Effects of ultraviolet-B stressed diatom food on the reproductive output in Mediterranean Calanus helgolandicus (Crustacea; Copepoda)

Recent predictions anticipate a solar ultraviolet-B increase of 5-10% over temperate latitudes within the next 15 years. As Mediterranean waters are clear, enabling deep UVB penetration, we used controlled experiments to test the effects of equivalent levels of UVB on trophic plankton interactions. We grew the diatom Skeletonema costatum in the laboratory under daily 4 h UVB exposure in doses of 10% higher than today in contrast to control cultures (no UVB). UVB-exposed algae showed modification in cell structure, volume increase and delay in cell division, eventually leading to lysis and cell death after five days of daily 4 h exposure. Both control and UVB-stressed S. costatum cultures were used as food for wild Calanus helgolandicus females collected in the NW Mediterranean. Those fed on control diatoms produced three times more eggs, healthier offspring with fewer lethal naupliar deformities than females fed on UVB-exposed diatoms. Our results provide the first experimental evidence of indirect UVB effects on reproductive output through trophic response in marine plankton. The sensitivity of the S. costatum strain is discussed as well as the reduced reproductive output of the female copepods. We suggest that the low egg numbers and high naupliar deformity ratios are possibly a consequence of UVB-induced deficiency of essential components in the UVB-stressed maternal diet.     

Inhibitory effect of Salicornia europaea on the marine alga Skeletonema costatum

Exploiting the negative biochemical interference between plants and algal species has been suggested as a method to control harmful algal blooms. In this work, we investigated the inhibitory effect of the salt marsh halophyte Salicornia europaea against the marine alga Skeletonema costatum. S. europaea suppressed the growth of S. costatum in a nutrient-sufficient co-culture system, indicating that the inhibition of algal growth was because of the phytotoxic effect of S. europaea, rather than nutrient competition. We tested aqueous and organic extracts from S. europaea roots against S. costatum. The organic extracts inhibited growth and affected the cell size and chlorophyll a content of S. costatum in a dose-dependent manner. Among the three tested organic extracts, the methanol extract had the greatest effects on S. costatum, followed by butanol extract, and then the chloroform extract. Two flavonoids, rutin and quercetin-3-??-D-glucoside, were identified in the methanol extract by high performance liquid chromatography. The concentration of rutin was much higher than that of quercetin-3-??-D-glucoside. In an algal bioassay, rutin inhibited the growth of S. costatum and the inhibitory effect increased with increasing rutin concentration and with decreasing initial algal density. Therefore, we concluded that S. europaea negatively affects the growth of S. costatum, and that rutin, a metabolite of S. europaea, may play a role in this inhibitory effect.     

Linking elements to biochemicals: effects of nutrient supply ratios and growth rates on fatty acid composition of phytoplankton species

Three species of marine phytoplankton, Rhodomonas sp., Isochrysis galbana Parke, and Phaeodactylum tricornutum Bohlin, were cultivated in semicontinuous cultures to test biochemical responses (fatty acids; FAs) to five nitrogen (N):phosphorus (P) supply ratios and four growth rates (dilution rates). The characteristic FA profile was observed for each algal species (representing particular algal class), which remained relatively stable across the entire ranges of N:P supply ratios and growth rates. For all species, significant direct effects of N:P supply ratios on FAs were found at lower growth rates. The highest saturated and monounsaturated fatty acid (SFA and MUFA) contents were observed under N deficiency at the lowest growth rate in all three species, while responses of polyunsaturated fatty acids (PUFAs) revealed no consistent pattern. Total FAs (and SFAs and MUFAs) in all species showed significant negative correlations with N cell quota (Q_N) under N deficiency, but PUFAs had species‐specific correlations with Q_N. The results show that characteristic FA profiles of algal genus or species (representing particular algal classes) underlie fluctuations according to culture conditions. The significant correlation between FAs and Q_N under N deficiency suggests that elemental and biochemical limitation of phytoplankton should be considered mutually as determinants of food quality for zooplankton in marine ecosystems.     

