automated mechanical stimulation and measurement of bioluminescence in marine dinoflagellates

Photrirhemisrq and Pho/obdrig.v. Vol. 61. No. 6. pp 6 2 7 4 3 1 , 1995 Printed in the United States. All rights reserved

003 I -X655/95 $5 0 + 0 00 C 1995 Amerncan Striety f'nr Photobmlogy

TECHNICAL NOTE

AUTOMATED MECHANICAL STIMULATION AND MEASUREMENT OF BIOLUMINESCENCE IN MARINE DINOFLAGELLATES

HANS-CHRISTIAN STOLZENBERG*, GUNTHER KALNOWSKI and WOLFGANG D0n-7 Fachgebiet Hygiene, Fachhereich 6, Technische Universitiil Berlin, Amrurner Str. 32, D- 13353 Berlin. Germany

(Received 17 November 1994; accepted 7 Februuy 1995)

Abstract-A new integrated system for reproducible, automated mechanical stimulation and measurement of bioluminescence (BL) in multiple samples of marine dinoflagellate cell suspensions is described. The system was designed to allow the application of standardized experimental routines to parallel test vials for the purpose of toxicity testing. A sample tray delivered test vials to the position of mechanical stimulation and BL measurement. Mechanical stimulation of BL was applied as sharp rotation-onset of the test vial about its vertical axis. Thus, any direct chemical or physical perturbation of the cell SUS- pension was avoided. A silicon photovoltaic cell measured the emitted light. Stimulation. measurement and recording of BL were integrated and controlled by specially developed software, which runs on a personal computer in the graphic environment of MS-Windows. Precise scheduling, flexible programming and identical repetition of experimental routines are possible in practice. For G'fmyUUkJX po!\'edrc1, details of BL, as stimulated and measured with the new system, are presented and discussed. We conclude that the system exhibits specific features that offer wide potential of application in several fields of research on dinoflagellate BL, particularly for toxicity testing.

INTRODUCTION

Unicellular marine dinoflagellates represent an important. sometimes predominant part of photosynthetically active plankton. Several well-studied species, for example Gonvau- lux polyedru. some Pyrocystis species and members of other genera, exhibit the striking phenomenon of bluish bioluminescence (BL,Q maximum emission at 475 nm). The most obvious expression of dinoflagellate BL are bright flashes of some 100 ms duration. These flashes originate from subcellular structures called scintillons and are naturally caused by gradients of shear forces at the cell surface. Key biochemical components are luciferin, luciferin-binding protein and luciferase. Comprehensive reviews on the details of the bio- chemistry and subcellular structures involved are available.',' In addition to stimulated flashes, cells emit spontaneous flashes (averaging about one flash per cell and day') and a continuous low-level luminescent glow. which displays an intensity maximum for a few hours each day. prob- ably due t o the breakdown of scintillons.'

The light emitted by dinoflagellates is a well-proven tool for studying various aspects of their physiology: light can be measured noninvasively. nondestructively, sensitively and precisely with minimal response time, the detectors being linear over a broad range.

Until now, research has focused on the biochemical. phys-

iological and genetic basis of dinoflagellate BL. Of special interest are daily rhythmic changes of BL, which are due t o changes of concentrations and activities of the components involved.' cI In early studies. the circadian course of BL was monitored by measurements of mechanically stimulated BL.' lo Later work on the rhythmic changes concentrated o n measurements of spontaneous BL and aimed at the expla- nation of the molecular mechanism\ of cellular clocks. In this respect, the unicellular dinoflagellates serve as promi\- ing models.",/'

Luminescent flashes can be triggered chemically.'' elec- trically." thermicallyl' and photochemically.'0 Mechanical stimulation is the most common stimulus in nature.'- For experimental application of mechanical stimulation numer- ous procedures are described. Depending on the aim of the research. the degree of technical expense is very ditferent. Simple "sharp raps on the phototube housing" have been applied by Colepicolo rt ol. l x and Hasting\.' lechniques commonly described are stirring the cell suspension with paper clips"'~'y or bubbling with air.: / " / y 2 " In other \tudies the cell suspension is pumped through capillaries," ?-'

splashed as a water jet or stirred with an impeller.'' Beside\. Anderson et ul.2/ submitted the cell suspension4 to pressure changes or rotating bodies. Most sophisticated are device\ for precise stimulation 01' large single cells. for example ot Noctilircri or Pyroc r;,y, immobiliyed with pipettes" ?'" or bent fluoroplastlc tube\.'" 27 Stimulation is by an electrical pulse." by a piezoelectric o r by a pulse triggered \olenoid.'h 2'

In order t o facilitate systematic and reproducible mea- \urernent\ of BL in a greater number of vessels, Ha\tings and co-u orkers developed automatcd experimental \y \ -

627

628 HANS-CHRISTIAN STOLZENLIERG er al.

tems,28r29 which are able to measure and record spontaneous BL over periods up to several weeks, distinguishing between luminescent flashing and glowing.

