Spatial and temporal distribution of pheromone biosynthesis-activating neuropeptide in Helicoverpa (Heliothis) armigera using RIA and in vitro bioassay.

A [3H]-PBAN (pheromone biosynthesis-activating neuropeptide) analog was synthesized, and binding of the radioligand to a specific PBAN-antiserum was achieved. The inhibition of binding of the radioligand by unlabeled PBAN, several PBAN analogs, and other competitors was studied and a specific radio-immunoassay was developed. Using this radioimmunoassay we found PBAN-like immunoreactivity in methanol extracts of hemolymph and neural tissues from females. Higher levels of PBAN-like immunoreactivity in extracts of brain-suboesophageal ganglion complexes, corpora cardiaca, thoracic ganglia, and abdominal ganglia were observed during the 4-5th h scotophase when compared to the PBAN-like immunoactivity levels during the 6-11th h photophase. On the other hand, the concentrations of PBAN-like immunoreactivity, in the terminal abdominal ganglion were higher during the photophase relative to minimal levels observed during the scotophase, indicating an accumulation before the onset of pheromone production. These differences in concentrations of PBAN were also reflected in the stimulation of in vitro pheromone glands, whereby significant stimulations were obtained by scotophase and photophase brain extracts, scotophase thoracic ganglia extracts, and photophase terminal abdominal ganglia extracts. No detectable levels of PBAN were found in hemolymph extracts during the sampling periods.     

Cell-density-dependent sensitivity of a mer-lux bioassay.

The sensitivity of a previously described assay (O. Selifonova, R. Burlage, and T. Barkay, Appl. Environ. Microbiol. 59:3083-3090, 1993) for the detection of bioavailable inorganic mercury (Hg2+) by the activation of a mer-lux fusion was increased from nanomolar to picomolar concentrations by reducing biomass in the assays from 10(7) to 10(5) cells ml-1. The increase in sensitivity was due to a reduction in the number of cellular binding sites that may compete with the regulatory protein, MerR, for binding of the inducer, Hg2+. These results show that (i) the sensitivity of the mer-lux assay is sufficient for the detection of Hg2+ in most contaminated natural waters and (ii) mer-specified reactions, Hg2+ reduction and methylmercury degradation, can be induced in natural waters and may participate in the geochemical cycling of mercury.     

The Daphnia bioassay: a critique

Daphnia magna is used widely as a standard ecotoxicological indicator organism, and protocols exist for its use in assessing the toxicity of substances under acute and chronic experimental conditions. Problems exist in repeatability of such bioassays between laboratories. Sources of variation are identified using a simple quantitative genetics model. Presenting specific examples, we conclude that these problems are tractable, but only if the genotype and culture conditions prior to and during tests are strictly controlled.     

Development and application of a nutrient-diffusing bioassay for large rivers

1. Laboratory and field experiments were performed to develop and then apply a nutrient-diffusing substratum (NDS) design suitable for use in large, fast-flowing rivers. 2. Initial laboratory experiments quantified diffusion of PO₄ and NO₃ from new and previously used clay pots, which were soaked in deionized distilled water. Mean release rates initially exceeded 2.4 and 725 μmol l^–1 day^–1 P and 0.22 and 18 μmol l^–1 day^–1 N from new and used pots, respectively, but declined rapidly with increasing time spent in deionized distilled water and were below detectable levels after about 18 and 29 days, respectively. 3. A phosphorus (P) dose–response experiment in a P-limited reach of the Athabasca River, Alberta, Canada showed that epilithic biomass and macroinvertebrate density on NDS increased with increasing concentrations of KH₂PO₄ up to about 0.5 m . Beyond this threshold, biomasses and densities were unaffected by initial KH₂PO₄ concentration. Coefficients of variation of epilithic biomass estimates declined with increasing KH₂PO₄ whereas invertebrate density appeared to be unaffected by KH₂PO₄ levels. 4. Release rates of both P and N from NDS filled with 0.5 m KH₂PO₄ or 0.5 m NaNO₃ declined at a log-negative rate from about 5000 μmol N-NO₃ l^–1 day^–1 and 3500 μmol P-PO₄ l^–1 day^–1 on day 2, to 200 μmol l^–1 day^–1 for both N and P on day 32. 5. After development, we used the diffusing substrata to identify spatial patterns in nutrient limitation at seven sites along a 120 km reach in the Athabasca River, that receives two known point-source nutrient inputs. NDS consisting of N, P, N + P and unenriched controls were attached to the river bottom for 22–23 days and then retrieved and sampled for epilithic chlorophyll a. Physicochemical parameters and epilithic biomasses on upper stone surfaces were also quantified when NDS were deployed and retrieved from each site. 6. Sites located immediately downstream of the two point source inputs had higher water column concentrations of PO₄ and epilithic biomasses than the site immediately upstream; epilithic biomass was positively related to PO₄ in the late autumn (r²⁼ 0.58) but not in early autumn. Sites located immediately below nutrient inputs were not nutrient-limited, whereas upstream reference sites were P-limited.     

