Elevated Na+/K+-ATPase responses and its potential role in triggering ion reabsorption in kidneys for homeostasis of marine euryhaline milkfish (Chanos chanos) when acclimated to hypotonic fresh water

The milkfish (Chanos chanos) is an economic species in Southeast Asia. In Taiwan, the milkfish are commercially cultured in environments of various salinities. Na⁺/K⁺-ATPase (NKA) is a key enzyme for fish iono- and osmoregulation. When compared with gills, NKA and its potential role were less examined by different approaches in the other osmoregulatory organs (e.g., kidney) of euryhaline teleosts. The objective of this study was to investigate the correlation between osmoregulatory plasticity and renal NKA in this euryhaline species. Muscle water contents (MWC), plasma, and urine osmolality, kidney histology, as well as distribution, expression (mRNA and protein), and specific activity of renal NKA were examined in juvenile milkfish acclimated to fresh water (FW), seawater (SW 35‰), and hypersaline water (HSW 60‰) for at least two weeks before experiments. MWC showed no significant difference among all groups. Plasma osmolality was maintained within the range of physiological homeostasis in milkfish acclimated to different salinities, while, urine osmolality of FW-acclimated fish was evidently lower than SW- and HSW-acclimated individuals. The renal tubules were identified by staining with periodic acid Schiff’s reagent and hematoxylin. Moreover, immunohistochemical staining showed that NKA was distributed in the epithelial cells of proximal tubules, distal tubules, and collecting tubules, but not in glomeruli, of milkfish exposed to different ambient salinities. The highest abundance of relative NKA α subunit mRNA was found in FW-acclimated milkfish rather than SW- and HSW-acclimated individuals. Furthermore, relative protein amounts of renal NKA α and β subunits as well as NKA-specific activity were also found to be higher in the FW group than SW and the HSW groups. This study integrated diverse levels (i.e., histological distribution, gene, protein, and specific activity) of renal NKA expression and illustrated the potential role of NKA in triggering ion reabsorption in kidneys of the marine euryhaline milkfish when acclimated to a hypotonic FW environment.     

Development of larval diets for milkfish (Chanos chanos)

This study aimed to develop nutritionally balanced and cost-effective processed diets for milkfish larvae ( Chanos chanos Forsskal). Two larval diets (feed A and feed B) were formulated and prepared to contain 45&percnt| protein and 10&percnt| lipid. Several larval diet preparations were tried such as microbound/ unpelleted (freeze-dried), microbound/pelleted (oven-dried) and microbound/flaked (drum-dried) and assessed in terms of feed particle size and buoyancy, water stability and feed acceptability. The preparation that gave the best particle size and buoyancy as well as good water stability was prepared as the microbound diet (using K -carrageenan as a binder) and flaked using a drum drier. A series of feeding experiments were conducted to determine the growth and survival of milkfish larvae reared on various feeding schemes using these processed larval diets which were fed either solely or in combination with live feed. Larvae in control treatments were reared on live foods such as Brachionus plicatilis and Artemia nauplii. Larvae were observed to ingest the diets, indicating that the feeds had suitable physical characteristics and were attractive to the larvae. The overall results of the feeding trials showed that the artificial diets could be fed to milkfish larvae in combination with Brachionus rotifers starting on day 2 or day 8, and could be fed alone starting from day 15. These promising results would reduce the dependence of milkfish larvae on live feed and would have significant economic benefits in the form of simplified milkfish hatchery procedures.     

The sulfide tolerance of milkfish and tilapia in relation to fish kills in farms and natural waters in the Philippines

