CONTINUOUS FLOW CULTURE OF BENTHIC DIATOMS AND ITS APPLICATION TO BIOASSAY1,2

The benthic diatom, Navicula seminulum var. hustedtii Patr, was isolated from the field and studied in laboratory cultures. Experiments were conducted to determine the suitability of Millipore membranes and solidified agar substrate for diatom colonization under continuous flow conditions. Diatom colonization showed large variation and low reproducibility on Millipore membranes of different materials and pore sizes. Solidified agar substrate supported stable and reproducible colonization and is nutritionally neutral, translucent, homogeneous and easy to sample. The diatom colonization process on agar substrate involved four growth phases: i) pioneer; ii) exponential; iii) steady state; and, iv) vanishing. The culture system was also used in bioassay, testing the toxic effect of copper on the growth of benthic diatoms. The proposed method provides a useful means for studying autecology of benthic diatoms as well as for bioassay work.     

A NEW LARVAL FISH BIOASSAY FOR TESTING THE PATHOGENICITY OF PFIESTERIA SPP. (DINOPHYCEAE)1

Water quality, microbial contamination, prior fish health, and variable results have been major impediments to identifying the cause and mechanism of fish mortality in standard aquarium‐format Pfiesteria bioassays. Therefore, we developed a sensitive 96‐h larval fish bioassay for assessing Pfiesteria spp. pathogenicity using six‐well tissue culture plates and 7‐day‐old larval cyprinodontid fish. We used the assay to test pathogenicity of several clonal lines of Pfiesteria piscicida Steidinger and Burkholder and P. shumwayae Glasgow and Burkholder that had been cultured with algal prey for 2 to 36 months. The P. shumwayae cultures exhibited 80%–100% cumulative mortality in less than 96 h at initial zoospore densities of approximately 1000 cells·mL^?1. No fish mortalities occurred with P. piscicida at identical densities or in controls. In a dose‐response assay, we demonstrated a strong positive correlation between dinospore density and fish mortality in a highly pathogenic culture of P. shumwayae, generating a 96‐h LD₅₀ of 108 zoospores·mL^?1. Additionally, we applied the assay to evaluate a 38‐L P. shumwayae bioassay that was actively killing fish and compared results with those from exposures of juvenile tilapia (Oreochromis niloticus) in a 500‐mL assay system. Water from the fish‐killing 38‐L assay was filtered and centrifuged to produce fractions dominated by dinoflagellates, bacteria, or presumed ichthyotoxin (cell‐free fraction). After 96 h, the larval fish assay exhibited 50%–100% cumulative mortality only in fractions containing dinoflagellates, with no mortalities occurring in the other fractions. The 500‐mL bioassay with tilapia produced inconsistent results and demonstrated no clear correlation between mortality and treatment. The new larval fish bioassay was demonstrated as a highly effective method to verify and evaluate dinoflagellate pathogenicity.     

GROWTH-INFLUENCING SUBSTANCES IN SEDIMENT EXTRACTS FROM A SUBTROPICAL WETLAND: INVESTIGATION USING A DIATOM BIOASSAY1

A Biological assay using Amphora coffeaeformis var. perpusilla (Grunow) Cleve and A. coffeaeformis (Agardh) Kützing was used to investigate the changes in the properties of soluble organic carbon in sediments taken from a coastal wetland. During January to May, sediment extracts became increasingly inhibitory to diatom growth After the onset of the spring rains, the inhibitory properties of the extracts disappeared. Substances capable of promoting mixotrophic growth and heterotrophic growth were found in extracts taken in July to December. These positive responses took place at the time of mangrove (Rhizophora mangle L.)leaf-fall.     

USE OF THE CRICKET ACHETA DOMESTICA L. AS A BIOASSAY ORGANISM FOR THE TOXIC EXTRACT FROM GONYAULAX MONILATA (DINOPHYCEAE)1

A toxic extract from unialgal cultures, of Gonyaulax monilata Howell was tested for insecticidal activity on the cricket Acheta domestica L The regression line of the activity was compared with that of ouabain in this insect as well as with that of the toxic extract in mice. Although mice are approximately 2.5°as sensitive as the cricket an a body weight basis, because of the insect's smaller mass‘(1/50 that of the mouse), one can treat 26° as many crickets with the same amount of toxic material. This fact, coupled with the simple rearing requirements of the insect, makes it an ideal bioassay organism as well as a useful subject far further investigations of the physiological properties of the toxic extract and other bioactive compounds available in limited quantities.     

