Evaluation of the Effects of Extremely Low Frequency Electromagnetic Fields on Movement in the Marine Diatom Amphora coffeaeformis

30 Pages·1998·11 MB·English

by  M S Davies

Reference: Biol. Bull. 194: 194-223. (April. 1998)
Evaluation of the Effects of
Extremely Low Frequency Electromagnetic Fields on
Movement in the Marine Diatom Amphora coffeaeformis
MARK S. DAVIES' -'*, RICHARD DIXEY'. AND J. C. GREEN-
^ Department ofMedical Electronics. St. Bartholomew's Hospital. West SmithfieUl. London. ECIA
7BE. UK; -Plymouth Marine Laboratoiy. Citadel Hill. Plymouth. PLl 2PB. UK: and 'Ecology
Centre. University ofSunderland. Sunderland. SRI 3SD. UK
Abstract. Published work has shown that population 100 kHz) electromagnetic fields (ELF EMFs) on biologi-
motility in the marine diatom Amphora coffeaeformis can cal systems, this branch of science is neither well known
be influenced by externally applied electromagnetic fields nor understood. The reasons for this are probably three-
(EMFs). Here we report attempts to repeat these experi- fold. Firstly, there is no well-accepted mechanism, based
ments, which have been proposed as a model forassessing on empirical studies, by which EMFs can influence and
the effects ofEMFs on biological systems. Susceptibility modify a biological or biochemical process. Any interac-
to EMFs was tested using five strains ofdiatoms on agar tion mechanism must describe how fields with energy
plates at a very broad range of field conditions, hut no levels less than that of thermal noise (A>) can exert an
effecton population motility was demonstrated. Exposure effect (Male. 1992). Secondly, experimentation paying
period to the EMFs, cell density, and position in the cell proper attention to both biological and physical variables
cycle had no effect on EMF susceptibility, and the direc- is necessary, but is not common. Thirdly, there is no
tion and distance moved by the diatoms were not affected '"tried-and-tested"" model system that can be used to test
by EMFs. When tested after at least a month of pre- new developments in bioelectromagnetic science. The ac-
incubation at 20 /iT. diatoms of strains #2038. 111b. and ceptance of empirical evidence is usually determined by
lllp did show an EMF-induced increase in population mo- the ability of others to replicate research findings, and
tility over control cells (up to ~207<-) at conditions pre- investigation to this end has yet to produce a robust sys-
dicted by the "ion cyclotron resonance"" model, but this tem, independently replicable in different laboratories.
effect was ephemeral. Later, 111b showed a similar in- Here we report attempts to replicate the findingsofexperi-
ccerlelassewetrheatusweads, a(2b)oltihseheEdMFw-hpernod(u1c)inngon-cpoirles-iwnecruebatneodt muleanttisondsesocfritbhiengmEaMrFi-niendduiacteodminAcmrpeahsoerdamotciolfifteyaeifnoprompi-s
energized, and (3) even harmonics were used. On observ- Agardh (McLeod et al.. 1987a; Smith et al. 1987a. b).
ing the response of diatoms to EMFs in real time, a sig- Amphora coffeaeformis is a common benthic marine
nificant increase (~2-fold) in diatom speed over control pennate diatom (Round et ai. 1990) whose motility is
cells was evident at "ion cyclotron resonance"" condi- well documented (Cooksey and Cooksey, 1980; Round
tions, using strain #2038 (pre-incubated at 20 /jT). The et al.. 1990). Motility can be observed by light micros-
effect was abolished at an even harmonic. We conclude copy in real time and is easily measured on agar plates
that EMFs can modulate diatom motility, but that the because the diatoms leave visible trails of mucus. The
system is, as yet. not consistently reproducible. mucus is produced via a channel, or raphe, running along
the long axis of the diatom (Edgar, 1980). In A. coffeae-
Introduction formis the raphe is restricted to one side of the diatom
Despite a vast literature describing the effects ofexter- only. Diatoms have many advantages as biological mod-
nally applied, non-ionizing, extremely low frequency (< els: they are easy to maintain in culture; have a short
generation time (<1 day. Round et al.. 1990); are single-
celled and eukaryotic; and commonly reproduce asexu-
R*eTcoeiwvehdom24cJournreesp19o9n7d;enaccecesphtoeudld5hDeecdiermebcteerd.1E9-9m7a.il: mark.davies ally. giving a large population ofcloned individuals. Mo-
@sunderland.ac.uk tility in A. coffeaeformis depends on external [Ca] (Cook-
194
RESPONSE OF DIATOMS TO EMFS 195
sey and Cooksey. 1980), and this property was exploited et al. (1987a, b) our methods follow theirs as closely as
by McLeod et ul. (1987a) and Smith et al. (1987a. b) possible.