Fish kills of milkfish Chanos chanos and tilapia Oreochromis spp. now occur frequently in brackish, marine, and freshwater farms (ponds, pens, and cages) in the Philippines. Aquafarms with high organic load, limited water exchange and circulation, no aeration, and high stocking and feeding rates can become oxygen-depleted and allow sulfide from the sediments to appear in the water column and poison free-swimming fish. The sulfide tolerance of 2–5 g milkfish and 5–8 g O. mossambicus was determined in 25-liter aquaria with flow-through sea water (100 ml min-1) at 26–30 °C and sulfide stock solutions pumped in at 1ml min-1. Total sulfide concentrations in the aquaria were measured by the methylene blue method and used in the regression against the probits of % survival. Four experiments showed that the two species have similar sulfide tolerance. In sea water of pH 8–8.5, about 163 ± 68 μM or 5.2 ± 2.2 mg l-1 total sulfide (mean ± 2 se) or 10 μM or 313 μg l-1 H2S was lethal to 50% of the fish in 4–8 h, and 61 ± 3 μM total sulfide or 4 μM H2S in 24–96 h (to convert all sulfide concentrations: 1 μM = 32 μg l-1). Earthen pond bottoms had 0–382 μM total dissolved sulfide (mean ± sd = 54 ± 79 μM, n = 76); a tenth of the samples had >200 μM. The water column may have such sulfide levels under hypoxic or anoxic conditions. To simulate some of the conditions during fish kills, 5–12 g milkfish were exposed to an abrupt increase in sulfide, alone or in combination with progressive respiratory hypoxia and decreasing pH. The tests were done in the same flow-through set-up but with sulfide pumped in at 25 ml min-1. The lethal concentration for 50% of the fish was 197 μM total sulfide or 12 μM H2S at 2 h, but 28–53 μM sulfide allowed fish to survive 6–10 h. Milkfish in aquaria with no aeration nor flow-through sea water died of respiratory hypoxia in 5–8 h when oxygen dropped from 6 to 1 mg l-1. Under respiratory hypoxia with 30–115 μM sulfide, the fish died in 2.5–4 h. Tests with low pH were done by pumping a weak sulfuric acid solution at 25 ml min-1 into aquaria with flow-through sea water such that the pH dropped from 8 to 4 in 5 h. Under these conditions, milkfish died in 7–9 h when the pH was 3.5. When 30–93 μM sulfide was pumped in with the acid, the fish died in 2–6 h when the pH was still 4.5–6.3. Thus, sulfide, hypoxia, and low pH are each toxic to milkfish at particular levels and aggravate each other's toxicity. Aquafarms must be well oxygenated to prevent sulfide toxicity and fish kills. This revised version was published online in July 2006 with corrections to the Cover Date.     

The expression of gill Na,K-ATPase in milkfish,Chanos chanos,acclimated to seawater,brackish water and fresh water

Juvenile milkfish Chanos chanos (Forssk?l, 1775) were transferred from a local fish farm to fresh water (FW; 0 per thousand ), brackish water (BW; 10 per thousand, 20 per thousand ) and seawater (SW; 35 per thousand ) conditions in the laboratory and reared for at least two weeks. The blood and gill of the fish adapted to various salinities were analyzed to determine the osmoregulatory ability of this euryhaline species. No significant difference was found in plasma osmolality, sodium or chloride concentrations of milkfish adapted to various salinities. In FW, the fish exhibited the highest specific activity of Na, K-ATPase (NKA) in gills, while the SW group was found to have the lowest. Relative abundance of branchial NKA alpha-subunit revealed similar profiles. However, in contrary to other euryhaline teleosts, i.e. tilapia, salmon and eel, the naturally SW-dwelling milkfish expresses higher activity of NKA in BW and FW. Immunocytochemical staining has shown that most Na, K-ATPase immunoreactive (NKIR) cells in fish adapted to BW and SW were localized to the filaments with very few on the lamellae. Moreover, in FW-adapted milkfish, the number of NKIR cells found on the lamellae increased significantly. Such responses as elevated NKIR cell number and NKA activity are thought to improve the osmoregulatory capacity of the milkfish in hyposaline environments.     

Natural spawning,egg and fry production of milkfish,Chanos chanos (Forsskal), broodstock reared in concrete tanks1

Gonadal maturation, rematuration, and spawning of nine- (1982 stock) and ten-year-old (1981 stock) milkfish reared in 150 and 200 m³ concrete tanks were observed in 1990 and 1991. From 23 September to 9 November 1990, the 1981 stock spawned 15 times with an average daily collection of 61,000 ± 21,000 eggs. From 4 larval rearing trials, the mean overall survival rate (from eggs to 21 -day-old fry) was 9.68 ± 4.3%. From 25 Aril to 31 October 1991, the stock spawned a totaf of 108 times. Mean daily egg collection increasef to 305,000 ± 32,000. Successful rearing trials conducted during the latter part of the spawning season had a mean overall survival rate of 17 ± 3% (n = 17) indicating that mass fry production techniques can be improved. Spawning of milkfish broodstock in concrete tanks provides an alternative to other existing methods of seed production.     