A BIOASSAY FOR COMPARING THE ACCEPTABILITY TO SLUGS OF PLANT EXTRACTS OR SOLUTIONS OF PURE CHEMICALS

A bioassay utilising a Y maze is described in which the responsesof slugs to aqueous plant extracts, or solutions of pure chemicalscan be evaluated. (Received 17 November 1977;     

Brine Shrimp致死率生物鉴定法对八种民间药活性成分的研究

采用ＴｈｅＢｒｉｎｅＳｈｒｉｍｐＬｃｔａｌｉｔｙＢｉｏａｓｓａｙ法对民间药翼蓼ＰｔｅｒｏｘｙｇｏｎｕｍｇｉｒａｌｄｉｉＤｕｍｍｅｒｅｔＤｉｅｌｓ）,金荞麦（Ｆａｇｏｐｙｒｕｍｄｉｂｏｔｒｙｓ（Ｄ．Ｄｏｎ）Ｈａｒａ）,齿果酸模（ＲｕｍｅｘｄｅｎｔａｔｕｓＬ．）巴天酸模（ＲｕｍｅｘｐａｔｉｅｎｔｉａＬ．）网果酸模（ＲｕｍｅｘｄｉｃｔｙｏｃａｒｐｕｓＢｏｉｓｓｅｔＢｕｈｓｅ）、掌叶大黄（Ｒｈｅｕｍｐａｌ     

MELANIN AND URATE ACT TO PREVENT ULTRAVIOLET DAMAGE IN THE INTEGUMENT OF THE SILKWORM,BOMBYX mori

The phenomenon that epidermal cells under the white stripes rather than black stripes contain many uric acid granules was found in larvae of several Lepidopteran species. However, the biological mechanism of this phenomenon is still unknown. In the present study, we take advantage of several silkworm (Bombyx mori) body color mutant strains to investigate the deposition patterns and biological mechanism of urate and melanin in the integuments of these mutant larvae. By imaging with transmission electron microscope, we found that there were some melanin granules in the larval cuticle in black body color mutant plain Black (p^B), but not in background strain plain (p) with white larval body color. In contrast, the larval epidermal cell of background strain had much more urate granules than that of black one. Furthermore, the uric acid content under the black stripes was significantly lower than that under the white stripes in a single individual of mottled stripe (p^S) with black and white stripes in each segment. Ultraviolet A (UVA) exposure experiments showed that the distinct oily (od) mutant individuals with translucent larval integument were more sensitive to the UVA damage than black body color mutant and background strain without any pigmentation in the larval cuticle. This is likely due to the absence of melanin granules and few urate granules in the integument of od mutant. Thus, both the deposited melanin granules in the cuticle and the abundant urate granules in the epidermis cells constitute effective barriers for the silkworm to resist UVA‐induced damage.     

A MODIFIED CELL WALL MUTANT OF THE RED MICROALGA RHODELLA RETICULATA RESISTANT TO THE HERBICIDE 2,6-DICHLOROBENZONITRILE1

The rigid component of the cell walls of red macroalgae, cellulose, is lacking in the red microalgae. Instead, the cells are encapsulated within an amorphous polysaccharide. These complex sul fated polysaccharides are composed of at least 10 different sugars, but their structure is not known, When the herbicide 2,6-dichlorobenzonitrile (DCB), a compound that specifically inhibits cellulose biosynthesis, was applied to cultures of the red microalga Rhodella reticulata upon inoculation, growth was inhibited. When added during the stationary phase of growth (after cell division had ceased), DCB did not affect cell number but it did inhibit polysaccharide production. A spontaneous mutant resistant to DCB was selected; it had physiological characteristics similar to those of the wild-type parent. The composition of the cell wall polysaccharide of the mutant was totally modified, being composed almost entirely (98% of its dry matter, as compared to 2.9% in the wild type) of methyl galactose, but retaining the same sulfate content. The molecular mass of the mutant polysaccharide was, however, similar to that of the wild-type parent (～6 × 10⁶ daltons), although its viscosity was significantly lower.     

