Davidovich, N.A. and S.S. Bates. 1998. Patterns of sexual reproduction in the pennate diatoms Pseudo-nitzschia
multiseries and P. pseudodelicatissima. In: B. Reguera, J. Blanco, M.L. Fernández, T. Wyatt [Eds.] Harmful microalgae.
Xunta de Galicia and the IOC of UNESCO, Paris, p. 152-155.
PATTERNS OF SEXUAL REPRODUCTION IN THE PENNATE DIATOMS PSEUDO-NITZSCHIA
MULTISERIES AND P. PSEUDODELICATISSIMA
Nickolai A. Davidovich* and Stephen S. Bates**
*Diatoms Biology Laboratory, Karadag Branch of the Institute of Biology of the Southern Seas, Feodosiya 334876
UKRAINE;
**Fisheries & Oceans Canada, Gulf Fisheries Centre, P.O. Box 5030, Moncton, New Brunswick, CANADA E1C 9B6
ABSTRACT
The pennate diatoms Pseudo-nitzschia multiseries and P.
pseudodelicatissima normally decrease in cell length in
culture until they eventually die without undergoing
sexual reproduction to regain the largest cell size.
However, we induced sexual reproduction by mixing
individual exponentially growing clonal cultures of the
appropriate minimal cell size under the same conditions
that are normal for vegetative growth. We observed:
pairing of parent cells (gametangiogamy); production of
four morphologically isogamous, non-flagellated
gametes per gametangial pair; rearrangement of the
gametes and their fusion to form zygotes, revealing
physiological anisogamy; enlargement of auxospores;
and formation of long initial cells. Our observations of
allogamous reproduction are consistent with those
reported for other dioecious pennate diatoms. Clones of
P. pseudodeli-catissima from the Black Sea and from the
CCMP culture collection failed to auxosporulate when
mixed together, although they are the same species
according to scanning electron microscopy. Knowledge
of the pattern and timing of sexual reproduction in
Pseudo-nitzschia spp. may provide insights into their
bloom dynamics.
INTRODUCTION
Diatom species of the genus Pseudo-nitzschia have
been actively investigated during the past decade, due in
part to their implication with Amnesic Shellfish
Poisoning (ASP) in humans caused by the neurotoxin
domoic acid [1,2]. One feature that has required study,
however, is their mode of sexual reproduction. This is of
interest because of its possible relationship to bloom
dynamics and cell toxicity.
As in most other diatoms, continued vegetative
division results in a decrease in mean cell size, which
must be restored to its maximal size by auxospore
formation, usually via sexual reproduction [3,4]. There
is only one other report of sexual reproduction in P.
multiseries [5], and it has provoked considerable debate
and criticism [6,7,8]. The main contention is that the
type of sexual reproduction reported was contrary to the
normal process known for pennate diatoms.
For
example, flagellated gametes and oogamy (production of
large, non-motile female gametes that are fertilized by
small, motile flagellated male sperm) were described [5].
This contradicts numerous other observations of
auxosporulation in pennate diatoms which show that
allogamy (accomplished by the pairing of gametangia, or
“parent cells”), not oogamy, is the most frequent mode
of their sexual reproduction [3]. The only other report of
sexual reproduction in a Pseudo-nitzschia species is for
P. subcurvata, which showed auxospore formation
consistent with other pennate diatoms [6].
Until recently, it was believed that most diatom
species were monoecious, involving production of both
male and female gametes by the same clone [9].
Therefore, investigators did not seriously consider the
possibility that clones could be made to reproduce
sexually simply by mixing them together. However, data
obtained during the last two decades indicate that some
pennate diatoms are dioecious (production of either
“male” or “female” gametes by two gametangia in
different clones) [10]. Here we show that species of
Pseudo-nitzschia have a dioecious type of mating and
that allogamy is their common mode of sexual
reproduction. A more detailed description of this
research is found in [11].