who demonstrated that populations of A. cojfeaeformis
on agar containing a [Ca| that normally checked motility Diatoms
could be induced to move when exposed to specific ELF
EMFs. Amongst the many studies of bioelectromagnetic Five strains of Amphora cojfeaeformis were used.
effects, these were important because they used a biologi- Mixed cell-size cultures of strains #2036, #2038. and
cal system that was potentially easy to re-create in other #2039 wereobtainedfromtheCulture Collection ofAlgae
A
laboratories, thus allowing replication studies, and they at the University of Texas (Starr and Zeikus, 1987).
demonstrated that, in common with most bioelectromag- mixed cell-size culture of strain Illg (referred to here as
neticeffects, adose-dependentresponse wasnotapparent; IIIp) was obtained from the University of Southern Cali-
ratherthe effect was maximal around specific field ampli- fornia. A narrow size-range (21-23 /jm along the long
tudes and frequencies, the latteras predicted by atheoreti- axis) culture of strain 111b was obtained from B. Cooksey
cal model ("ion cyclotron resonance"; Liboff, 1985). In at Montana State University. These latter two strains are
effect, McLeod et al. (1987a) and Smith et al. (1987a, b) claimed to be descendants of the strain referted to in the
putatively 'tuned' the applied fields to stimulate Ca'^ ion original publications (McLeod et al., 1987a; Smith et al..
activity. Thusthestudiesprovidedaclue tothe interaction 1987a, b) on diatom/EMF interaction (B. Cooksey, pers.
mechanism between living systems and EMFs. comm.).
"ion cyclotron resonance" theory can be used to pre- Although these strains are conspecific, there is consid-
dict the frequency of EMF that will stimulate any ion, erable variation in their gross moiphology (Fig. 1); they
dependent on its charge-to-mass ratio, according to the show differences in upper lethal temperature (Davies, un-
formula publ. data), and they produce differently shaped growth
mats in liquid culture (Davies, unpubl. data). Gallagher
f B, (1986 and pers. comm.) advocates a reexamination ofthe
taxon Amphora cojfeaeformis with a view to splitting it
where/is the stimulation frequency, q/m the charge-to- into numerous species. Thus, it is important that strain
mass ratio and B the static magnetic field (Liboff, 1985). types and their origin be specified in studies such as this
(see Wood and Leatham, 1992).
Extrapolating from the model, stimulation at even har-
monics should abolish the effect, and in terms of diatom Diatoms were grown in the ASP2 medium ofProvasoli
aImrtonetdai"uldicidoteinytd,ioctdnyhi,cialsMtowotcamrLosenmwoorhtdiealsteitotwnyaala,.sncw(feh1o"i9uc8nc7hdobn)(adgMifatcoiiuLnonendcosedtnfhtoaerettrastelth.d.eimo1uKn9l*8a7tpbiir)noe.ng- earwlte/..nvael.1S9ti(8u1g7b9mae5)sa7.)a',tRAe'ms1o5ead°rgiCvaf.reieDisdnitaotmctookodscmiosfoniftefadodirinaAet0Sx.oPp2me25srirwsmmleeaWrnnettCsakalienp(utSpsmoeolinytwshe2try%ee-t
grown either (a) in an incubator at 20°C at 3000 lux
dmiocdCtleeeldarfalinyedldstahuemgpgle"is"ttdueiddaetsaonmainnsdtyesfrrtaeecqtmui"eonncpimreeesc.sheanntiedsm.a Ntoetstasbulre- o(n~5a8.128-^hEsligh' tm-da-r)kiclylculmeinawthieorneftrhoemhofrliuzoornetsaclentcolmipghot-s
nentofthe magnetic field, Bh = 4.7 /l/T; the vertical com-
prisingly, several groups then attempted to replicate the ponent. Bv = 14 /iT; and the oscillating fields were negli-
original findings (Reese etai. 1991; Parkinson and Sulik.
gible; or(b) in awaterbath at25°C at 3000lux continuous
1992; Saalman et al.. 1992; Prasad et al.. 1994; Florig, fluorescent illumination (Bh = 23 ^T, By = 20 pT\ when
pers. comm.) and. with one exception, all failed. Reese
eptopaul.la(t1i9o9n1m)otrielpiotrytewdasa pianrctrieaalseredploincaEtiMoFn isntitmhuatladtiioantoimn a=th13erpmoTstpaetiacka-lpleyakcoanttr5o0llHezd,hBeva.t,.er=wa3s/joTpepreaatki-ngp,eaBkh^act
mM 50 Hz). Cultureswereperiodically (aboutevery4months)
some, but not all, experiments (15/19 at 0.25 Ca).