The cockroach heart as a bioassay organ

Detailed procedures are presented for denervation of the American cockroach heart, Periplaneta americana L., by removal of the lateral cardiac nerve cords. Results of bioassay with the innervated heart are also presented and compared to responses obtained from identical assay conditions with the denervated heart.The response of the innervated heart to 10^-3 M sodium azide, slight changes in the concentration of sodium ion, reduced glutathione, saline dilutions, and low amounts of ethanol and acetone was characterized by the immediate appearance of irregularities in the heartbeat. In contrast, the responses of the denervated heart to the first three of these compounds were always characterized by a smooth even heartbeat changing gradually in amplitude and/or rate and/or contractile state of the alary muscles. All assay conditions examined caused qualitatively less response in the beat of the denervated heart.Irregularities in the beat of the innervated heart which were induced in bioassay were found to be due to extreme sensitivity of the spontaneously active cardiac neurons.

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Zusammenfassung Das isolierte abdominale Herz von Periplaneta americana L. reagiert auf Durchspülung mit 10^-3 Mol Natriumazid in physiologischer Kochsalzlösung mit einem unmittelbaren Anstieg des Tonus bis zu fast systolischer Hemmung. Danach verringert das Herz den Tonus allmählich, schlägt unregelmäßig und tritt in eine kurze Periode diastolischer Pause ein, bevor es in Diastole stehen bleibt. Die Herzschlagaktivität wurde wieder aufgenommen, wenn das Natriumazid durch Spülung mit physiologischer Kochsalzlösung ersetzt wird.Nach Entfernung beider Paare der Herzseitennerven gab das Herz von P. americana auf Bespülung mit 10^-3 Mol Natriumazid in physiologischer Kochsalzlösung keine Initialreaktion. Statt dessen stieg der Herzschlag nach 2 min allmählich leicht an und die Herzschlagamplitude nahm im Verlaufe von 6 min sehr allmählich ab. Nach 6 min blieb das Herz in Diastole stehen.Die Herzreaktionen der amerikanischen Küchenschabe waren bei Auftropf-Tests mit anormaler Natriumchloridkonzentration, herabgesetztem Glutathion und Lösungsmitteln ähnlich den für Natriumazid beschriebenen. Die Reaktion des denervierten Herzens ist durch das Fehlen von Unregelmäßigkeiten im Herzschlagmuster gekennzeichnet.Sowohl das denervierte wie das innervierte Herz reagieren auf Testtropfen von nur etwas anormalen Calciumchloridkonzentrationen.

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Supported by USPHS Training grant PHS GM 1076.     

Characterization of a rat anterior pituitary cell bioassay

Summary We have described the protocols and characterization of a pituicyte culture, which became established as a reliable and reproducible bioassay for the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). The bioassay was used to measure the bioactivity of factors that inhibit and stimulate gonadotrophin secretion. The protocol that was used involved the culling of female Wistar rats (200 to 250 g weight), at random stages of their cycle, and dispersal of their pituicytes in a concentration of 0.4 × 10⁶ cells · ml⁻¹ · well⁻¹ in serum-free medium (Dulbecco’s modified Eagle’s medium/Ham’s F12 mixture, supplemented with insulin and transferrin) in Falcon 3047 24-well culture plates. After 24 h of pre-culture, the medium was changed and the cells cultured for a further 48 h. The supernatant was removed and assayed for basal secretion of FSH and LH. The cells were then stimulated with 10⁻⁸ M GnRH for 4 h and the supernatant assayed for gonadotrophin-releasing hormone (GnRH)-stimulated FSH and LH secretion. All samples were assayed as pairs of duplicates (i.e. quadruplicate samples) which were randomly added to the plates to minimize plate effects. Random number tables were used to achieve this randomization.     