Environmental capacity of receiving water as basis for regulating intensity of milkfish (Chanos chanos Forsskal) culture

This paper presents the results of the assessment of the environmental condition of a receiving water and demonstrates how the environmental capacity of the system can be estimated based on effluent discharge from milkfish ponds and water criteria from scientific literature and other studies. An estuary (average volume, 295 333 m³; average depth, 1.9 m) in Punta Pulao, Dumangas, Iloilo, Philippines served as discharge and irrigation system for commercial milkfish ponds and experimental/verification ponds owned by the Southeast Asian Fisheries Development Center. Total ammonia nitrogen, nitrite, nitrate, phosphate, and chlorophyll a (chl a) were determined monthly (during spring tide) for 4 months. Samples were taken at low and high tides that coincided with the draining and flooding of ponds, respectively. Monthly concentrations of these water quality variables generally increased from the first to fourth month of sampling (April–July 2001) during low tide. Very slight increases were observed during high tide. The magnitude of changes in the diurnal patterns of phosphate, chl a, and dissolved oxygen appeared to be higher at spring tide than at neap tide. This indicates that the inflowing river at low tide (during spring tide) brought effluents containing high amounts of nutrients from ponds located upstream. The water, salt, dissolved inorganic nitrogen (DIN), and phosphate (DIP) budgets of the entire estuary were determined following the one‐box model by LOICZ‐IGBP (2000, LOICZ biochemical budgeting procedure: a tutorial pamphlet. L. T. David, M. L. San Diego‐McGlone, C. J. Crossland and S. V. Smith (Eds). Publ. for LOICZ International Project Office, the Netherlands, 29 pp.). The budgets indicate that the system is net heterotrophic and nitrogen (N) fixing during the dry months, but that there could be no environmental impact during the rainy months because of short water exchange time. Results further suggest that the system is a source of N and phosphorus (P) during the dry and rainy months; the condition is largely influenced by the high amount of nutrient inputs in to the river. Linear regression analysis was performed to determine relationship between nutrient concentrations in the system and total DIN or DIP input to the system at low tide. Environmental capacity in terms of the maximum amount of DIN or DIP input to the system was predicted using regression analysis and following set criteria for nutrients, i.e. nitrite, nitrate, and phosphate. At present, the estuarine water quality has already reached its environmental capacity during the dry months. About 945 ha of commercial milkfish ponds are operating upstream, mostly as extensive systems. If these ponds are converted to semi‐intensive or intensive systems, it is recommended that the pond area be reduced to 122 ha if the DIP criterion is to be followed so as not to exceed the environmental capacity. Exceeding this environmental capacity may affect production through reduction of fish growth, occurrence of diseases, and fish mortalities.     

Biological evaluation of frozen zooplankton as food for milkfish (Cbanoschanos) fry

Milkfish fry with an average standard length and weight of 13.88 mm and 3.95 mg, respectively, were reared for 30 days using live and frozen Moina macrocopa and Brachionusplicatilis at feeding densities of 10–20 individuals per ml. Growth, survival and yield were used as indicators of the overall performances of the various treatment groups. Fry fed live M. macrocopa showed gains (both length and weight), growth and survival rates and yields significantly higher than fry fed with other treatment groups (P < 0.05). However, significant reductions in growth and survival rates resulted when fry were fed frozen M. macrocopa. On the other hand, there were no significant differences in growth and survival rates (P < 0.05) in fry fed live or frozen B. plicatilis. The results of the current study showed that although milkfish fry could be grown successfully using B. plicatilis, feeding with live Moina significantly improved growth, survival rate and yield (P lt; 0.05). Frozen Moina was found to be unsuitable as a feed for rearing milkfish fry because it reduced growth rates and increased mortality. Comparisons between live and frozen rotifers have proven the suitability of frozen rotifers as feed for rearing milkfish fry. By freezing surplus rotifers this would permit short term storage in anticipation of high hatchery demand and overcome any unpredictable failures with live cultures.     