Fatty acids’ profiles as indicators of stress induced by of a common herbicide on two marine bivalves species: Cerastoderma edule (Linnaeus, 1758) and Scrobicularia plana (da Costa, 1778)

In Europe, mainly the Mediterranean region, intensive use of fertilizers and pesticides has been recorded over the past 30 years, exceeding, in some cases, the limits of contamination authorized by the European Union. The intensive use of pollutants in fields near ecological coastal wetlands has led to implementation of pesticide monitoring programs to recover aquatic systems such as the Mondego estuary (Figueira da Foz, Portugal). According to information from the agricultural cooperatives of the Mondego valley, Primextra^® Gold TZ is the most-used herbicide in corn crop fields. Biomarkers, such fatty acids (FAs), proved to be new and potentially powerful tools to detect, illustrate, and evaluate exposure to and the effects of contamination hazards. They play important roles in establishing neural levels in organisms’ biochemical and physiological responses and are considered good bio-indicators of stress and potential indicators of ecosystem health. Bivalves are currently used in ecotoxicological bioassays because of their ecological importance, wide geographic distribution, ease of handling in the laboratory and in the field, and their ability to filter and ingest large volumes of water and sediment particles. Thus, the main goal of this work was to determine the toxic and biochemical (namely fatty acid profiles) responses of two size classes (small and big) of the two marine bivalve species Cerastoderma edule and Scrobicularia plana to the herbicide Primextra^® Gold. Furthermore, we aimed to compare the fatty acid contents, and thus the nutritive values, of both species and size classes collected in the field with those under laboratory conditions. Results show S. plana is more sensitive to the herbicide than C. edule. In general, among the larger-sized specimens in the field, S. plana is more nutritive than C. edule, but among the smaller-sized specimens, the opposite tendency is seen, where C. edule presents a greater abundance of FAs.     

Algicidal activity against Skeletonema costatum by marine bacteria isolated from a high frequency harmful algal blooms area in southern Chinese coast

Four marine bacterial strains P1, P5, N5 and N21 were isolated from the surface water and sediment of Mirs Bay in southern Chinese coast using the liquid infection method with 48-well plates. These bacteria were all shown to have algicidal activities against Skeletonema costatum. Based on morphological observations, biochemical tests and homology comparisons by 16S rDNA sequences, the isolated strains P1, P5, N5 and N21 were identified as Halobacillus sp., Muricauda sp., Kangiella sp. and Roseivirga sp., respectively. Our results showed that bacterial strain P1 killed S. costatum by release of heat labile algicide, while strains P5, N5 and N21 killed them directly. The algicidal processes of four bacterial strains were different. Strains P1, N5 and N21 disrupted the chain structure and S. costatum appeared as single cells, in which the cellular components were aggregated and the individual cells were inflated and finally lysed, while strain P5 decomposed the algal chains directly. We also showed that the algicidal activities of the bacterial strains were concentration-dependent. More specifically, 10?% (v/v) of bacteria in algae showed the strongest algicidal activities, as all S. costatum cells were killed by strains N5 and N21 within 72?h and by strains P1 and P5 within 96?h. 5?% of bacteria in algae also showed significant algicidal activities, as all S. costatum were killed by strains N5, P5 and N21 within 72, 96 and 120?h, respectively, whereas at this concentration, only 73.4?% of S. costatum cells exposed to strain P1 were killed within 120?h. At the concentration of 1?% bacteria in algae, the number of S. costatum cells continued to increase and the growth rate of algae upon exposure to strain N5 was significantly inhibited.     

Uptake and Metabolism of 2,2-bis-(p-Chlorophenyl-1,1,1-trichloroethane (DDT) by Marine Phytoplankton and Its Effects on Growth and Chloroplast Electron Transport

The effects of DDT (2,2-bis-(p-chlorophenyl)-1, 1, 1-trichloroethane) on the growth of seven marine phytoplankters, representative of five algal divisions, were studied. At a concentration of 80 parts per billion (0.23 μm) DDT, growth of Dunaliella tertiolecta was unaffected, and there was slight, if any, influence on the development of Cyclotella nana, Thalassiosira fluviatilis, Amphidinium carteri, Coccolithus huxleyi, and Porphyridium sp. Skeletonema costatum exhibited a 9 day lag before cell division commenced, the rate of growth subsequently being the same as in the control (no DDT). A further inoculation of this culture of S. costatum into 80 parts per billion DDT gave another 9-day lag before initiation of normal growth.     