treated with an antibiotic solution (see Stein. 1973) to
Here we report the most thorough attempt at repeating
maintain axenicity.
tahweiodreigriananlgefionfdibnogtshobfiotlhoegidciaaltoamndsEysMtFemctoonddiattieo,nst.estOiunrg polAyssteycroenned winastuelratbiaotnhwcaosnstursuecdtefdorofEaMcrFylpircep-liansctuibcawtiitohn
aim is to assess the repeatability of the original experi- experiments and was enclosed by near-Helmholtz coils
ments, implicating apotential interaction mechanism, and
toexamine the usefulness ofthe diatom system asa model (see Exposureapparatus). Waterwas pumped to this bath
for bioelectromagnetic phenomena in general. bwya.tearndbartehcisrictueldate1d.5tmo, afrLoamudath-ethaecrrmyolsitcatbatrhe.cirDciualattoimnsg
Materials and Methods wilelruemignratoiwonnhferroematD2C5°lCamapts1f0o0r0altulxea(s~t 119.m6onptEhspr'imor t"o)
Because our primary aim was to investigate the repeat- experimentation.
ability ofthe results of McLeod et al. ( 1987a) and Smith Aseptic techniques were practiced throughout.
196 M. S. DAVIES ET AL
Figure 1. Strains #2036. #2038. #2039. IIIb and Illf of Amphora coffeaeformis. Left, views of the
raphed surface; right, lateral views. Scale bar is 1 ^m in each case.
Exposure apparatus near-Helmholtz configuration. One of the pairs (260-mm
separation) had its magnetic axis along the c-axis and
Two pairs of coils (each 295-mm internal diameter) controlled the vertical (static) component (Bv) of the
were mounted in a PVC frame at 90° to each other in a EMF within the coils. This pair was supplied with current
RESPONSE OF DIATOMS TO EMFS 197
from a Kingshill 50V2C constant-voltage supply. The
second pair (135-mm separation) had its magnetic axis
along the .v-axis (north-south) and controlled the hori-
zontal (both static. Bh. and alternating, Bh^c) components
of the EMF within the coils. This pair was driven by a
Farnell stabilizedpowersupplyE30/2coupledtoaFamell
synthesized digital signal generator DSG2 whose outputs
were fedthrough a Hewlett-Packard 6826A bipolarpower
supply/amplifier acting as a variable gain amplifier. The
output of this latter device was used to energize the coil
pair. The EMF in the y-axis (east-west) was brought to
zero by aligning the .v-axis along the earth's north-south
magneticmaximum. Thus wecouldcontrol themagnitude
04
of the vertical (static) component (By), the magnitude of Logn+1 [Ca](mM)
the horizontal static component (B„). and the magnitude
and frequency of the sinusoidally oscillating component Figure2. Dialommotility responsetoexternal |Ca|onDifcoNoble
EMF agar. Ineachcasen = 3 agarplateswith KKldiatotiiscountedperplate.
(Bh.c) of the within the coils.
The EMFs generated were measured at the center of
the coils using a Domain SAM3 single-axis fluxgate mag-
netometer, whose analog display was used to record the though McLeod, Smith, and K. Cooksey (pers. comms.)
static field components. This magnetometer was coupled maintain that Difco Noble agarwas used, ourexperiments
to an Iso-tech ISR420 oscilloscope that was used tocheck with this agar demonstrated that its Ca content was too
the magnitude and frequency ofthe oscillating field com- high to produce the prerequisite Ca-limiting effect on
ponents. motility (Fig. 2). Figure 2 also shows aCa-curve prepared
Experimental samples were placed at the center of the using Difco Noble agar from the sarne container as was
coils on a platform that could be raised or lowered, de- claimed to be used in the original experiments (McLeod,
pending on the nuiTiber of samples introduced. The coils Smith, and K. Cooksey, pers. comms.). This latter curve
were placed on a table at least 1 ni away from any metal was produced by one of us (MD) on a study visit to
object.Thegeomagnetic fieldcomponents withinthecoils Montana State University. Subsequent spectrophotomet-
were identical to those at the site chosen for the control ric analysis showed Difco Noble agar (UK batch) to con-
(sham) exposure 2 m away (Bh = 16.5 ^l^, Bv = 44 /jT). tain ~62 niM Ca. For routine work we have therefore
mM
Lighting over both coil and sham sites was provided by used Sigma 'A' agar (~6 Ca) which gives Ca-curves
DC lamps producing a flux density of 500 lux consistent with those ofMcLeod etal. (1987a) and Smith
(—9.8 f/E s ' m -) at sample level. Experimental (room) et al. (1987a, b).