Adoptive γδT-cell transfer alone or combined with chemotherapy for the treatment of advanced esophageal cancer

Background aimsAfter therapy with platinum, 5-fluorouracil and taxane, no further recommended therapy is available for recurrent or metastatic esophageal cancer (r/mEC). Here the authors report two phase 1 trials of adoptive γδT-cell therapy, one for treatment-refractory r/mEC (γδT-monotherapy-P1, UMIN000001419) and the other for r/mEC with no prior systemic therapy (DCF-γδT-P1, UMIN000008097).MethodsFor γδT-monotherapy-P1, patients received four weekly and four biweekly injections of autologous γδT cells. For DCF-γδT-P1, patients received docetaxel, cisplatin and 5-fluorouracil (DCF) chemotherapy consisting of docetaxel (60 mg/m²) and cisplatin (60 mg/m²) on day 1 and continuous injection of 5-fluorouracil (600 mg/m²/day) on days 1–5 of each 28-day cycle; additionally, they received autologous γδT-cell injections on day 15 and day 22 of each cycle.ResultsTwenty-six patients were enrolled for γδT-monotherapy-P1. No severe adverse events were associated with γδT-cell therapy. Median overall survival was 5.7 months (95% confidence interval [CI], 4.3–10.0), and median progression-free survival was 2.4 months (95% CI, 1.7–2.8). Eighteen patients received DCF-γδT-P1. All treatment-related adverse events were associated with DCF chemotherapy, not γδT injection. Median overall survival was 13.4 months (95% CI, 6.7–not reached), and median progression-free survival was 4.0 months (95% CI, 2.5–5.7). The response rate and disease control rate were 39% and 78%, respectively.ConclusionsThe use of γδT-cell immunotherapy with or without chemotherapy was safe and feasible for r/mEC patients. Although the authors failed to demonstrate any clinical benefit of γδT-monotherapy-P1, survival benefits were observed in the DCF-γδT-P1 trial.     

Towards automation: Radiata pine shoot hedges in vitro

A novel system for in vitro shoot production has been developed whereby shoot hedges are maintained in one vessel. Monthly crops of shoots are produced for rooting. Radiata pine shoot hedges were maintained on Lepoivre (LP) nutrient agar medium for 18 months using a weekly liquid-nutrient replenishment system. In a separate experiment liquid-LP-nutrient replenishment of shoots twice weekly without transfers (D) resulted in better shoot growth and health than monthly transfers to fresh agar medium (B), monthly transfers to fresh agar medium plus aeration twice weekly (C), or no transfers and no liquid nutrient addition (A). Liquid nutrient replenishment twice weekly was better than 2 weekly or 4 weekly replenishment. The percentage of normal waxy (abundant tubular epicuticular wax) shoots harvested monthly increased significantly over the culture period from 41% at the first harvest to 93% at the eight harvest, and remained high at 97% from the ninth to twelfth harvest. The percentage of wet (no tubular epicuticular wax, small amounts of globular epicuticular wax) shoots harvested showed a corresponding decline—from 59%, to 7% at the eighth harvest. Shoots were harvested at a rate of 672/h (1.19 cents/shoot at a labour cost of NZ$8.00/h) and approximately 1100 shoots were produced per square metre of agar surface per month. Initial problems of contamination and crowding were overcome. These results will greatly facilitate progress towards automation of shoot production and reduction of costs of micropropagated trees. An automated system used in combination with other cost-saving techniques or robotics could potentially result in a substantial reduction in costs. This is the first report of a method of culturing shoots as hedges for a period of up to 18 months without manual subculturing.     

Growth, daily ration, and gastric evacuation rates of milkfish (Chanos chanos) fed supplemental diet and natural food

Growth, daily ration, and gastric evacuation rates of milkfish ( Chanos chanos ) that fed on natural food and supplement diet were evaluated. Milkfish fingerlings (5.5g) were stocked at 1.5 fish/m² in ten 12 m² concrete tanks layered with 15-cm thick earthen bottoms. All tanks were regularly fertilized (16–20–0 and chicken manure) to maintain natural food production; 4 of the tanks additionally received a supplemental diet containing 34.3% protein and 4290 kcal/kg gross energy. Estimates or daily ration (based on dry weight of stomach contents) were calculated using the E lliot and P erson (1978) and E ggers 1977) models. Gastric evacuation rate was lower in fish that fed on natural food (1.57) compared to fish fed a supplemental diet (1.79). Consequently, the lower rate resulted in lower food intake and slower fish growth. When fish were provided a high quality supplemental diet, daily rations for fingerlings (35 g) to marketable size (116 g) ranged approximately from 0.60 to 19.68 kcal/fish/day. The deviation in daily ration (kcal/fish/day) from the above estimates may indicate the insufficient quantity of dietary energy taken by fish from natural food alone, which could be provided by supplemental diet.     