Black soldier fly as dietary protein source for broiler quails: meat proximate composition,fatty acid and amino acid profile,oxidative status and sensory traits

In the perspective of improving the sustainability of meat production, insects have been rapidly emerging as innovative feed ingredient for some livestock species, including poultry. However, at present, there is still limited knowledge regarding the quality and sensory traits of the derived meat. Therefore, the present study tested the effect of a partial substitution of soya bean meal and oil with defatted black soldier fly (Hermetia illucens) larvae meal (H) in the diet for growing broiler quails (Coturnix coturnix japonica) on meat proximate composition, cholesterol, amino acid and mineral contents, fatty acid profile, oxidative status and sensory characteristics. To this purpose, three dietary treatments were designed: a control diet (C) and two diets (H1 and H2) corresponding to 10% and 15% H inclusion levels, respectively, were fed to growing quails from 10 to 28 days of age. At 28 days of age, quails were slaughtered and breast meat was used for meat quality evaluations. Meat proximate composition, cholesterol content and oxidative status remained unaffected by H supplementation as well as its sensory characteristics and off-flavours perception. Differently, with increasing the dietary H inclusion, the total saturated fatty acid and total monounsaturated fatty acid proportions raised to the detriment of the polyunsaturated fatty acid fraction thus lowering the healthiness of the breast meat. The H2 diet increased the contents of aspartic acid, glutamic acid, alanine, serine, tyrosine and threonine thus further enhancing the biological value of the meat protein. As a direct result of the dietary content of Ca and P, the meat of quails fed with the highest H level, displayed the highest Ca and the lowest P values. Therefore, meat quality evaluations confirmed H to be a promising insect protein source for quails. The only potential drawback from feeding H to broiler quails regarded the fatty acid profile of the meat, therefore requiring further research efforts to understand to what extent the fatty acid profile of H can be improved.     

Antifouling potential of the marine microalga Dunaliella salina

Marine organisms have usually been viewed as sources of environmentally friendly compounds with antifouling activity. We performed a series of operations to investigate the antifouling potential of the marine microalga Dunaliella salina. For the ethyl acetate crude extract, the antialgal activity was significant, and the EC₅₀ value against Skeletonema costatum was 58.9 μg ml^?1. The isolated purified extract was tested for antifouling activity, the EC ₅₀ value against S. costatum was 21.2 μg ml^?1, and the LC₅₀ against Balanus amphitrite larvae was 18.8 μg ml^?1. Subsequently, both UHR–TOF–MS and GC–MS were used for the structural elucidation of the compounds, and a series of unsaturated and saturated 16- and 18-carbon fatty acids were detected. The data suggested that the fatty acid extracts from D. salina possess high antifouling activity, and could be used as substitutes for potent, toxic antifouling compounds.     

Does allelopathy affect co-culturing <Emphasis Type="Italic">Haslea ostrearia</Emphasis> with other microalgae relevant to aquaculture?

Haslea ostrearia is a marine diatom known to produce marennine, a water-soluble blue-green pigment responsible for the greening of oysters in ponds along the French Atlantic coast. This phenomenon occurs seasonally when H. ostrearia blooms in oyster ponds, and it increases the economic value of cultured oysters. From an ecological perspective, H. ostrearia blooms are accompanied by a decrease in the abundance of other microalgae, suggesting that this diatom produces allelochemicals. Recent studies showed that purified marennine has other biological activities, for instance antioxidant, antibacterial, and antiviral activities, which could be used in aquaculture to promote this pigment as a natural antipathogen agent. One important issue regarding the possible use of H. ostrearia in aquaculture as a mixed algal diet, however, is the importance of marennine allelopathy. In this study, we investigated the allelopathic effect of H. ostrearia on the growth of five microalgal species relevant to aquaculture: Chaetoceros calcitrans, Skeletonema costatum, Phaeodactylum tricornutum, Tetraselmis suecica, and Tisochrysis lutea. Allelopathic tests were realized by co-culturing these microalgae with H. ostrearia in batch and in semi-continuous mode, based on initial biovolume ratios. Our findings showed that inhibition of the growth of microalgae due to the presence of H. ostrearia and marennine was species dependent. Skeletonema costatum, C. calcitrans, and T. lutea were significantly more sensitive, whereas T. suecica and P. tricornutum appeared to be more resistant. Growth irradiance significantly influenced the allelopathic effect against the sensitive species S. costatum, and the H. ostrearia production of marennine increases with irradiance. Data presented in this study partly support the hypothesis that marennine released into the culture medium possibly acts as an allelochemical compound, thus explaining the dominance of H. ostrearia and the loss of sensitive algae in oyster ponds, but also that some species are insensitive, which allows co-culturing and use in a mixed algal diet in aquaculture.     