temperature was 23 ± TC. The laboratory chosen for Agar (Sigma 'A') was introduced at 2% (w/v) to vari-
experimental work (at the Plymouth Marine Laboratory, ous maintenance media (the minimal medium [M-M] of
Citadel Hill, Plymouth) was electromagnetically quiet: no Smith et al. [1987a|) each containing 308 mA/ NaCl,
EMF interference could be detected at nT levels. 8.1 mM KCl, 20.2 mM MgSOj. 8.3 mM Tris-HCl and
A second set of two coil pairs (each 300-mm internal adjusted to pH 7.6-7.7. but varying in theirCa (as CaCL)
diameter) was employed in EMF pre-incubation of cells content (0-lOniM). After each mixture was autoclaved
and enclosed a water bath. The coils were mounted in a for 5 min at 12rC, agar plates were made by pipetting
wooden frame at 90° to each other in a near-Helmholtz 2 ml of the molten agar into each 50-mm-diameter poly-
configuration. The coils controlling the horizontal field styrene petri dish required. Thus a series of plates could
were separated by 150 mm; those controlling the vertical be prepared containing 0-10 mM added Ca. Plates were
field by 210 mm. The EMF in the y-axis was again wrapped in aluminum foil, stored at 4°C, and used 3-21
brought to zero utilizing the geomagnetic maximum. Both days after pouring. Plates used less than 3 days after
coil pairs were driven by a Famell E30-1BT dual power pouring gave poor diatom tracks.
supply such that the EMF in the center of the coils was Diatoms were harvested by centrifugation (1000 X g)
set to Bh = 20 ^T, By = /jT, negligible oscillating for 5 min at incubation temperature before being both
components. washed and recentrifuged twice in Ca-free maintenance
medium to ensure a Ca-free pellet of diatoms. The cells
were then adjusted, using a hemacytometer, to a density
Agarplate experiments
of 1.5 X 10' ml'. In later experiments, in accordance
There is some confusion over the type of agar used by with arevised protocol supplied from Montana State Uni-
McLeod et al. (1987a) and Smith et al. (1987a, b). Al- versity (McLeod, pers comm.), cells were then returned
198 M. S. DAVIES ET At.
to the incubator for 30 min prior to plating. This step were harvested when in theirmost motile phase ofgrowth
was introduced to the revised protocol putatively to allow asdeterminedfromtheresultsoftheage-dependentmotil-
diatom membranes ruptured during centrifugation to heal ity tests (3 days for #2036. #2038. and #2039. and 2 days
(Smith, pers. comm.). for lllp and IIIb) and introduced to three plates at each
McLeod etal. (1987a) and Smith etal. ( 1987a. b) used [Ca]. Motility was scored after 1 h in the incubator at
a paintbrush to introduce diatoms to the agar. The volume 20°C. Diatom cultures were grown in the water bath
of suspension applied in this way was not constant, and (25°C).
it was difficult not to disturb the agar surface. We substi- f/i'j Effect of light inten.sity. McLeod (pers. comm.)
tuted a technique in which a 5-^\ drop ofdensity-adjusted and Parkinson and Sulik (1992) found that light intensity
cell suspension was introduced to the agar on each plate during diatom exposure influenced the proportion ofdia-
at one end of a 30-mm line marked on the underside of toms showing motility. To test this we harvested a 6-day-
the plate. The drop was then drawn along the length of old incubator-grown#2038 culture and seeded itsdiatoms
the line with an inoculation loop whose tip had been onto 15 agar plates containing 5 mM added Ca. Plates,
squared to present a 3-mm bar to the agar surface. Care in triplicate, were placed in illumination at <0.625 (inside
was taken not to touch the loop to the agar. a drawer), 21.4, 500, 1190, and 2740 lux (<0.01, 0.4,
werAeftkeirlleedxpbeyriimnetnrtoadlucienxgpoasufreew,drdoipastoomfs2o%n(wt/hve)pOlsatOejs 9e.x8p,os2u3r.3e,,atnhedd5i3a.t7o^msEwse"'rem"k"i,llerdesapnecdtisvceolrye)d.fAofrtemrotiIlhityo,f
to the inside of the lid of each plate. This procedure fv) EMF experiments. In all cases we searched for
ensured that there was no additional time after the termi- EMF-induced motility using plates with 0.25 mM or
nation of plate exposure during which the diatoms could 0.5 mM added Ca. Diatoms in logarithmic growth were
move.
seeded onto six replicate plates for each experiment.