THE TETRAHYMENA HOMOLOG OF BACTERIAL AND MAMMALIAN 4-HYDROXYPHENYLPYRUVATE DIOXYGENASES LOCALIZES TO MEMBRANES OF THE ENDOPLASMIC RETICULUM

The expression and intracellular localization of the Tetrahymena homolog of 4-hydroxyphenylpyruvate dioxygenase (HPPD) were investigated in wild-type Tetrahymena thermophila strain B1868 VII and the mutant strains IIG8, defective in food vacuole formation, MS-1, blocked in secretion of lysosomal enzymes, and SB 281, defective in mucocyst maturation. Immunoelectron microscopy and confocal laser scanning microscopy demonstrated that Tetrahymena HPPD primarily localized to membranes of the endoplasmic reticulum. In addition, Tetrahymena HPPD was detected in association with membranes of the Golgi apparatus, and transport vesicles in exponentially growing wild-type and mutant strains. In starved cells, Tetrahymena HPPD localized exclusively to membranes of small vesicles. Since no de novo synthesis ofTetrahymena HPPD takes place in cells starved for more than 30min, these results suggest that there is a flow ofTetrahymena HPPD from the endoplasmic reticulum to small vesicles, possibly via the Golgi apparatus, and thatTetrahymena HPPD contains a signal for vesicle membrane retrieval or retention.     

ESTIMATION OF THE PHYSIOLOGICAL PARAMETERS OF A MEMBRANE CHANNEL EXPOSED TO ELECTROMAGNETIC FIELDS USING THE GPOF APPROACH

A method is proposed for the estimation of kinetic parameters of ionic channels in the cell membrane. The method is based on the generalized pencil-of-function approach, which exploits transient current signals from single channels to derive the frequency of the system poles. The proposed approach is validated for the well-known potassium channel by comparing the estimated values with the theoretical values given by Hodgkin and Huxley. The approach is superior to previous spectral approaches, both for its accuracy and for its robustness. It is especially useful for parameter estimation when the channel is exposed to electromagnetic fields. Results are given for exposure to 200-Hz and 915-MHz signals, to demonstrate the effect of fields on the kinetic parameters of the channel.     

用新的计量方法研究黑腹果蝇的求爱歌

对雄果蝇求爱脉冲歌的研究已有多年的历史,但对脉冲间隔的计量方法并不方便。本文介绍我们采用微机终端计时新技术,自动对脉冲进行计数,可在范围更为广泛的数据信息内,对果蝇脉冲歌进行测量统计,对ipi分布中纵数与平均值的关系提出我们的看法,并首次对ipf及npb的分布进行了研究。     

THE ROLE OF ENDOMYCORRHIZAE IN REVEGETATION PRACTICES IN THE SEMI-ARID WEST. II. A BIOASSAY TO DETERMINE THE EFFECT OF LAND DISTURBANCE ON ENDOMYCORRHIZAL POPULATIONS

Populations of the endomycorrhizal fungus Glomus fasciculatus were significantly reduced following land disturbance in western Colorado soil. A bioassay was developed to measure changes in the endomycorrhizal population. In the bioassay, inoculum levels were measured by comparing the percentage infection in corn (Zea mays) root systems thirty days after planting in undisturbed or disturbed soils. The percentage infection was 2% in the disturbed soil compared to 77% in the adjacent undisturbed soil. Glomus fasciculatus was identified as the endophyte in both soils. Considering the importance and function of endomycorrhizal fungi to their plant hosts the reduction of active inoculum in the disturbed soil may be an important ecological factor in subsequent succession.     