Allelopathic effects of the dinophyte Prorocentrum minimum on the growth of the bacillariophyte Skeletonema costatum

We investigated growth interactions between the dinophyte Prorocentrum minimum and the bacillariophyte Skeletonema costatum using bi-algal cultures under axenic conditions. When low cell densities of P. minimum and high cell densities of S. costatum were inoculated into the same medium, growth of P. minimum was suppressed. Other inoculum combinations resulted in reduced S. costatum maximum cell densities. A mathematical model was used to simulate growth and interactions of P. minimum and S. costatum in bi-algal cultures. The model indicated that P. minimum always outcompeted S. costatum over time. Enriched filtrate from low-density P. minimum cultures significantly stimulated S. costatum growth, but enriched filtrate from high-density P. minimum cultures notably inhibited the growth of S. costatum. Growth of P. minimum was not affected by enriched filtrate from cultures of P. minimum at any density. Filtrates of P. minimum cultures were fractionated by ultrafiltration (molecular weight cutoff >3000 Da), and retentate that included polysaccharide(s) significantly inhibited the growth of S. costatum.     

Characterization of the triacylglycerol profile in marine diatoms by ultra performance liquid chromatography coupled with electrospray ionization–quadrupole time-of-flight mass spectrometry

Diatoms are considered to have great potential as new biofuel sources because they can effectively accumulate triacylglycerols (TAGs). Detailed structure information of TAG in diatoms is much needed not only for the assessment of biofuel quality such as fatty acid chain length and unsaturation degree but also for the tracing of biosynthetic precursors because the biosynthesis of TAG is typically completed by utilizing the diacylglycerol acyltransferase in the cytoplasm. In this report, a comprehensive characterization of TAGs in marine diatoms was performed using ultra performance liquid chromatography–electrospray ionization–quadrupole time-of-flight mass spectrometry. Many types of major TAGs were identified for the first time in these diatoms: 12 TAGs in Chaetoceros debilis, 9 TAGs in Phaeodactylum tricornutum Bohlin, 16 TAGs in Nitzschia closterium f. minutissima, 16 TAGs in Thalassiosira weissflogii, 13 TAGs in Thalassiosira sp., 16 TAGs in Stephanodiscus asteaea and 7 TAGs in Skeletonema costatum. Semi-quantification of TAGs in these diatoms was also carried out, and it was found that the contents of individual TAGs ranged from 0.5?±?0.1 to 217.9?±?8.1 nmol mg^?1 total lipids. In addition, the total lipid contents in diatoms ranged from 143.6?±?16.3 to 201.1?±?16.3 mg g^?1 dry microalgae and the total TAG contents ranged from 36.8?±?9.5 to 793.2?±?54.4 nmol mg^?1 total lipids. By comparative analysis of the compositions and concentrations of major TAGs in the seven algal strains, N. closterium f. minutissima with high abundance of TAGs containing the most monounsaturated fatty acids (mainly palmitoleic acid) was considered as one of the most promising diatom strains for microalgal biofuel production. Additionally, based on the information of sn-2 fatty acid obtained (mainly C16 in the sn-2 position), we propose the hypothesis that TAGs in diatoms are mainly derived from lipids in chloroplasts through the prokaryotic biosynthesis pathway, including monogalactosyldiacylglycerol and digalactosyldiacylglycerol.     