MATERIALS AND METHODS
Clones of Pseudo-nitzschia pseudodelicatissima
(ND-1, ND-2, ND-3) were isolated in Spring 1996, from
plankton samples collected in the Black Sea (Crimean
peninsula). Clones of P. pseudodelicatissima (CCMP1562, CCMP-1564) were received from the ProvasoliGuillard National Centre for Culture of Marine
Phytoplankton (CCMP), Maine, USA. Clones of P.
multiseries (KP-103, KP-104, KP-105) were isolated by
K. Pauley from Cardigan Bay, Prince Edward Island,
Canada, in December 1993. The clones were maintained
and experiments carried out in f/2 medium [12]. Culture
conditions suitable for sexual reproduction were those
used for vegetative growth. The clones were kept in
exponential growth at a photon flux density of 20-120
µmol⋅m-2⋅s-1, either at 14°C and 12:12 h light:dark, or
20°C and 10:14 h light:dark.
Parent clones were mixed during the period of
exponential growth (days 3-7) in borosilicate Petri dishes
containing 15 mL of f/2 medium. Triplicate mixtures
were made, containing 0.15, 0.30, or 0.45 mL of each
parental clone. The mixed cultures were examined after
2-4 days for signs of sexual reproduction. Cultures were
observed in situ by light microscopy with a x30 waterimmersion objective. Apical cell length was measured
with an ocular micrometer (ca. 0.8 µm precision).
RESULTS
Scheme of Sexual Reproduction
Gamete production started 2-3 days after mixing
either all clones of the same species or certain pairs from
different clonal cultures. Mixture experiments thus
showed that there were only two types (sexes) of clone,
designated as “+” and “-”. Each type was unable to
of the auxospore (Fig. 1J) represent the remnants of the
wall secreted by the zygote, and could remain until
formation of the initial cell. The restored, initial cell was
formed inside the auxospore (Fig. 1J-K) as a result of a
two-step process of frustule construction. Finally, the
initial cell exited the auxospore and divided vegetatively,
forming long chains (Fig. 1L). The entire process of
sexual reproduction, from gamete formation to the
appearance of the initial cells, took 2-4 days.
Differences were observed among clones and
between species in particular features of sexual
reproduction. The degree of attachment between a pair
of parent cells, or between a parent frustule and the
auxospores, differed between isolates. Gametangia were
most tightly attached in the ND- clones of P.
pseudodelicatissima, resulting in a high percentage of
successful fusions of the two pairs of gametes to yield
eventually two auxospores per gametangium (Fig. 1F-I).
In contrast, for P. pseudodelicatissima clones CCMP1562 and CCMP-1564, and P. multiseries, the parent
frustules had only a weak attachment. This resulted in
gametogenesis but often without subsequent gamete
fusion, or only the first pair of gametes fused
successfully, thus producing only one auxospore.
The weak attachment between the auxospores and
parent frustules sometimes led to an irregular
configuration between the two in P. multiseries and
CCMP- clones of P. pseudodelicatissima. In the NDclones of P. pseudodelicatissima, on the other hand, the
auxospores generally laid parallel with one another and
perpendicular to the parent frustule (Fig. 1H-I).
When the attachment of the auxospores to a parent
frustule was strong, we could ascertain the activity (i.e.,
physiological isogamy or anisogamy) of the gametes. In
such cases we concluded that the auxospores were
derived from gametes that had remained attached to the
“mother” frustule, and hence those gametes were
passive. The gametes therefore displayed behavioral
anisogamy. Furthermore, when the clones had cells of a
different size, it was possible to determine which parent
clone provided the active (“-”) or the passive (“+”)
gametes.
cross with clones belonging to the same type (intraclonal
breeding not observed), but able to mate with clones
belonging to the other type (interclonal breeding). Thus,
P. multiseries clone KP-105 was determined to be the
opposite sex of clones KP-103 and KP-104. For P.
pseudodelicatissima, clone ND-3 entered into sexual
reproduction with clones ND-1 and ND-2. The two
clones of P. pseudodelicatissima (CCMP-1562 and
CCMP-1564) were of the opposite sex. Because each
clone had a different cell size, it was possible to
determine the clone of the gametangium to which the
auxospores remained attached. All attempts to cross the
ND- clones with the CCMP- clones failed to result in
auxosporulation, in spite of the fact that all of these
clones are P. pseudodelicatissima, according to their
morphology as seen by scanning electron microscopy
(SEM) [11]. The ND- clones were wider (2.1-2.5 µm)
than the CCMP-clones (1.4-1.6 µm), although they were
still within the acceptable range for P. pseudodelicatissima [13].
