 gene as significant predictors for total DNA adduct levels. Exposure to ambient air pollution, smoking, and polymorphisms of XPD repair gene in exon 6 were significant predictors for B[a]P-“like” DNA adduct. To sum up, this study suggests that polymorphisms of DNA repair genes involved in nucleotide excision repair may modify aromatic DNA adduct levels and may be useful biomarkers to identify individuals susceptible to DNA damage resulting from c-PAHs exposure.