东海原甲藻与中肋骨条藻的种间竞争数值模拟

研究探讨了两个零维箱式模型在东海典型赤潮藻东海原甲藻和中肋骨条藻竞争与演替研究中的应用。模型在采用不同接种密度下的单种培养实验数据进行参数校正后,被用来模拟不同N/P条件下单种培养实验以及两藻种共培养竞争实验,并以实验数据对其结果进行了验证。模拟结果表明,在单种培养条件下,模型能够较好地重现两种藻在不同N/P环境中的生长及对营养盐的利用;共培养实验的模拟结果显示,在所有初始细胞密度比例条件下,中肋骨条藻的最终密度均会超过东海原甲藻,且PO4的消耗主要源于中肋骨条藻的利用,与实验结果一致,表明模型能够很好地体现两种藻的竞争结果及对营养盐的竞争关系;由于模型不足以模拟除营养盐竞争以外的藻间相互作用,模拟结果未体现东海原甲藻细胞数迅速衰减这一现象,有待进一步研究。     

Lipid quality of the diatoms Skeletonema costatum and Navicula gregaria from the South Atlantic Coast (Argentina): evaluation of its suitability as biodiesel feedstock

Since the fatty acid ester profile of a given biofuel is relatively consistent with the source’s fatty acid profile, the properties of the biodiesel produced from a particular feedstock exhibit predictable quality. Thus, lipid fractions and the fatty acid composition of stationary growth-phase cultures of the local strains of the diatoms Skeletonema costatum and Navicula gregaria were analysed to evaluate their suitability as biodiesel feedstock. Total lipid content was 20.83 pg cell⁻¹ in S. costatum and 19.17 pg cell⁻¹ in N. gregaria. Neutral lipids were the main fraction of total lipids in both species, accounting for ca. 65% and 76%, respectively. S. costatum was predominant in saturated fatty acids (SFAs; 43.48 %) and monounsaturated fatty acids (MUFAs; 40.11%), while N. gregaria was predominant in MUFAs (54.85%), followed by SFAs (33.42%). In S. costatum, the main fatty acids in neutral lipid fraction were myristic, palmitic, palmitoleic and oleic acids, while the main ones in N. gregaria were palmitic and palmitoleic acids. The oils extracted from these species presented linolenic acid contents within biodiesel’s quality specifications. However, in neutral lipid fraction both species showed eicosapentaenoic acid levels higher than the required limit. The lipid quality analysed in both species suggests that a biodiesel derived from these oils may present an acceptable cetane number, but likely poor cold-flow properties. This baseline information is useful for future research tending to find more suitable conditions in order to improve oil yield. In addition, both estuarine species neither compete with agriculture for food nor require farmland nor fresh water.     

Effect of growth conditions on accumulation of internal nitrate,ammonium, amino acids,and protein in three marine diatoms

The capacity of marine phytoplankton to change their cellular content of nitrate, ammonium, amino acids, and protein in response to different growth conditions was systematically investigated. Cellular concentrations of these compounds were measured in N-starved, N-deficient, and N-sufficient Skeletonema costatum (Grev.) Cleve and in N-deficient Chaetoceros debilis Cleve and Thalassiosira gravida Cleve, both before and after the addition of a pulse of nitrogen.N-sufficient Skeletonema costatum contains high concentrations of protein, large persistent pools of amino acids, and, if it is growing on nitrate, sizeable amounts of nitrate. As it becomes N-starved, the total cellular nitrogen decreases, the internal nitrate and amino acids become entirely depleted, and the protein content is drastically reduced. After nitrogen additions to N-deficient and N-starved cultures, transient pools of unassimilated nitrogen form which can account for a large fraction of newly taken up nitrogen. The size and kind of pool which accumulates is determined by the preconditioning of the cells, the nitrogen compound which is added, and the species identity. The pools which form in S. costatum indicate that nitrate reduction is the slowest step in nitrogen assimilation, the synthesis of protein from amino acids is the next slowest, and the incorporation of ammonium into amino acid is the fastest. However, the rate limiting steps may vary between diatom species.For the first time, measurements of the variation in cellular nitrogen compounds over a wide range of environmental conditions reveal the ability of some phytoplankton to buffer the effects of a changing, and sometimes growth-limiting, nitrogen supply. They accomplish this by utilizing stored internal nitrogen for growth when the external supply is low and by quickly storing unassimilated nitrogen when the external supply is suddenly increased beyond their ability to immediately assimilate it. The accumulation of large pools of unassimilated nitrogen compounds can explain the often observed difference between nitrogen uptake rates and growth rates.     

