FEMS Microbiology Letters 37 (1986) 263-268
Published by Elsevier
263
FEM 02599
Effects of two Chitin synthesis inhibitors on Thalassiosira fluviatilis
and Cyclotella cryptica
(Thalassiosirafluviatilis; Cyclotella cryptica; chitin synthesis; effect of inhibitors)
Linda G. Morin a R i c h a r d A. S m u c k e r a and Werner Herth b
a University of Maryland, Center for Environmental and Estuarine Studies, Chesapeake Biological Laboratory,
Solomons, MD 20688-0038, U.S.A., and b Zellenlehre, University of Heidelberg, Heidelberg, F.R.G.
Received 11 August 1986
Accepted 12 August 1986
1. SUMMARY
The effects of two commercial chitin synthesis
inhibitors, dimilin and polyoxin D, on chitin fiber
formation and cell sedimentation for the diatoms
Thalassiosira fluviatilis and Cyclotella cryptica
(Bacillariophyceae) were investigated. Dimilin
treatments for both diatom species were indistinguishable from controls in terms of chitin fiber
productions, cell density and sedimentation. Polyoxin D-treated cells of both diatom species completely lacked the characteristic chitin fibers. Polyoxin D cultures were also characterized by a significant decrease in population density, increased
sedimentation rates and a strong tendency to
clump in comparison with control and dimilin
treatments. It was concluded that (1) dimilin does
not directly inhibit chitin synthesis in diatoms; (2)
polyoxin-D inhibits r-chitin fibril formation, and
(3) chitin fibers play an important role in cell
separation and cell buoyancy.
2. INTRODUCTION
T. fluviatilis, C. cryptica, and other diatoms are
unique among the plant kingdom for their produc-
tion of chitin fibers [1,2]. These fibers, which
radiate from pore structures on the valve surfaces
of both diatom species, are composed entirely of
crystalline fl-(1,4)-linked poly-N-acetylglucosamine [1-5]. McLachlan et al. [1] calculated that
31-38% of the total dry weight (including frustule)
of T. fluviatilis was due to chitin fiber production.
Both diatom species produce chitin fibers under
both CO2-1imiting and NO3-1imiting conditions
[2]. It is evident that both diatoms expend considerable energy and resources for chitin fiber production. Walsby and Xypolita [6] reported a doubling in the sinking rate of T. fluviatilis cells after
chitinase digestion of the chitin fibers, demonstrating their importance as a flotation mechanism. Gooday et al. [7] also observed increased
sedimentation rates for T. fluviatilis cultures in
which chitin fiber production was blocked by nikkomycin.
We have also observed chitin fibers in Chesapeake Bay natural diatom populations. These
fibers, visible by phase contrast microscopy and
electron microscopy, are consistent in their point
of origin with chitin fibers reported by other investigators [1,2,8-11]. Calculations were made for
chitin production levels in Chesapeake Bay waters
based upon available data for chlorophyll a and
0378-1097/86/$03.50 © 1986 Federation of European Microbiological Societies
264
carbon assimilation numbers [12]. Using the conservative estimate of 30% for weight content of
chitin [1] in the organic carbon pool for Cyclotella,
production estimates for chitin in central Chesapeake Bay during May for a single species of
diatom, Cyclotella caspia [13], is 34 mg C/m3/h.
Diatom chitin may represent a significant contribution to the total chitin pool, especially during
seasonal blooms.
A suite of compounds which block chitin
synthesis have become available [14]. Polyoxin-D
occurs as a component of the uracil-antibiotic
polyoxin complex produced by Streptomyces cacoai
var. asoensis [15]. This antibiotic, a structural
analogue of UDP-N-acetylglucosamine (UDPGlcNAc), functions as a competitive inhibitor for
the enzyme chitin synthetase (UDP-N-acetylglucosamine:chitin N-acetylglucosaminyl-P-transferase) [16], and is a potent inhibitor of a-chitin
synthesis in fungi [17-20] and arthropods [21].
Dimilin ® (1-(4-chlorophenyl)-3-(2,6-difluorobenzoyl)-urea) is an effective inhibitor of a-chitin
formation in arthropod post-molt cuticle development [14,22]. Although dimilin is considered a
chitin synthesis inhibitor, there is no evidence that
it directly inhibits isolated chitin synthetase in
insect systems [21].
The present study documents some effects of
polyoxin-D and dimilin on chitin fiber production
and growth of the r-chitin-producing diatoms, T.
fluviatilis and C. cryptica.