Our aim was to evaluate mechanically stimulated BL as an endpoint in short-term experiments within the scope of ecotoxicological studies, testing the effects of toxic sub- stances on dinoflagellate BL and growth. Luminescent flashes are easily detectable because of their brightness. As a working hypothesis, we consider mechanically stimulated BL to be a significant indicator of cell vitality, because luminescent flashes are the result of several metabolic activities: biosynthesis of luciferin, luciferase and luciferin-binding protein, and energy-supplying reactions necessary to maintain a membrane potential.

Experimental design in toxicity testing requires identical treatment of a series of aliquot test samples to which grad- uated concentrations of potentially toxic substancdsample are added. The requirements for designing a statistically co- herent and reproducible toxicity test based on dinoflagellate BL are fulfilled with the system described here. The system allows automated and reproducible mechanical stimulation of BL in multiple samples of cell suspensions with standardizable experimental routines. It is also flexible, being open to developmental modifications of testing procedures, with- out large-scale alterations of the hardware. Data acquisition and storage provide the possibility for detailed further anal- yses of data plots.

MATERIALS AND METHODS

Culrures. Unialgal but not axenic batch cultures of G. polyedra, Pyrocystis lunula (strains provided by R. Hardeland, Gottingen), Pyrocysris acuta. and Pyrocysris nocriluca (strains BAH ME 9, 10, provided by M. Elbrachter, List/Sylt) were grown in light incubators at 20-21°C. The growth medium was prepared with seawater (North Sea off Helgoland), enriched with 21.5 mUL of additional nutrient solutions: 10 m U L NaNOl (stock solution 88 mM), 10 mL/L NaH,P0.,.H20 (stock solution 3.63 mM), 0.5 m u trace metal solution (concentrations as referred to as f/2 by McLachlan,-i'l original formula of f-medium by Guillard and Ryther-") and 1 mL/L vitamin mix.'* The medium was membrane-filtered (0.22 km, Sterivex-GS, Millipore. Bedford, MA, USA). Culture volumes were 100 mL in 250 mL flasks, 50 mL in 1 0 0 mL flasks and 10 mL in cylindrical vials (25 mm diameter, 60 mm height). The cultures were grown in 12 h:12 h 1ight:dark (L:D) cycles with white fluorescent lamps (spectral quality Lumilux I I and Universal White 25, Osram, Ger- many), either from two opposite sides or from above. The irradiance of culture vessels depended on their position in the incubator; the overall range was 80-170 pWm2 s, measured by a photometer (PRC Krochmann, Berlin, Germany). The G. polyedra cells, which were used in most experiments, were subcultured every 14 days by di- luting the suspensions I : 10. The Pyrocvsris species have comparatively large cells (hundreds of pm in at least one dimension); they are nonmotile and surrounded by a comparatively thick continuous wall and thus less suitable for counting and sizing.

Handling of G. polyedra cell suspensions. For experiments, cell suspensions were derived from standardized maintenance. Cultures were grown for 2-12 days; i n several cases the cell suspensions were diluted with medium or seawater. In suspensions for BL rneasure- ments. the range of cell numbers was 103-104 cells/mL. Cell numbers and distributions of cell sizes were determined with an elec- tronic particle analyzing system (CASY 1. Schiirfe System, Reut- lingen, Germany). Pipetting of cell suspensions and addition of RO-

lutions were done during photophase. when luminescent flashing is photoinhibited.'Y For all experiments described here, the cell suspensions were transferred to darkness at circadian time (CT) I2:OO.