Development and applications of a yeast-based bioassay for the mycotoxin zearalenone

Zearalenone (ZON) is a non-steroidal estrogenic mycotoxin produced by plant pathogenic species ofFusarium. As a consequence of infection withF. culmorum andF. graminearum, ZON can be found in cereals and derived food products. Several countries have established monitoring programs and guidelines for ZON levels in grain intended for human consumption and animal feed. We have developed a sensitive yeast bioassay allowing detection of the estrogenic activity of ZON in cereal extracts without requiring further clean up steps. The high sensitivity makes this assay suitable for low cost monitoring of contamination of small grain cereals with estrogenicFusarium mycotoxins, but also attractive as a tool for basic research. We have successfully used yeast indicator strains to screen for mutants ofF. graminearum which no longer produce detectable amounts of ZON, and have identified a plant cDNA encoding a ZON detoxification enzyme. Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003     

Creation of a non-mycorrhizal control for a bioassay of AM effectiveness

and application times 2 weeks before sowing, at sowing and 1 week after sowing were investigated. Various Finnish field soils with their indigenous AMF communities were used. The main test plant species was oil-seed flax (Linum usitatissimum). In a comparison of sampling time, barley (Hordeum vulgare) was also used and phytotoxicity was studied additionally on red clover (Trifolium pratense), barley and pea (Pisum sativum) mutants. Sampling in the spring after the thaw resulted in the highest infectivity and AM response and the clearest differences between soils with varying AM potential. No evidence of temporal variation in benomyl effectiveness on mycorrhiza was found. The dose of benomyl sufficient to create a control with suppressed mycorrhization was 20 mg per kg soil at target moisture incorporated in the soil. Plant growth reduction in irradiated soil was observed with benomyl application 1 week after sowing only with flax and red clover. The most effective application time for benomyl was immediately before sowing.     

Creation of a non-mycorrhizal control for a bioassay of AM effectiveness

γ -irradiation of soil by 10 and 3 kGy, and the use of a myc⁻ mutant. The methods were examined on clay and loam. Two management histories were included with both soils to study the ability of the methods to differentiate AM effectiveness. For each soil type, two pot experiments were conducted in field soil, one to investigate the effects of the methods on soil nutrient status, and the other to study the effects on mycorrhization and plant response. The test plants, flax (Linum usitatissimum) and pea (Pisum sativum) myc⁺ and myc⁻ mutants, were grown in 1-l pots for 4 weeks in a growth chamber. To test the ability of the bioassay to reflect differences in AM effectiveness in the field, the mutants and benomyl were also studied in the field from which the loam for the pot experiments was obtained. The bioassay accurately represented the situation in the field and the use of benomyl appeared to be the most appropriate method currently available. The advantages were the ability to use a test plant responsive to AM, the use of less elevated nutrient concentrations than with irradiation, and thus the possibility to use untreated soil as the mycorrhizal treatment. The pea mutants proved unresponsive to AM, and reinoculation to irradiated soil resulted in only half the colonization rate in untreated soil. Benomyl may, however, lead to an underestimation of AM effectiveness because the control is not totally non-mycorrhizal. Its use also carries with it health and environmental risks. Received: 9 February 1999 / Accepted: 27 September 1999     

Bacterial bioluminescence as a bioassay for mycotoxins.

The use of bacterial bioluminescence as a toxicological assay for mycotoxins was tested with rubratoxin B, zearalenone, penicillic acid, citrinin, ochratoxin A, PR-toxin, aflatoxin B1, and patulin. The concentrations of mycotoxins causing 50% light reduction (EC50) in Photobacterium phosphoreum were determined immediately and at 5 h after reconstitution of the bacteria from a freeze-dried state. Generally, less toxins were required to obtain an EC50 at 5 h. The effects of the above mycotoxins on bioluminescence were determined after 5, 10, 15, and 20 min of incubation with the bacterial suspensions. The concentration of rubratoxin B necessary to elicit an EC50 increased with time, whereas the concentration of citrinin, penicillic acid, patulin, and PR-toxin necessary decreased with time. There was very little change in the concentration of zearalenone, aflatoxin B1, and ochratoxin A required to elicit an EC50 with time. The bacterial bioluminescence assay was most sensitive to patulin and least sensitive to rubratoxin B.     