Diatom movement on each plate was scored by count- Three plates served as treatment (EMF-exposed) plates
ing 100 diatoms in random fields viewed with a phase- and three as control (sham-exposed) plates. Control and
contrast microscope. Diatoms were scored as motile or
treatment plates for each experiment were always run
nonmotile depending on the presence or absence of a
simultaneously and were seeded from the same diatom
track visible at either pole of the frustule. Only spatially
culture at its most motile phase ofgrowth (Parkinson and
individual diatoms were counted: diatoms that appeared
Sulik, 1992). Using diatoms from more than one culture
in clumps or whose track originated in a clump were not would have introduced confounding factors. A positive
scored. Ifthese latter diatoms had been scored as motile, control in the form of a plate with 5 mM added Ca was
then all clumped diatoms would need to be scored as
always placed with the sham-exposed plates to check that
nonmotile. Diatoms were also not scored if they could be
the diatoms were motile at high [Ca]. Each exposure
clearly seen to be lying with a lateral surface touching
the agar. These diatoms are incapable ofmovement (pers. lasted 1 h. To allow for geomagnetic disturbance, experi-
ments conducted on days when large fluctuations in the
obs.) because the raphe system is not in contact with the
earth's magnetic field occurred (data from Hartland
substratum.
Monthly Bulletin, British Geological Survey) were re-
(/) Growth cun'es. Growth curves were produced for
each strain of A. cojfeaeformis by introducing a 1.5 x peated atalaterdate. "Large fluctuations" was arbitrarily
10''-cell inoculum in modified ASP2 into a polystyrene defined as a variation greater than 30 nT in either hori-
tube and making the total volume up to 5 ml with modi- zontal or vertical intensity during any experimental pe-
fied ASP2. Growth, measured daily by hemacytometer riod.
(twochamberscounted) was monitoredfor8 days in three All strains were exposed to calcium "ion cyclotron
tubes for each diatom strain. Diatoms were incubated at resonance" conditions, which produced the greatest field
either 20°C in the incubator or 25°C in the water bath. effect for McLeod et al. ( 1987a) and Smith et al. (1987a,
(//) Age-dependent motility. To investigate the rela- b). Thesewere: Bv = iil. Bh = 20.9 /jT, Bwac = 41.8 ^T
tionship between age ofculture and diatom motility, cells peak-peak at 16 Hz.
of each strain were harvested (see abovel at 1-8 days Subsequently a set of permutations of field conditions
after inoculation (1.5 x 10'' cells in 5 ml) and streaked was developed that covered all possible avenues of ap-
onto three agar plates each containing 5 niM added Ca. proach in attempting to reproduce the results of Smith et
Motility on the plates was scored after 1 h in the incubator al. (1987a, b) and was designed to allow the detection of
at 20°C. Again diatoms were used from both incubator any ELF-EMFeffectondiatommotility. Theseconditions
(20°C) and water bath (25°C). were tested both with strain lIIp, which was claimed to
(///) Ca cun'es. The relationship between extracellu- be the same strain as used by Smith et al. (1987a), and
lar [Ca] and motility was investigated for all five strains strain #2038, with which a partial replication ofthe origi-
of diatom by preparing a series of agar plates containing nal results has been achieved (Reese et al.. 1991). Each
a range of [Ca] from 0-5 mM. Diatoms of each strain permutation was tested twice. In all experiments, except
RESPONSE OF DIATOMS TO EMFS 199
those in (7) below, the vertical component of the earth's motility as above, and the plate was returned to the expo-
field was nulled (Bv = 0). sure or sham-exposure site.
The following permutations were used (the amplitude {vii) Ejfectofcelldensity. To test whetherthe density
of Bhsc is expressed as peak-peak). of the diatom cells on the agar plates had an effect on
motility or susceptibility to EMFs (see Aarholt et al..