System hurclwure. The complete system for stimulation and detection was housed in a benchtop incubator, composed of a universal

1

Figure 1. Representation of the sample changing and stimulating system. (a) Mechanical components: removable sample tray (360 mm diameter) supplied with 20 units of test vial and basal plate; the vial elevating and gripping device is shown on the right below the tray; diagram supplied with the kind permission of H. Kunst (Tech- nomed GmbH, Berlin). (b) Organization of the complete system: I , incubation chamber; 2, sample tray; 3, unit of test vial and basal plate; 4, gripping device; 5 , microcontroller unit; 6, photocell; 7. picoammeter; M, motor; PC, personal computer; arrows indicate motor-driven movements. (c) Scheme of software organization (see text for details).

incubation unit (Certomat HK, Braun Melsungen, Germany; temperature set to 21°C. accuracy + 0.2"C. air circulation 80 m'h) and an aluminum container with about 1 0 0 L volume, painted black inside and carefully shielded against ambient light.

Mechunical stirnutarion and sample chunging. Cylindrical glass vials. 25 mm in diameter and 60 mm high, usually supplied with 10 mL of cell suspension and closed with foam stoppers, were used as standard vessels for measurements of BL. Figure 1 gives a schematic representation of design, hard- and software components employed for the stimulating and sample changing system. The vials were fixed with the bottom to basal aluminum plates by a special foil ("sticky stuff," lnfors GmbH. Einsbach, Germany), that allowed frequent firm attachment as well as easy removal of the vials. Up to 20 units of attached vials could be placed on a sample tray ( 3 6 0 mm diameter. made of light metal). each unit exactly recessed in an appropriate hole of the tray. Completely supplied with vials, the sample tray could be handled as a single unit. Mounted on the driv- ing axle of a stepper motor, the revolving sample tray could deliver any vial to the position of stimulation and measurement. Mechanical

Technical Note 629

stimulation was (repeatedly) applied by short-term rotation of the vial about its vertical axis at 500 rpm (=52.4 radians X s I angular velocity) by a D.C. motor, thus avoiding any direct physical contact to the suspension. A rigid linkage between stimulation drive and the vial selected was provided &via the attached basal plate by a mechanical gripping device, which was driven with an elevating stepper motor. For stimulation the vial in position was elevated above the tray by the gripper, which simultaneously linked to the vial basal plate and enabled transmission of the stimulating rotation to the vial. The actual stimulation was a sequence of (one or more) pause- rotation cycles. After stimulation the gripper was lowered back to its starting position, thus leaving the vial in its position on the tray.

Light detection and darn acquisition. Bioluminescence was measured with a highly sensitive photocell (silicon photovoltaic cell, model P30 OOO, Lichtmeatechnik Berlin, Germany; window 17.5 mm in diameter. sensitivity 331 n N l x for standard illuminant A). mounted in front of the rotating vial. An aperture shielded 17 mm distance between the window of the photocell and the vial in position. All data reported in this paper were ohtained using an identical measuring geometry and are therefore directly comparable. The current output of the photocell was measured with a high performance ammeter (LichtmeStechnik Berlin. Germany; measuring range I X 10 ‘3-0.5 X 10 measurementds, integration constant 4 ms). Data were transferred to the system controlling PC I J ; ~ an IEEE488 (IEC625) interface bus.

Controlling softucire. Special software was developed in TurhoPascal as programming language in order to integrate data acquisition, ammeter control as well as control and coordination of the three motors involved ( ~ j Fig. Ic). The automated running process of an experiment was controlled with run tiles, written as plain text sequences of keyword commands. A range of parameters could be set for an experiment and each single measurement, respectively: order and selection of vial number. selection of ammeter range and time constant, rate of data transfer to the controlling PC (maximum rate one value every 5 ms, adjustable in ms-steps). starting time of a measurement, durations of pause and rotation (adjustable in ms- steps). number of pause-rotation cycles, determination of sample detinition and data files. Each measurement was followed by Storage of all respective settings in a protocol file. Loading the data files into any commercially available spreadsheet software, the effective use Of programming allowed a high degree of automation in standardized processing. analysis and graphical presentation of the data stored.

A, conversion rate of the An, converter

RESULTS

Busic elements of N BL plot

Figure 2 displays the basic elements of a BL plot repre- sentative for measurements done between C T 14 and C T 20. Five vials supplied with aliquot samples of the same cell suspension were measured at CT 14:30, CT 1S:30 and CT 16:30. with the same routine of stimulation and measurement at each time. After onset of vial rotation. the subsequent fast increase of photocurrent occurred after 30 ms delay. and the maximum photocurrent was detected after a further 50 ms. This burst of BL actually represents the indiscernible sum of all individual luminescent Hashes emitted from the single cells of the suspension. In Fig. 2 , the burst emitted upon the vial’s first rotation displayed a rise-time (10-90%) of 30 ms with a half maximum width of 80 ms and a decay time (9(k 10%) of 9s ms. Only small amounts of BL, <2 .S% of the preceding first burst. were detected upon rotation offwt, upon on\et of the \econd rotation period or even if the v ~ a l wa\ wbniitted t o further pause-rotation cycles iminediately following ( n o t \hewn in Fig. 2).