SEASONAL SUCCESSION OF ALGAL PERIPHYTON FROM A WASTEWATER TREATMENT FACILITY1

Attached algal populations were sampled at weekly or biweekly to characterize successional changes in the secondary clarifiers of a wastewater treatment plant. Three communities were compared from areas of slow, medium and rapid current velocities. In general, the algae resembled those reported for other hypereutrophic flowing water. Of the twenty-three algae recorded, Stigeoclonium, Oedogonium, Oscillatoria, Lyngbya, and Pleurocapsa were dominant at some point in the 15 month sampling period. Nutrient concentrations were consistently high (N = 1.1–21.4 mg·L⁻¹; P = 0.1–10.4 mg·L⁻¹); therefore, changes in temporal distribution of algae were probably dependent on seasonal changes in light and temperature. Colonization of artificial substrates was also observed. Small unicellular algae were the first autotrophs to attach and these were followed by larger filamentous forms.     

Gradients of coastal fish farm effluents and their effect on coral reef microbes

Coastal milkfish (Chanos chanos) farming may be a source of organic matter enrichment for coral reefs in Bolinao, Republic of the Philippines. Interactions among microbial communities associated with the water column, corals and milkfish feces can provide insight into the ecosystem's response to enrichment. Samples were collected at sites along a transect that extended from suspended milkfish pens into the coral reef. Water was characterized by steep gradients in the concentrations of dissolved organic carbon (70-160 microM), total dissolved nitrogen (7-40 microM), chlorophyll a (0.25-10 microg l(-1)), particulate matter (106-832 microg l(-1)), bacteria (5 x 10(5)-1 x 10(6) cells ml(-1)) and viruses (1-7 x 10(7) ml(-1)) that correlated with distance from the fish cages. Particle-attached bacteria, which were observed by scanning laser confocal microscopy, increased across the gradient from < 0.1% to 5.6% of total bacteria at the fish pens. Analyses of 16S rRNA genes by denaturing gradient gel electrophoresis and environmental clone libraries revealed distinct microbial communities for each sample type. Coral libraries had the greatest number of phyla represented (range: 6-8) while fish feces contained the lowest number (3). Coral libraries also had the greatest number of 'novel' sequences (defined as < 93% similar to any sequence in the NCBI nt database; 29% compared with 3% and 5% in the feces and seawater libraries respectively). Despite the differences in microbial community composition, some 16S rRNA sequences co-occurred across sample types including Acinetobacter sp. and Ralstonia sp. Such patterns raise the question of whether bacteria might be transported from the fish pens to corals or if microenvironments at the fish pens and on the corals select for the same phylotypes. Understanding the underlying mechanisms of effluent-coral interactions will help predict the ability of coral reef ecosystems to resist and rebound from organic matter enrichment.     

Effects of sodium chloride on water quality and growth of Oreochromis niloticus in earthen ponds

A 2 × 2 factorial experiment was conducted between April and August 2001 to evaluate the effects of NaCl on Oreochromis niloticus growth and water quality in twelve 0.015 ha limed ponds. The design involved fertilizer and salt as factors with two treatments for each factor. Each salt‐fertilizer combination was replicated three times and fish were not offered external food during the 98 days culture. Growth of O. niloticus was significantly enhanced by salt at higher fertilizer level but not at the lower fertilization level. Although salt had no direct effect on fish growth, a significant salt‐fertilizer interaction was demonstrated. Water quality variables, with a few exceptions, were similar among the salted treatments. Total ammonia increased significantly with fertilization level, but the values were similar in salted and unsalted treatments at the same fertilizer level. Total nitrogen was higher in the salted than unsalted treatments while the organic matter content was lower in the salted treatments. The reasons for the better growth of O. niloticus, are discussed with respect to water quality variables. The present results suggest that fertilization rates of 20 kg N ha^?1 may have negative effects on fish growth. However, presence of sodium chloride seems to reduce these negative effects.     