(1) Frequencyresponse. Bh = 20.9 ^T; Bh.„ = 41.8 /jT 1981; Carson et al.. 1990), a 6-day-old incubator-grown
at frequencies from 1 to 24 Hz in 1-Hz steps. This was #2038 culture was harvested and its cells were seeded
an attempt to demonstrate the frequency window for cell onto 5 plates with mM added Ca. Quadruplicate plates
motility shown by Smith et al. (1987a). were prepared at each cell density used (5 X 10"*, 1 X
(2) Amplitude response. Bh = 20.9 /jT; Bhuc varied 10\ 1.5 X 10\ 1 X 10^ 2 X lO"- cells ml '). Two ofeach
from 5 to 140 fxT (in 5-^T steps to 30 pT, 10-^T steps four were placed in Ca ion cyclotron resonance EMF
to 80 ;uT. and 20-/iT steps to 140 ^T) at 16 Hz. This was conditionsandtwowere sham-exposed. After hofexpo-
1
an attempt to demonstrate the amplitude window for cell sure, the diatoms were killed and scored for motility.
motility shown by Smith et al. (1987a). (viii) Ejfect ofposition in cell cycle. To test for EMF
(3) Amplitude response. Bh = (Bh.,J/2: Bh.,, varied susceptibility of diatoms at different stages of their cell
from 5 to 140 ^T (in 5-/iT steps to 30 ^T. 10-pT steps cycle, cells (IIIp) were acclimatized (2 weeks) to aregime
to 80 /xT, and 20-^T steps to 140 ^/T) at 16 Hz. of 12 h light/dark at 20°C in the incubator. Following
(4) Amplitude response. Bh = ambient: Bh^c varied this, on one day cells were inoculated in the usual manner
from 5 to 140 ^T (in 5-pT steps to 30 ^T, 10-/uT steps into polystyrene tubes at intervals of 2 h for a total of 16
to 80 luT. and 20-/uT steps to 140 ^T) at 16 Hz. h. Each culture was then simultaneously harvested 4 days
(5) Amplitude response. Bh = //T; Bh.„ varied from later when in logarithmic growth and seeded on to tripli-
5 to 140 /jT (in 5-/jT steps to 30 ^T. 10-^T steps to cate sham-exposed and triplicate Ca ion cyclotron reso-
80 fjJ, and 20-/uT steps to 140 fjT) at 16 Hz. nance-exposed plates with 0.5 mM added Ca plus a plate
mM
In (2). (3). (4), and (5). plates with 5 mM added Ca ewxiptohsu5re, thaedcdeeldlsCwaeraes sacoporseidtiivnetchoentursoula.lAwfatye.r 1 h of
were also used as treatment and control plates (again in (a) Ejfect of EMF on distances moved. During the
triplicate) in an attempt to reproduce the reductioEnMiFn frequency-response test above, the distances moved by
motility reported by Smith et al. (1987a) at high IIIp cells during the 1-h exposure period were recorded
amplitudes.
using a calibrated eyepiece graticule attached to the mi-
(6) Bh = (BHac)/2 = ambient at 16. 12.5, and 6.25 Hz. croscope. Thedistances moved by 10motile diatoms were
These latter frequencies are the predicted cyclotron reso- recorded on each triplicate sham-exmpoMsed and each tripli-
nance frequency and an even harmonic, respectively, of cate EMF-exposed plate (each 0.5 added Ca) in each
the unhydrated calcium ion in the ambient horizontal field experiment. The frequency of Bho, was varied from 1 to
in our laboratory. This follows from Blackman et al. 24 Hz in 1-Hz steps, Bh^c = 41.8 ^T, Bh = 20.9 /jT, By
=
t(h1e98a5m)biaenndtLfeiaelldetisali.mp(o1r9t8a6n)t, iwnhoinsdeucwionrgkasnuEggMesFteedfftechta.t (0..x) Effect ofEMF on direction ofmovement. The di-
(7) Switched field axes at ion cyclotron resonance con- rection of cell movement was also recorded during the
ditions. Bh = 0/jT; Bv = 20.9 /jT; Bv., = 41.8pT at frequency-response test using strain IIIp. An eyepiece
16 Hz. Switching field axes altered field quantities within graticule was divided into twelve 30° sectors. Each motile
the plates (see McLeod et al.. 1983). diatom scored was assigned to a sector depending on its
initial direction of movement (0° = north). In this way,
mM
(vi) Ejfectofe.xpo.siireperiod. McLeod et al. (1987a) 100 diatoms per triplicate plate with 0.5 added Ca
and Smith et al. (1987a, b) used an exposure period of 1 were scored in EMF-exposure and sham-exposure condi-
h. In our search for an EMF effect we tried extending tions at each frequency tested. Direction of movement
this period. We harvested a 6-day-old #2038 culture and was tested for nonrandomness using \- tests.