The mea\urements presented in Fig. 2 were made with a resolution of one value every 5 ms. In this case the curve peak was represented by four consecutive value5 >YS% of

- bioluminescence

rotation periods -

0 0 095 1 195

time [s] Figure 2. Plot of original BL measurements; mean of luminescent emissions at CT 14:30, CT 15:30 and CT l6:30. at each time for the same tive test vials; each vial was supplied with a 10 mL aliquot of cell suspension (4 day grown batch culture of G. polyedrcr. diluted t o 1460 cellslmL, mean cell diameter 33.55 bm); rate of data transfer = 200 values per s (maximum of the system); the horizontal bars relate to the x-axis only and indicate the rotation periods o f the stimulation drive as recorded according to the photocurrent values.

the absolute maximum value. Thus, a resolution of one value every 20 ms was chosen for routine measurements with con- sequently less storage capacity required. The main parameters for further analysis of routine measurements were peak maximum [nA] and curve area [nAs].

Reproducibilitv of stimulation arid r,J’ stiniulated BL

In all experiments, which were done with a wide range of parameter settings, the technical process of stimulation and measurement was highly reproducible, the starting times of data acquisition and of stimulating rotation being precise at the ms level.

lntensity of BL in aliquot samples was more variable than cell counts and cell sizes, BL peaks showing higher variability than BL curve areas. Triplicate measurements in 24 suspensions of G. polyedrti from different cultures gave coefficients of variation (CV) for peak heights from 1.4 to

23.4% (mean 8.7%) and for curve areas from 1.3 t o 13.W- (mean 5.9%). In the same suspensions. numbers and sizes of cells were distributed with C V from 0.7 to 7.9% (mean 4.1 %). This also applied to the tive aliquot samples represented in Fig. 2. Here the mean BL intensities (peak and area) were nearly constant (CV <2%). when measured in I h intervals at CT 13:30. 15:30 and 16:30. In other experiments it was found. that i n the middle of scotophase (CT 14 t o CT 30) a recovery time interval of about 2 0 rim between subsequent stimulations was sufficient to restore the capacity for bursts identical in size and \hape.

Variability of burst kinetics alway\ displayed a sharp min- imum coinciding with the inauiiiiuiii of luminewmt eniis- \ion 80-85 ms after rotation onset (Fig. 3). Even more con- \tan1 wa\ the 30 ms delay troin onwt ot’ vial rotation t o the t int increased photocurrent value. whereas the pattern\ of further burst decay below 40% of the curve inaxiinuni were niost variable, e\peci;illq anionp different cell \u\peiisions.

630 HANS-CHRISTIAN STOLZENBERC et d.

100

80

60

40

20

0

coefficients of variation 1 ~ . ~~~

100 200 300 400 (msl

Figure 3. Kinetics of mechanically stimulated BL; data of Fig. 2, normalized; drawn is the section of the plot between rotation onset ( I(x) ms) and decay of photocurrent values to 5% of maximum intensity; CV are given for normalized values >S%.

DISCUSSION

Mode of stirnulation and luminescent response

Details of mechanical stimulation of G. polyedra BL by shear, acceleration and pressure were carefully investigated by Anderson et uL2’ Of the several stimulating configura- tions tested in their study, an ellipsoid rotating with angular velocities of 24, 40, 56 and 120 radians X s I in a cell suspension compares best with the situation in the rotating vial of our system. As Anderson and co-workers observed by image intensification, the emission of BL is restricted to regions of sharp shear gradients at the surface of the rotating body. Bioluminescence is most intense when the rotation is initiated, and the crucial component of the stimulus is its change. Applied to the principle of stimulation in our system, these findings point to the importance of sharp starts and stops of the stimulation drive. The inner surfaces of the test vial are expected to be the main sites of BL emission. The actual rpm-value should be less important, as was observed with manually lowered velocities of the drive (data not shown). The patterns o f flow in the test vial were highly complex. but with KMnO, crystals it was obvious that the suspension was completely mixed not later than during the second period of rotation; sharp shear gradients occurred at the vial surfaces every time the rotation stopped or started. This suggests that nearly all cells that were able to flash did 40 during the first rotation period, presumably just once, because scarcely any further BL was detected upon additional rotation peritds (cj Fig. 2).