Hertwig effect caused by UV-irradiation of sperm of Oryzias latipes (teleost) and its photoreactivation

When sperm of the fish Oryzias latipes were irradiated with ultrviolet light and allowed to fertilize normal egges, the so-called “Hertwig effect” was observed, with a dose-dependent decrease in survival rate at low doses (0–27 J · m^?2) but a better survival rate at higher dose ranges. Illumination with visible light after fertilization (10–70 min after insemination) showed the existence of photoreactivation (PR), demonstrating that pyrimidine dimers are a lesion in sperm DNA that is mainly responsible for the UV-caused Hertwig effect. Genetic analysis, in which sperm from a wild-type of this fish was used, showed that, after UV-irradiation at the high dose range, male nuclei did not participate in embryonic development (a gynogenetic haploid condition). Embryos having only a maternal set of chromosomes could develop no further than stage 27. Only the visible light during the early part (until around 20–30 min after insemination, at 25°C) of the single-cell stage was effective for PR; illumination thereafter was not.     

Evaluation of water productivity and fish yield in sewage-fed vis-à-vis fertilized based carp culture

Reuse of wastewater in aquaculture provides a scope to enhance water productivity of the system. Quantification of nutrient inputs incorporated through treated domestic sewage with varying dosages viz. 79.3 x 10(5)lha(-1) and 67.7 x 10(5)lha(-1) and water productivity in a controlled carp culture system were assessed in comparison to those involved in a fertilized based one, with a view to correlate among physical, chemical and biological processes involved in fish yield under the systems. The net water productivities were measured on the basis of net return values (in Indian rupees; INR) from the carp production systems at a stocking density @ 5000 per ha with four species combination. Selected relevant water parameters such as dissolved oxygen, total alkalinity, biochemical oxygen demand (BOD5) in sewage effluent and fertilizer based systems were monitored along with certain biological parameters viz. gross primary productivity, fish production and water productivity. The nutrient inputs in terms of total ammonia-nitrogen (TAN) in effluents, total nitrogen (TN) in fertilizers and phosphorus (P2O5) in both effluents and fertilizers were found significantly correlated with biological production. The results of the experiment revealed that the sewage incorporation at 79.3 x 10(5)lha(-1) yielded similar gross fish production as recorded from fertilizer based system, whereas net water productivity using sewage as nutrient source was found 64% higher than that of a fertilizer based system.     

Pathology and biochemical genetic variation in the milkfish, Chanos chanos

Milkfish, Chanos chnos , were sampled from two geographical areas in the Hawaiian Islands approximately 200 miles apart, from Pearl Harbor on Oahu, and Puako on Hawaii. Differences in the incidence of gastritis, a stomach inflammation, and electrophoretic patterns of nuclear lens proteins from fish of the two areas suggested they were separate populations. Genetically, the pattern frequencies indicated homogeneity in the Pearl Harbor fish and heterogeneity in the Puako fish. The former may be a small population in which homozygousity was produced through genetic drift. The genetic heterogeneity of the Puako fish may indicate a single large population or a mixture of populations. These observations support the hypothesis, previously deduced from field studies, that milkfish have many nearshore spawning areas over a wide geographical range and, therefore, little opportunity for genetic interchange between the fish from widely separated areas. Pearl Harbor fish had the highest incidence of gastritis, which appeared to be due to a combination of polluted water and a possible genetic susceptibility to disease from inbreeding.     

An Analysis of the Nutrient Contribution by Phytoplankton Primary Production to the Food Web of a Small Lake