a 4-day-old 111^ culture (both grown in the incubator) f.v/J Effect of EMF pre-inciibation. After at least a
and seeded their diatoms onto six replicate plates with month of pre-incubation (subculturing every 4-5 days)
0.25 mM added Ca and one plate with 5 mM added Ca. in a water bath at Bh = 20 fjT (see E.xpositre apparatus),
mM
Three ofthe plates with 0.25 added Ca plus the plate 1-, 2-, 3-, 4- and 5-day-old cells of each strain of A.
with 5 mM added Ca were placed at the sham-exposure cojfeaeformis wereexposed at Ca ioncyclotron resonance
site; the remainder were placed at the EMF-exposure site EMF conditions. Exposures lasted for 1 h on triplicate
and subjected to Ca ion cyclotron resonance EMF condi- agar plates for each treatment. Treatment plates ranged
mM
tions. At 1, 2, 4, 6, 8, 12, 16, 24, 34, and 58 h after from to 5 in added Ca. Those experiments that
the exposure commenced, each plate was removed from indicated a positive response to the EMFs in terms of
exposure or sham-exposure, its diatoms were scored for motility were repeated. Five replicate experiments were
200 M. S. DAVIES ET At.
Figure3. Equipmentused in perfusionexperiments. Near-Heimholtzcoilsarrangedaroundmicroscope
stage. Video display ofdiatoms in perfusion chamber.
also conducted with 3-day-old diatoms (in logarithmic maximum (see above) and. when energized, the field at
growth) of strain Ills under three conditions: (1) using stage level was at Ca ion cyclotron resonance conditions.
cells that were not pre-incuhated (grown at ambient; Bh The sensing head ofthe fluxgate magnetometer (56 X 48
= 16.5 fjT, Bv = 44 /iT); (2) where the near-Helmholtz X 22 mm) was too large to fit on the stage with the
coils were disconnected from their supply (which re- objective lens cluster in place, so the latter was removed
mained switched on); and (3) at the even harmonics of 8 during EMF measurement. However, the introduction of
and 32 Hz (other field conditions remained the same). the objective lens cluster next to the sensing head within
According to Smith et al. (1987a). shifting to even har- energized coils caused no more than 1 ^T deviation in
monics should abolish any EMF effect. The experiments magnetic flux density. During perfusion, diatoms were
were conducted not in the order listed but in a random viewed through a long-working-distance 20x phase-con-
order, determined using random number tables. trast objective lens and displayed on the video monitor.
A perfusion chamber was created from two small glass
Peifitsion experiments plates separated by a30-mmdiameterO-ring andclamped
together with bulldog clips. Two hollow (hypodermic)
All perfusion experiments were performed on cultures needles (0.55-miTi diameter) were inserted through the O-
of strain #2038 that had been incubated at Bh = 20 fjT ring at points 180° to each other to act as fluid entry and
for at least one month (see above). Perfusion media were exit ports. Polyethylene tubing was connected to these
at room temperature (23 ± 1°C). ports. Two 60-ml syringes acted as reservoirs from which
(i) Equipment. The near-Helmholtz coils used for ex- perfusion fluid flowed undergravity through silicone rub-
perimental EMF exposure were placed around a Reichert ber tubing and then polyethylene tubing to the perfusion
Zetopan inicroscope so that the microscope stage was at chamber on the microscope stage. Flow from each reser-
the center ofthe coils. The microscope was set for phase voir was controlled using a clip on the silicone rubber
contrast and was liiiked to a U-matic video recorder and tubing. An acrylic plasticjunction unit allowed flow into
monitor via a Panasonic CL700 CCD video camera (Fig. the chamber from one reservoir only. In a chamber
3). The coils were again aligned along the geomagnetic flooded with water, the time taken from switching to a
—
RESPONSE OF DIATOMS TO EMFS 201
flow offood coloring until the centra! areaofthe chamber formis was grown in polystyrene tubes or glass flasks
was flooded with color was 50 s. from30000cells mP' until stationary phase was attained.
(ii) Effects ofCa levels on motility: Since the examina- Diatoms were harvested by centrifugation (1000 X g for
tion ofdiatom motility in a perfusion chamber was a novel 5 min) a minimum of2 weeks after inoculation and were
technique, we initially assessed the effects of [Ca] in the then washed and recentrifuged four times in deionized
perfusion medium. Three-day-old cells were harvested by (17.4 Mfi cm ') water. Diatom pellets were then lyophi-
a single centrifugation (5 min at 1000 X g) at 25°C and lized for 2 days. At no time did the diatoms come into
resuspended in 5 mA/ added Ca minimal medium (M-M). contactwith any metal object. To minimizecontamination
A dropofthiscell suspension was introduced intothe center from ferrous particles, both the modihed ASP2 and the
of the perfusion chamber. Following a 5-min period deionized waterused were kept inglass flasks atop a large
allowing for diatoms to adhere to the glass substratum, the strong permanent magnet for at least I month prior to use
chamber was perfused for 10 min with 5 mM added Ca (a technique employed by J. Kirschvink, pers. comm.).