青蛙蝌蚪微核试验——一种水体诱变剂检测系统的建立

以青蛙蝌蚪为实验生物,利用甲基磺酸乙酯(EMS)和亚硝基胍(MNNG)探讨其化合物浓度、暴露时间和蝌蚪发育阶段等因素对诱发青蛙蝌蚪红细胞微核的影响,不同统计单位的特点和相互关系;提出了青蛙蝌蚪微核试验作为一种水体诱变剂检测系统的基本实验程序和一般原则。此外,还描述了“小体M”——一种特殊的细胞学现象,并初步讨论了微核代谢机制。     

NOTE MOLECULAR ANALYSIS OF THE AHAS GENE OF PORPHYRIDIUM SP. (RHODOPHYTA) AND OF A MUTANT RESISTANT TO SULFOMETURON METHYL

Acetohydroxyacid synthase (AHAS) is the target enzyme of the sulfonylurea herbicides, and here we report the sequence of the gene from wild-type and herbicide-resistant Porphyridium sp. (Rhodophyta). The resistant mutant has a single residue substitution at a position known to confer herbicide resistance in E. coli and in plants. The rhodophyte gene is of cyanobacterial origin and distinct from the nuclear-encoded chlorophyte gene, which may be of mitochondrial origin.     

USE OF THREE RAPID DETECTION SYSTEMS TO EVALUATE THE PREVALENCE AND DISSEMINATION OF SALMONELLA IN A BRAZILIAN POULTRY SLAUGHTERHOUSE

Prevalence and dissemination of Salmonella in a Brazilian poultry slaughterhouse were evaluated by three rapid detection systems (SS/SV^TM, VICAM, OSRT^TM, Unipath/Oxoid, and REVEAL^TM, Neogen), plus the conventional procedure. The carcasses were sampled after bleeding (P1), defeathering (P2), evisceration (P3), washing (P4), chilling (P5) and the packaged end-product (P6). In the first set of carcasses, the Salmonella incidence determined by the conventional method was 38.3% and 22.5% by SS/SV^TM. In the set for evaluation of OSRT^TM, the number of positive samples was the same detected by the cultural procedure (49.0%). In the third set, the positivity by the conventional procedure was 33.3%, and 5.0% by REVEAL^TM. The comparisons of positives in the first and third sets of carcasses were significantly different (P < 0.05). The positivity for Salmonella, in carcasses at P1 to P6, as determined by at least one of the methods, was 47.5%, 47.5%, 32.5%, 30.0%, 30.0% and 37.7%, respectively.     

酵母细胞生物转化反式—肉桂酸生产L—苯丙氨酸的研究

据文献调查,搜集了国内可能相关的30株酵母,进行生物转化反式-肉桂酸(t-Ca) 生产L-苯丙氨酸 (L-Phe) 的微生物筛选研究,并对部分菌株生物转化能力,即苯丙氨酸解氨酶 (PAL,EC _(4、3、1、5) 活性水平进行了初步评估。筛选结果是:22株酵母具有转化 t-Ca 生成 L-Phe 的能力,转化率在2—67％范围。选出7株酵母研究在液体培养条件下细胞生长和PAL活性的时间过程关系,PAL 活性范围在 2.3—14.4x10~(-s)u/m g细胞干重。深红酵母 (Rhodotorularubra) AS2.166作为生物转化制备实经菌株,在静止细胞和固定化细胞批式反应条件下,结果获得L-Phe分离产率分别为42.0％,28.7％。     

A RAPID AND SIMPLE METHOD FOR THE DETERMINATION OF PICOGRAM LEVELS OF SEROTONIN IN BRAIN TISSUE USING LIQUID CHROMATOGRAPHY WITH ELECTROCHEMICAL DETECTION

A rapid and simple technique using solvent extraction and high pressure liquid chromatography with electrochemical detection has been developed for the determination of serotonin in small brain tissue samples (1-20 mg). The method has a reasonably good specificity and a very low experimental error (less than 3%s.e ., calculated from six samples processed and analysed from the same brain homogenate). The recovery of authentic 5-HT added is 80-90%. The 5-HT levels of rat whole brain was found with the present technique to be 690 ± 17.5 ng/g and of mouse neocortex 304 ± 16 ng/g. Monoamine oxidase inhibition with pargyline (2 h) increased 5-HT levels in mouse neocortex to 194 ± 15% (N = 5) of control, while reserpine depleted 5-HT to 13 ± 4% of control. The method has a sensitivity level of about 20 pg (0.1 pmol) per brain sample.