Occurrence and bioassay responses of G

A growth regulator (G) occurs at high concentrations in adult leaves of E. grandis Maiden. Low concentrations of G are present in juvenile leaves of this species and also in adult leaves of some other Myrtaceae. Low concentrations of G (5×10^–6 and 10^–5 M) promote rooting in mung-bean cuttings and elongation in Avena coleoptile sections; high concentrations (5×10^–4 M) inhibit. These and other bioassay results indicate that G may have auxin-like activity.     

The bioassay of gibberellins

Summary A bioassay is described which is dependent upon the fact that gibberellin induced -amylase release from barley half-seeds is proportional to the logarithm of gibberellin concentration applied. This bioassay has been successfully applied to the estimation of gibberellin-like substances in plant extracts. The bioassay has the following advantages: 1) Release of -amylase is one step closer to the primary site of action of GA; 2) release of -amylase is not affected by solvent residues and is apparently completely specific for gibberellin; 3) release of -amylase is not affected by substances other than gibberellins present in crude plant extracts.Supported by U.S. Atomic Energy Commission Contract No. AT (11-1)-1338.     

Tradescantia micronucleus bioassay

Four coded chemicalsm azidoglycerol (AG), N-methyl-N-nitrosourea (MNU), sodium azide (NaN₃), and maleic hydrazide (MH), were tested with the Tradescantia micronucleus (Trad-MCN) bioassay by five independent laboratories from five different countries. The purpose of this international collaborative study was to evaluate four plant bioassays, of which the Trad-MCN assay was one, for their sensitivity, efficiency and reliability. The study was carried out under the sponsorship of the International Programme on Chemical Safety. All laboratories adhered to a standard Trad-MCN protocol which suggested that three replicate tests be conducted with each chemical. The results reported by all laboratories, although not equal, showed good agreement among the laboratories. In fact, all five laboratories obtained positive results with MH and MNU, while four of the five laboratories achieved positive results with NaN₃. AG was tested in only three laboratories. Two reported negative results, while one reported positive results but only at a single high dose. The data from this study suggest that under normal conditions, the Trad-MCN bioassay is an efficient and reliable short-term bioassay for clastogens. It is suitable for the rapid screening of chemicals, and also is specially qualified for in situ monitoring of ambient pollutants.     

Evaluation of a high throughput toxicity biosensor and comparison with a Daphnia magna bioassay

A high throughput toxicity biosensor has been designed and constructed using recombinant Escherichia coli cells, containing stress specific promoters (recA, fabA, or katG) or constitutive promoters (lac) fused to luciferase genes originating from Vibrio fisheri. These genetically engineered cells were immobilized in 96 well plates. By optimizing cell immobilization conditions and the strains' response specificity to toxic chemicals, bioluminescent outputs decreased or increased dose-dependently upon adding test chemicals. However, to date the toxicity data obtained using this biosensor have not been compared with the results of other toxicity tests. Phenolics were chosen to evaluate the correlation between the LD50 and the EC50 (GC2) or EC120 (DPD2540) of Daphnia magna and E. coli, respectively. Toxicity data obtained from constitutive strains by bioluminescent level decrements were compared with the results from D. magna as a standard. LD50 values were used as parameters of D. magna toxicity and EC50 of EC120 values were used for the immobilized biosensor. In the DPD2540 test, phenolics, membrane damaging toxic chemicals, for testing immobilized stress specific bacterial strains trigger dose-dependant bioluminescence increase within specific concentration. Although the stress specific responsiveness from the strains could not be compared with D. magna's LD50 values, these responses offer additional information, such as upon the mode of toxic action in the sample, in addition to the cellular toxicity results as indicated by the EC50. This novel high throughput toxicity biosensor can be implemented to investigate the toxicity of any other soluble materials, and can be used as a standardization tool for the evaluation of toxicity.