The general pattern of sexual reproduction for P.
multiseries and P. pseudodelicatissima is shown in Fig. 1.
Pairing of parent cells (gametangiogamy), one from each
of the two clones, was the first stage (Fig. 1A-C).
Gametangia contacted each other valve-to-valve, lying
parallel. No mucilage envelope or copulation tube were
observed. Pairing was possible both between two single
cells (Fig. 1B-C) and between one single cell and
another still linked in a chain (Fig. 1D-G). More than
one single cell at a time could form a pair with others in
the chain.
The next stage was gametogenesis. Each cell in a
pair divided meiotically. The contents of the cells then
divided along the apical plane, and after rearrangement
(Fig. 1A-B), spherical gametes were formed (Fig. 1C-D).
Each gametangium thus yielded two morphologically
isogamous non-flagellated gametes, but whose behavior
was anisogamous (physiological anisogamy). In each
pair, one gametangium produced two active gametes and
the other produced two passive gametes (= cisanisogamy; [4]). The frustules opened completely,
permitting both gametes in one cell to move by
amoeboid action toward the passive gametes in the other
gametangium. Single gametangia, that is not in a pair,
were occasionally found (Fig. 1A).
Only one pair of gametes fused at a time. Which two
gametes did fuse depended on the relative positions of
the gametangia; each gamete fused with the nearest one
in the other gametangium. Plasmogamy of two gametes
to form a spherical zygote (Fig. 1D-G) took only 1-2
min. Gametes were unable to fuse if they had lost
contact with their parent frustule; they apparently
required a substrate for amoeboid movement. Many
free-floating, single gametes could thus be observed in a
culture.
The resulting zygotes then started to expand to form
auxospores (Fig. 1F-J), although not necessarily
synchronously. As a rule, the two auxospores eventually
attained a similar length (Fig. 1I). The auxospores
remained attached to only one of the parent frustules
(Fig. 1H). Fully expanded auxospores contained two
unfused nuclei and usually four chloroplasts in a thin
layer of peripheral cytoplasm (Fig. 1I). Caps on the ends
Change in Cell Size
The ND- clones of P. pseudodelicatissima were
unable to reproduce sexually just after isolation in early
1996, when their cell length was >80 µm. The apical
cell length of all the clones has since declined in culture,
and sexual reproduction was observed. For example, the
cell length of P. multiseries clone KP-103 has decreased
from 94.9 ± 1.2 µm to 43.0 ± 2.1 µm during an 18month period (or about 2.5 µm per month), although the
transapical width has remained constant at ca. 5 µm.
Gametes of the opposite sex had a similar form and
dimensions (i.e., they are morphologically isogamous).
Zygotes had a volume about twice that of the gametes.
The length of the initial cells formed within the
auxospores was about twice that of the parent cells.
Only the apical length was restored as a result of
auxosporulation; the transapical width in parent and
initial cells was similar.
153
Fig. 1. Stages in the sexual reproduction of Pseudo-nitzschia multiseries (P.m.) and P. pseudodelicatissima (P.ps.),
ND- clones. A) paired gametangia (P.ps.) in a chain, along with one unpaired gametangium, on left, showing
rearrangement of cell interior during gamete formation; B) single pair of gametangia (P.m.), showing rearrangement;
C) two of the gametes in the larger cell of (B) have become rounded, and the frustule has opened wider; D) fusion of
two gametes (P.ps.) to form a zygote, on right; E) two zygotes (P.ps.), middle, with gametangia undergoing
rearrangement, on right; F-G) start of auxospore expansion (P.ps.), on left, also showing fusion of gametes to form two
zygotes, middle; H) continued expansion of auxospores (P.ps.), showing that the attachment is only to one (“mother”)
gametangium; I) fully expanded auxospores (P.ps.); J) initial cell (P.ps.) forming inside the auxospore, on left, and
another expanded auxospore attached to a different cell, on right; K) initial cell (P.m.) forming inside the auxospore;
L) long, initial cells (P.m.), having exited the auxospore envelope, and now dividing vegetatively and forming chains.