Kinetic4 of BL burst\ in our measurements (Fig. 3 ) resem- ble those o f previously reported measurements in G. p o l y d - ru suspensions, wJhich were stimulated by tapping’ or stirring.’” For cell w\pen\ions. our experimental design allowed for the tint time the preciw time allocation o f stimulation onset t o the suhsequent luminescent emis\ion. However. at the present \tate o f our 4tudies it is not clear t o what extent the cellular reaction time, the time o f physical transmission o f the \timulu\ to the \u\pension and software processing delays (at lea\t 15-20 m\) contribute to the 30 ms delay recorded.

Krasnow et a1.j reported results from extensive measurements of spontaneous G. polyedra BL. Half maximum widths of spontaneous flashes of single cells differ from 10 to 100 ms, about 27 ms being the most frequent value. Re- garding the half maximum width of about 80 ms for a suspension’s burst of flashes in our experimental configuration, and assuming the same kinetics for stimulated as for spontaneous cell flashes, we conclude that it takes only an order of 100 ms for at least 95% of the cells to get a stimulus sufficient for a flash.

Variability of suspension bursts exceeded the variability of cell distribution in aliquot samples as determined by particle analysis. This confirms the presence of differences in luminescent behavior among single cells as previously stat- ed:’ Effects of scattering (“inner filter effect”-’-7) and flow patterns within the rotating vial need further investigation and are beyond the scope of this study.

Mechanically stimulated BL as a purameter measured

We favored the mechanically stimulated form of BL for measurements. Compared to the low intensities of spontaneous BL, the ease of measuring bright bursts of flashes from mechanically stimulated cell suspensions is obvious. Mechanical application of shear forces has the advantage of representing a natural stimulus. Moreover, the linkage of stimulated BL to cell vitality is more clearly defined than of spontaneous BL, which comprises the glow and spontaneous flashes, the latter’s cause remaining unclear..’ Both forms of spontaneous BL, flashes and glow, are not strictly parallel, so that in experiments using this parameter data analysis was necessary to achieve a reliable distinction between flashes and glow.-’.2x

Because results of toxicity tests depend to a high degree on the test organism selected, on the endpoint measured and on the sum of test conditions, the validation of dinoflagellate BL data for ecotoxicological assessment of aqueous samples needs further study. Our findings point at the importance of establishing defined test conditions, e.g. CT of measurements. exposure time or BL parameter(s) analyzed. Presently we are using such standardized testing procedures to evaluate the impact of several groups of chemicals on dinoflagellate BL.

Asse.c.cment of general system features

The new system exhibited several features that proved to be valuable for several kinds of experimental procedures. The test vessels are completely independent of each other. as n o invasive application of stirring or bubbling devices. neither pumping as in flow systems were required. Precise scheduling and reproducibility of the measuring and stimulating process allowed exact analysis o f time courses and chronological relations in stimulus-burst sequences and allowed access t o identical repetitions for statistical valida- tions. Nearly all experimental system settings were per- formed o n the \oftware level. This permitted very flexible determination of experimental schedules, which could be stored and recalled as standardiLed routines. The data measured and recorded could also be processed in a flexible and standardizable manner. Routine experiments were easy t o carry o u t due t o the software operating i n a common \tan-

Technical Note 63 I

dard environment. Besides other potential uses, the devel- opment of standardized automated routines for toxicity tests based on bioluminescence is the most immediate application of our system.

AcknoH.ledgement,s-This work was supported by the Deutsche For- schungsgemeinschaft (grant Ka 863/1). We thank R. Hardeland and M. Elbrachter for kindly supplying cultures of dinoflagellates. H. Kunst and J. Paulick (Technomed GmbH, Berlin) designed and as- sembled the mechanics of the sample changing and stimulating system with great skill. N. Schmidt (1ng.-Bur0 N. Schmidt, Berlin) did the programming and upgraded it with many worthwhile prudentials. G . Geutler, H. Pieper, G . Gottscholl and G. Kupler provided essen- tial technical support concerning light in black boxes. We are greatly indebted to M. Rayner for his valuable critical comments On the manuscript.

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automated mechanical stimulation and measurement of bioluminescence in marine dinoflagellates

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