3. MATERIALS AND METHODS
3.1. Cultures
T. fluviatilis Hustedt and C. cryptica Reimann,
Lewin and Guillard were chosen for their welldocumented chitin production [1,2]. T. fluviatilis
and C. cryptiea strains were maintained as described by Herth and Barthlott [8]. Other strains
of T. fluviatilis (ACTIN) and of C. cryptica (T. 13
• L) were obtained from the Bigelow Laboratories
(Bar Harbor, ME).
3.2. Chemicals
Polyoxin-D was kindly donated by Dr. Hiromi
Kuzuhara of the Institute of Physical and Chem-
ical Research, Sattaria, Japan. Technical grade
dimilin (> 99% purity) was provided by Richard
Cannizzaro of Thompson Hayward Co. (Duphar).
All other chemicals were reagent grade from J.T.
Baker, Sigma or Fluka.
3.3. Media and growth conditions
Cells were maintained and experiments performed in Guillard's f / 2 medium [23] at 18°C,
and 3500 lux fluorescent lighting (GE Soft-fluorescent light tubes) with a 14 h/10 h light/dark
cycle. All cells were grown in 50 ml of f / 2 medium
in 250-ml Erlenmeyer flasks. Aeration was achieved by gently swirling the flasks once daily. Experimental conditions differed only by the addition of
25 ppm dimilin or polyoxin-D (25 mg- 1 l).
Control and experimental flasks were inoculated with 1 ml of actively growing cultures.
Duplicates were performed for each species and
each treatment. Experiments were repeated 4
times. At the end of 7 days, the flasks were gently
swirled to suspend the cells uniformly. Samples
were removed for cell enumeration, sedimentation
characteristics and scanning electron microscopy.
3.4. Cell density
Cell density was determined using an A / O
hemocytometer. Each culture flask was sampled in
triplicate. Differences between treatments were
analyzed using Duncan's Multiple Range Test.
3.5. Sedimentation characteristics
Sedimentation characteristics were measured by
monitoring changes in absorbance (A) at 627 nm
[24] with a Bausch and Lomb Spectrophotometer.
Cuvettes (1 cm) were filled 5 mm above the light
path and absorbance read periodically for 50 min.
3.6. Electron microscopy
A 15-ml sample from each of the 7-day algal
cultures was fixed for 30 min using the methods of
Herth and Barthlott [8]. Each set was dehydrated
in a graded ethanol series (10 min in 15%, 30%,
50%, 70%, 80%, 90%, 95% and then 2 changes of
100%) and air-dried directly onto polished brass
stubs under a vented bell jar with a stream of dry
N 2 gas (2.5 psi). The samples were coated with
Au-Pd in a diode coater (Technics Hummer V).
265
All samples were viewed and photographed in a
JEOL JSM-U3 scanning electron microscope using
an accelerating voltage of 15 or 25 keV.
4. RESULTS
4.1. Cell density
Cell-density results are summarized in Fig. 1.
These data show no significant difference between
dimilin-treated cells and the controls for both
diatom species. Polyoxin-D treatment for both
species resulted in significant reduction (95% confidence) in cell density as compared with controls
and dimilin treatments.
4.2. ~Sedimentation characteristics
Spectrophotometric sedimentation data are
summarized in Fig. 2. Dimilin had no apparent
effect on the sedimentation of either T. fluviatilis
or C. cryptica when compared to the respective
controls. Polyoxin D-treated cultures showed a
dramatic increase in sedimentation for both T.
fluviatilis and C. cryptica in comparison with controls and dimilin treatments.
4.3. Scanning electron microscopy
T. fluoiatilis and C cryptica produced characteristic chitin fibers in the control cultures (Figs.
3a and 4a). Both diatom species also produced
chitin fibers in the presence of 25 ppm dimilin
with no apparent reduction in fibril size or number (Figs. 3b and 4b). Controls and dimilin-treated
cells of both diatom species were characterized by
cells occurring singly, in pairs or in short chains of
up to 4 cells.
Chitin fiber formation was completely blocked
in 25 ppm polyoxin D-treated T. fluoiatilis and C.
cryptica (Figs. 3c and 4c). Similar results were
observed with 12.5 ppm polyoxin D. Polyoxin
D-treated T. fluoiatilis often formed chain lengths
of 15-20 cells (Fig. 3c). Under similar conditions,
Co cryptica tended to form aggregates which were
apparent even to the unaided eye (Fig. 4c).