Biochemical compositions of two dominant bloom- forming species isolated from the Yangtze River Estuary in response to different nutrient conditions

Variations of cellular total lipid, total carbohydrate and total protein content of two dominant bloom-forming species (Skeletonema costatum and Prorocentrum donghaiense) isolated from the Yangtze River Estuary were examined under six different nutrient conditions in batch cultures. Daily samples were collected to estimate the cell growth, nutrient concentration and three biochemical compositions content during 7 days for S. costatum and the same sampling procedure was done every other day during 10 days for P. donghaiense. Results showed that for S. costatum, cellular total lipid content increased under phosphorus (P) limitation, but not for nitrogen (N) limitation; cellular carbohydrate were accumulated under both N and P limitation; cellular total protein content of low nutrient concentration treatments were significantly lower than that of high nutrient concentration treatments. For P. donghaiense, both cellular total lipid content and total carbohydrate content were greatly elevated as a result of N and P exhaustion, but cellular total protein content had no significant changes under nutrient limitation. In addition, the capability of accumulation of three biochemical constituents of P. donghaiense was much stronger than that of S. costatum. Pearson correlation showed that for both species, the biochemical composition of three constituents (lipid, carbohydrate and protein) had no significant relationship with extracellular N concentration, but had positive correlation with extracellular and intracellular P concentration. The capability of two species to accumulate cellular total lipid and carbohydrate under nutrient limitation may help them accommodate the fluctuating nutrient condition of the Yangtze River Estuary. The different responses of two species of cellular biochemical compositions content under different nutrient conditions may provide some evidence to explain the temporal characteristic of blooms caused by two species in the Yangtze River Estuary.     

细叶小羽藓营养成分的测定分析

该研究以细叶小羽藓为材料,测定了基本营养成分总糖、粗脂肪、粗蛋白、灰分的含量,采用气相色谱、氨基酸自动分析仪分别检测了脂肪酸和氨基酸的组分含量,并通过化学分析法和扫描电镜明确了细叶小羽藓矿质元素含量及分布情况。结果表明:细叶小羽藓中主要成分是糖类,占16.11%,粗蛋白质、灰分含量分别为11.20%、23.34%。氨基酸组成均衡,必需氨基酸(EAA)占氨基酸总量(TAA)的27.70%,必需氨基酸与非必需氨基酸的比值(EAA/NEAA)为0.38;限制性氨基酸为组氨酸,比值系数分(SRC)为12.96;药用氨基酸占总氨基酸的62.12%;天冬氨酸、谷氨酸、精氨酸、亮氨酸等含量相对较高。不饱和脂肪酸与饱和脂肪酸的比值为0.74,不饱和脂肪酸花生四烯酸(AA)和二十二碳六烯酸(DHA)相对含量较高。富含矿物质,茎叶元素均匀分布,其中Al、Ca、Fe、K、Mg等元素含量很高。综上认为,细叶小羽藓是一种含糖量较多、矿质丰富、低脂、低蛋白的苔藓,作为生物资源具有开发潜力。     

Publisher's note

During the late spring and early summer 1976, laboratory cultured cells of five marine algae were enclosed in dialysis sacks and grown in situ, in Ligurian coastal waters.Skeletonema costatum (Greville) Cleve did not grow at all. Hemiselmis virescens Droop stayed alive, but did not grow significantly. Platymonas suecica (Butcher) Manton &; Parke grew on cell reserves. Only Phaeodactylum tricornutum Bohlin and Thalassiosira pseudonana Hasle &; Heimdal took up nutrients from sea water when all reserves were exhausted. When division ended, all cells were capable of growing when sub-cultured, but physiologically were only slightly active. During immersion in oligotrophic waters, cell contents adapted quickly from nutrient-rich to nutrient-limited growth conditions. During adjustment to equilibrium with natural conditions, the three species became impoverished mainly in phosphorus (67–88% of initial values), ATP (54–92%), and chlorophyll a (59–89%); losses of nitrogen were lower (24–53%).To obtain algal material similar to that in the wild, the data suggest that the cells have to be kept in situ only until they divide five times, which usually requires no more than a week. Counting the cell density each day is probably the most convenient method of control, and each culture should be counted, because the difference of growth between aliquot dialysis cultures can be important (up to 28%).Data obtained for “wild-like” cells of Platymonas suecica, Phaeodactylum tricornutum, Thalassiosira pseudonana, and a wild contaminant population of Cylindrotheca closterium Rabenhorst with biochemical analysis used to run the Droop's model and with enrichment bioassays demonstrate that, during the period studied, phosphorus was the limiting factor for the algal growth. Bioassays also demonstrate that there were some specific variations; some algae were permanently limited by phosphorus, while for a few others nitrogen and phosphorus could be equally limiting.     