M-M. Perfusion was then switched to mM added Ca These liquids were carefully pipetted from the flasks for
M-M for 5 min; switched back to 5 mM added Ca M-M use.
for 5 min; to mM added Ca M-M for 5 min; and finally Samples of freeze-dried diatoms of all strains were
to5 xnMadded Ca M-M for 5 min. Video recordings ofone sent for analysis to the laboratory of Dr J. Kirschvink
fieldofview were made during the whole ofthis procedure. (University of California Institute of Technology, USA)
Diatom speed was estimated by measuring, on screen, the where a superconducting quantum interference device
distance moved by each motile diatom in the 10-s period (SQUID) was used to detect the presence of biogenic
prior to each switching of perfusion medium and in each magnetite or ferrous material in the diatoms. For further
10-s period following I min ofperfusion (except the initial information on this method, see Kirschvink et al. ( 1992).
perfusion with 5 mM added Ca M-M). We had hoped to obtain a sample of diatoms from the
(iii) EMF experiments. The procedure for EMF ex- group (Reese et al.. 1991 ) that achieved a partial replica-
periments closely followed the Ca-response experiments tion of the results of McLeod et al. (1987a) and Smith et
above. Cells (3 days old) were again pelleted by a single al. (1987a, b), but none were available.
centrifugation and resuspended in 5 mM added Ca M-M.
A drop of diatom suspension was introduced into the Results
perfusion chamber. Perfusion with 5 mM added Ca M-M Agar plate experiments
commenced after 10 min. Diatoms that showed motility
werethenobservedduringtheremainderofthe procedure. (i) Growth and motility. During growth in culture,
After 10 min, perfusion was switched to 1 mM added Ca each strain ofdiatom exhibited maximal motility on agar
M-M (aCalevel suboptimal fordiatom motility). Motility during the logarithmic phase of its growth. This was true
was measured as before, after 2 min. The EMFs were whether cultures were grown at 20°C (Fig. 4) or 25°C
applied and motility was remeasured aftera further3 min. (Fig. 5). although the time taken to reach logarithmic
EMFs were then discontinued and motility was remea- growth, and hence maximal motility, was shorter at 25°C
sured after a further 3 min. than at 20°C. These data were used to determine when
The above procedure was repeated until a total of 30 to harvest cells for maximal motility in the rest of the
diatoms had been recorded during each treatment, with experiments.
3-10 diatoms (depending on the number visible on the The maximum motilities of #2036, #2038, and #2039
monitor screen) recorded during each procedure. Control seem independent of growth temperature, whereas those
experiments were performed by following the procedure ofIIIb and III, increase with increased temperature (maxi-
but not applying any EMF. Each treatment and its control mum mean motilities: IIIb at 20°C = 51% ± 7 SE, 25°C
were performed during one 3-h period on diatoms from = 78% ± 9 SE; IIIp at 20°C = 42% ± 4 SE, 25°C =
the same culture. Two treatments were used. One em- 72% ± 6 SE). The maximum motility recorded on any
ployed EMFs at Ca ion cyclotron resonance conditions. agar plate during the experiments was for IIIb (98%).
The second (conducted2 dayslater) involved amanipula- The higher growth temperature also apparently allows a
tion of frequency to an even harmonic which should, greatercarryingcapacity foreach strain in culture. Within
according to ion cyclotron resonance theory, abolish any each temperature treatment the growth curves can be split
EMF effect. We used Bv = a'T, Bh = 20.9 /jT, B„ac = into two groups: IIIb and IIIp showed a short logarithmic
41.8 ^T peak-peak at 32 Hz i.e.. the same conditions phase and a small carrying capacity; #2036, #2038, and
for ion cyclotron resonance but at double the frequency. #2039 showed a longer logarithmic phase and a higher
carrying capacity (Figs. 4 and 5).
Paramagnetic investigations (//) Ca curves. In Figure 6, the motility response of
To assess whether the response of diatoms to EMFs our diatoms to external [Ca] is compared with the results
was owing to paramagnetism, each strain of A. coffeae- obtained by Smith et al. (1987a). Our resuUs show that
202 M^ S. DAVTES ET AL
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RESPONSE OF DIATOMS TO EMFS 203