5. DISCUSSION
The concentration of dimilin used in these experiments was several orders of magnitude greater
than the residue concentrations found in natural
0.3"
2.5"
5
2.0
CD
I
O
•
4
---- Control
u')
I
0
~-
f~
Cq
(D
0.2"
1.5
-
T
T
~
~
Jr
~
"
4~
~
Control
- " 1 DlmtNn
II Dimilin
"
CO 1.0
(9
o
0
2
0.1"
0.5-
~
Polyoxln-D
--- Polyoxin-D
-
0
Control
DJmg~
Polyoxin-D
C. cryptica
Control
Dimilln P o l y o x l n - D
T. fluviatilis
Fig. 1. Effect of chitin synthesis inhibitors on average cell
density after 7 days of growth. Treatments were performed in
duplicate; each culture flask was sampled in triplicate. N = 6.
Bars represent standard error.
0
1~3 2'0
3'0
4C)
5'0
]]me in Minutes
Fig. 2. Effect of chitin synthesis inhibitors on sedimentation
expressed as a function of changes in absorbance over a
50-min interval. B, T. fluviatilis; e, C. cryptica. Each point
represents an average of 4 readings.
266
!i~ ¸
!
Fig. 4. C. cryptica (T-13-L). (a) Control; (b) dirnilin treatment; (c) polyoxin D treatment. Abundant chitin fibers are
present in the control and dimilin cultures (arrows). Cells
typically occur singly or in pairs. Polyoxin D-treated cultures
(c) showed complete lack of fibers and tended to form large
aggregates.
Fig. 3. T. fluviatilis. (a) Control; (b) dimilin treatment; (c)
polyoxin D treatment. Note the abundant chitin fibers present
in both the control and the dimilin-treated cultures (arrows)
and the tendency for cells to occur singly or in pairs. Polyoxin
D-treated cultures (c) produced no fibers, and cells formed
long chains.
systems after pesticide application [25]. Our dimilin experiments with diatoms show no effect on
chitin fiber formation or buoyancy and support
other studies [26-28] which have shown that dimilin does not inhibit chitin synthetase directly.
Cell-free chitin synthetase experiments by Mayer
et al. [26,27] and Cohen and Casida [28] have
shown that dimilin does not directly inhibit chitin
synthesis in insect models. Mayer and Meola [22]
267
have correlated chitin synthesis inhibition with
inhibition of imaginal epidermal cell proliferation
in dimilin-treated stable fly (Stomoxys calcitrans
L.) pupae.
Our diatom sedimentation results, when using
the U D P - G l c N A c analogue polyoxin D, to block
chitin fiber production, corroborate the results of
Walsby and Xypolita [6]. In that study, chitinase
was used to digest the fibers of T. fluviatilis. Lack
of chitin fibers resulted in significantly increased
sedimentation rates due to a reduction in form
resistance. G o o d a y et al. [7] reported similar resuits using another U D P - G l c N A c analog, nikkomycin. Nikkomycin-treated T. fluviatilis also
lack chitin fibers and sediment more rapidly than
controls. This observation provides further evidence that chitin fibers play an important role in
cell suspension.
Fiber production also appears to play a role in
cell separation. We observed chain formation
greater than 4 cells per chain only in cultures in
which chitin fiber production was blocked by
polyoxin D. In contrast, Eppley et al. [29] observed an increase in sinking velocity in older T.
fluviatilis cultures, which they attributed to aggregate formation due to chitin fiber 'entanglement'. Our T. fluviatilis and C. cryptica controls
did not form chains or aggregates, even after 7
days of growth. Increased chain length results in
increased sinking rates for a number of diatom
species [30-33]. Chain formation in polyoxin Dtreated T. fluviatilis cultures and C. cryptica cultures may have also contributed to the dramatic
increase in sinking rates observed for both diatom
species. We were careful to preserve cell chains
and aggregates before measuring sinking rates.
Therefore, we are unable to distinguish the effects
of chain formation, from lack of chitin fibers, on
sinking velocity.
The cell count data address the effect chitin
fiber production may have on growth rates. When
compared to control and dimilin treatments, polyoxin D treatments showed a significant reduction
in growth (cell number) for both diatom species.
This reduction in total cell number may be the
result of concomitant reduction in surface area
due to the observed increase in chain length. Exchange of nutrients and metabolites may be limited
due to the effective reduction in surface a r e a /
volume.
Although dimilin is a potent inhibitor of
arthropod chitin synthesis, it has no apparent
effect on diatom growth or chitin fiber production. Polyoxin D, on the other hand, is an effective
inhibitor of diatom chitin fiber synthesis and, as
such, may provide us with a useful tool in developing a primary production-based chitin synthesis
assay.
ACKNOWLEDGEMENTS
The authors are grateful to Ms. Billie Little and
Ms. Pam Blancato for secretarial assistance in
preparing this manuscript.
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