Involvement of an Extracellular Protease in Algicidal Activity of the Marine Bacterium Pseudoalteromonas sp. Strain A28

The marine bacterium Pseudoalteromonas sp. strain A28 was able to kill the diatom Skeletonema costatum strain NIES-324. The culture supernatant of strain A28 showed potent algicidal activity when it was applied to a paper disk placed on a lawn of S. costatum NIES-324. The condensed supernatant, which was prepared by subjecting the A28 culture supernatant to ultrafiltration with a 10,000-M_w-cutoff membrane, showed algicidal activity, suggesting that strain A28 produced extracellular substances capable of killing S. costatum cells. The condensed supernatant was then found to have protease and DNase activities. Two Pseudoalteromonas mutants lacking algicidal activity, designated NH1 and NH2, were selected after N-methyl-N′-nitrosoguanidine mutagenesis. The culture supernatants of NH1 and NH2 showed less than 15% of the protease activity detected with the parental strain, A28. The protease was purified to homogeneity from A28 culture supernatants by using ion-exchange chromatography followed by preparative gel electrophoresis. Paper-disk assays revealed that the purified protease had potent algicidal activity. The purified protease had a molecular mass for 50 kDa, and the N-terminal amino acid sequence was determined to be Ala-Thr-Pro-Asn-Asp-Pro. The optimum pH and temperature of the protease were found to be 8.8 and 30°C, respectively, by using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a substrate. The protease activity was strongly inhibited by phenylmethylsulfonyl fluoride, diisopropyl fluorophosphate, antipain, chymostatin, and leupeptin. No significant inhibition was detected with EDTA, EGTA, phenanthroline or tetraethylenepentamine. These results suggest that Pseudoalteromonas sp. strain A28 produced an extracellular serine protease which was responsible for the algicidal activity of this marine bacterium.     

Nitrate Utilization by the Diatom Skeletonema costatum: II. Regulation of Nitrate Uptake

Nitrate utilization has been characterized in nitrogen-deficient cells of the marine diatom Skeletonema costatum. In order to separate nitrate uptake from nitrate reduction, nitrate reductase activity was suppressed with tungstate. Neither nitrite nor the presence of amino acids in the external medium or darkness affects nitrate uptake kinetics. Ammonium strongly inhibits carrier-mediated nitrate uptake, without affecting diffusion transfer. A model is proposed for the uptake and assimilation of nitrate in S. costatum and their regulation by ammonium ions.     

The essential dietary fatty acid requirement of the tufted apple budmoth, Platynota idaeusalis

The tufted apple budmoth, Platynota idaeusalis (Walker), was reared non-axenically for two successive generations on a casein-based semisynthetic diet. The qualitative essential fatty acid requirement for growth, development and normal pupal-adult ecdysis was studied using the non-axenic casein-based semisynthetic diets with and without various 99% pure fatty acids. Linoleic or linolenic acids caused accelerated larval development; linoleic, linolenic and arachidonic acids showed similar activity in body weight gain and survival to pupal-adult ecdysis. Linoleic or linolenic acids were active in alleviating wing deformities; arachidonic acid was partially active in alleviating wing deformities at the one dietary concentration evaluated. Activity of arachidonic acid as an essential fatty acid for P. idaeusalis is unique among insects, except for mosquitoes. The essential fatty acid deficiency syndrome of the adult, resulting from the larvae feeding on fat-deficient diets, was greatly reduced when larvae were fed on a diet adequate in essential fatty acid during either their early or late development.