Scale bar = 100 µm.
154
cells in field samples and by knowing the environmental
conditions conducive for sexual reproduction. Initial
cells produce substantially more domoic acid than the
parent cultures, which have virtually lost their toxicity
over a period of several years [unpublished results].
Changes in toxicity over the life history of the cells
could therefore account for an interannual variability in
bloom toxicity.
The genus Pseudo-nitzschia was recently revised and
separated from the genus Nitzschia because of a set of
morphological and molecular peculiarities [14]. We can
now add to this list the ability for like diatoms to enter
into sexual reproduction, a major biological criterion of a
species. This is made possible by a collection of clones
now characterized as to their sexuality. In the case of P.
pseudodelicatissima there was successful auxosporulation, separately, among the ND- clones and the CCMPclones, demonstrating that the cells were sexually
capable. That the ND- and CCMP- clones did not
interbreed raises questions concerning their taxonomic
relationship. It may also explain contrary observations
regarding the ability of P. pseudodelicatissima from
different parts of the world to produce domoic acid [2].
DISCUSSION
Sexual reproduction has been documented in only a
few diatom species [3,4]. In the case of Pseudo-nitzschia
spp., it would be difficult to find sexually reproducing
cells in the field because 1) in each generation, only cells
of a suitable apical size can sexually reproduce (in
Pseudo-nitzschia, this may occur only once every three
years [11]), 2) there is a low probability that cells of the
opposite sex can encounter each other in the plankton,
and 3) the entire process takes only 2-4 days. Therefore,
not all cells in a population will enter into sexual reproduction, even under favourable conditions. Moreover,
the association between sexually reproducing pairs may
be so weak as to be disturbed during normal sampling.
Finally, the gametes and auxospores may not preserve
well after collection. Our documentation of sexual
reproduction in Pseudo-nitzschia spp. may nevertheless
help others recognize sexual stages in field samples.
Making mixtures of different clones of Pseudonitzschia spp. permitted us to determine mating types.
Because auxosporulation was observed only in mixtures
containing clones of the opposite sex, not in separately
growing clones, these Pseudo-nitzschia spp. exhibit a
dioecious type of sexual reproduction. Dioecy seems to
be usual in pennate diatoms [10] in spite of the recently
held belief that diatoms were mostly monoecious [9].
Our description of allogamous sexual reproduction in
Pseudo-nitzschia spp. is contrary to the results of Subba
Rao et al. [5] for the reasons given by Rosowski et al.
[7], but is consistent with a preliminary description given
by Fryxell et al. [6] for P. subcurvata. Among many
other objections, Rosowski et al. [7] suggest that sexual
reproduction was not even observed. Rather, they
propose that contaminating chytrids were mistaken for
flagellated gametes. The Pseudo-nitzschia spp. gametes
we observed were morphologically indistinguishable
from each other and had no flagella, consistent with
studies of other pennate diatoms [3,10].
The observation of two active or two passive gametes
produced by the same gametangium, and the connection
of auxospores with the parent frustules of strictly
“mother” gametangia (which contain passive gametes)
argue for the placement of these Pseudo-nitzschia
species in the type IA2 anisogamous sexual
reproduction. Type IA2 is shown mainly in araphid
pennate diatoms, and thus is believed to be primitive
[3,4].
Pseudo-nitzschia is a geographically wide-spread
enus restricted to marine environments [13]. Cells of
this genus are capable of active motility, which is used to
form stepped chains after vegetative division. This
feature also appears to be adaptive for sexual
reproduction, allowing the two gametangia to line up in
close proximity with each other in order to copulate.
An understanding of the life history of P. multiseries
may provide some insights into the reasons for the
interannual variability in bloom intensity and toxicity of
this species in eastern Prince Edward Island, Canada [2].
For example, knowledge of the cell size suitable for
sexual reproduction and of the rate of decrease in cell
length would allow one to predict when auxosporulation
can take place. This may be accomplished by measuring
the size-frequency distribution of Pseudo-nitzschia spp.
ACKNOWLEDGMENTS
We thank C. Léger for excellent technical assistance.
Funding by NATO Collaborative Research Grant
OUTR.CRG 960380 is gratefully acknowledged.
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