Phytoplankton primary production was measured for 148 days, from May 31 to October 25, 1971, in Sunfish Lake, Ontario, using the ¹⁴C light and dark bottle technique. The purpose of the experiment was not only to measure the rate of production but also to give some indication of the fates of the material produced by the algae. The following data were calculated for the sampling season: total gross production 57,770.6 mg C m ⁻²; algal respiration 31.74% of gross production; total net production 39,895.4 mg C m ⁻²; extracellular production 9.2% of total net production. The efficiencies of both gross and net total production were inversely correlated with light intensity. The rate of extracellular production was directly correlated with the rate of total production. The rate of respiration was not significantly correlated with either the rate of gross production or with the algal biomass as calculated from differential cell counts. The weekly rate of phytoplankton turnover was calculated by combining measurements of algal biomass with net particulate production measurements. The turnover rates for the phototrophic zone ranged from plus 6,788.5 mg C m ⁻² to minus 1,104.4 mg C m ⁻² The negative figure indicates an increase in algal biomass which can not be accounted for by primary production. The rate of phytoplankton turnover was compared with fluctuations in the standing crop of zooplankton but the correlation was not significant. Gross and net rates of extracellular production were estimated and compared. There was no significant difference between the two measurements and the implications of this are discussed with respect to other work in the field.     

Nitrate leaching and soil nitrous oxide emissions diminish with time in a hybrid poplar short‐rotation coppice in southern Germany

Hybrid poplar short‐rotation coppices (SRC) provide feedstocks for bioenergy production and can be established on lands that are suboptimal for food production. The environmental consequences of deploying this production system on marginal agricultural land need to be evaluated, including the investigation of common management practices i.e., fertilization and irrigation. In this work, we evaluated (1) the soil‐atmosphere exchange of carbon dioxide, methane, and nitrous oxide (N₂O); (2) the changes in soil organic carbon (SOC) stocks; (3) the gross ammonification and nitrification rates; and (4) the nitrate leaching as affected by the establishment of a hybrid poplar SRC on a marginal agricultural land in southern Germany. Our study covered one 3‐year rotation period and 2 years after the first coppicing. We combined field and laboratory experiments with modeling. The soil N₂O emissions decreased from 2.2 kg N₂O‐N ha^?1 a^?1 in the year of SRC establishment to 1.1–1.4 kg N₂O‐N ha^?1 a^?1 after 4 years. Likewise, nitrate leaching reduced from 13 to 1.5–8 kg N ha^?1 a^?1. Tree coppicing induced a brief pulse of soil N₂O flux and marginal effects on gross N turnover rates. Overall, the N losses diminished within 4 years by 80% without fertilization (irrespective of irrigation) and by 40% when 40–50 kg N ha^?1 a^?1 were applied. Enhanced N losses due to fertilization and the minor effect of fertilization and irrigation on tree growth discourage its use during the first rotation period after SRC establishment. A SOC accrual rate of 0.4 Mg C ha^?1 a^?1 (uppermost 25 cm, P = 0.2) was observed 5 years after the SRC establishment. Overall, our data suggest that SRC cultivation on marginal agricultural land in the region is a promising option for increasing the share of renewable energy sources due to its net positive environmental effects.     

Induction of meiotic gynogenesis in Atlantic cod,Gadus morhua (L.)

Gynogenesis is one of several chromosome‐manipulating techniques used in fish. In gynogenesis the male does not contribute to the genetic material of the offspring, and the sperm cells act only as stimulators in order for the egg to start development. This technique has several applications, both in aquaculture and in biological research: gynogenetic fish may be used as a step in the production of all‐female populations, the production of isogenetic‐ and inbred lines, revealing of the sex determination mechanism, construction of genetic maps, and testing of environmental vs genetic control of different traits. The aim of this study was to develop a simple protocol for production of gynogenetic cod (Gadus morhua L.) for further use in aquaculture research. Various milt dilutions and UV‐irradiation doses were tested, in order to inactivate the sperm without destroying its ability to induce egg development. This was followed by pressure treatment of the eggs shortly after ‘fertilization’ to suppress the completion of meiosis II, and thereby restoring diploidy. A dose of 9000 erg mm^?2, followed by a 5‐min pressure treatment (58.6 MPa) 180 min‐degrees after fertilization gave 100% gynogenetic larvae. Histologically, sexual differentiated fish were all females, possibly confirming female homogamety in Atlantic cod. No particular signs of reduced growth, survival or enhanced deformity rates were observed after the fish had reached the juvenile phase. Mortality was, however, high during the egg and larval stages. This protocol has made capable the production of gynogenetic cod juveniles in significant amounts using relatively simple means; the next step will be to elaborate on the technique in order to produce mitotic gynogenetic (double haploid) individuals, which are 100% homozygous.