Plant
Physiol. (1968) 43,
13l9-I4i
Short Communication
The Response of Wild Type Male Gametes of Allomyces
to Sirenin
Leonard Machlis
Department of Botany, University of California, Berkeley, California 94720
Received May 13, 1968.
The hermaphroditic, haploid, sexual generation
of the watermolds Allomyces macrogynus and A.
arbuscula bear orange male gametangia and colorless female gametangia which discharge motile male
and female gametes into an ambient liquid environment (1,8). The female gametes, beginning prior
to their release from the gametangia, secrete sirenini,
a sperm-attractant (3, 4), whose structure was recently established (6) and which is depicted in
figure 1. The production of sirenin (7) was done
with the almost entirely female isolate #F-1 (3)
derived from a cross between A. macrogynus
(n=28) and A. arbuscula (n=16) and the bioassay,
during the time it was needed, with an almost entirely male isolate derived from the same cross.
These particular hybrids have not been studied
cytologically or genetically but the chromosome
behavior of various crosses between the parents has
been described (2).
The purpose of this communication is to show
that m-ale gametes from the parent species do respond
chemotactically to sirenin which was synthesized by
the female hybrid strain. At the same time, the
experiments raise the possibility that there may be
species-specific sirenins.
H
HOH2C
C C
OH3
H2
H
OCH3
K
H
\-1~ H2
C
C
c
H
CH?OH
FIG. 1. The structure of sirenini. C15H240.,. MA'
236. Bond representation:
in the plalle of the
paper; p, coming out of the plane toward the
reader; ----, going back of the plane away from the
reader. Two sites of potential isomerism occur. In the
attachments to the apex of the cyclopropyl ring, it is
the methyl that projects out over the cyolohexyl ring
rather than the isohexn -l side chain. The attachment
of the hydroxymethyl group to the double-bond in the
isohexenyl side chain is trans to the ethylene group.
The gametophytic generation of L. mnacrogynus,
strain Burma 31 and A. arbuscula, strain iCeylon 1
were grown on Difco YpSs nutrient agar plates.
Gametangia were scraped from these plates and
placed in DS solution (3) in a small glass petri
dish. Male gametangia were then picked up in a
micro pipette and 500 placed in 0.6 nil of DS in
containers of a proper size to take the assay apparatus (fig 2). Discharge of the gametes was then
allowed to take place for 3 hours. The male
gametangia must be separated in approximately 40
minutes, the time between immersion in DS solution
and the first emergence of gametes. In this period
it was possible to select 1500 gametangia thus per1 The original cross used isolate Burma lDa (1).
Professor Ralph Emerson has informed me that isolate
Burma 3 was collected in the same locality as Burma
1 Da and that both isolates are almost certainly completely equivalent.
FIG. 2. The apparatus for the bioassay. The glass
ring is 10 mm deep and 22 mmn in diameter. In practice,
12 of these are cemented to a glass plate for ease of
manipulation under the microscope. The apparatus will
be described in detail elsewhere (5). Tihe basic feature
is that through the center of it is a hole one-fourth
inch in diameter and three-eighth inch long which is
closed at its lower end by a piece of dialyzing tubing
held in place by a small dental rubber band. The sirenin
solutions are placed in the well above the membrane.
A sperm suspension is placed in the ring up to a level
just above the membrane. The membrane itself is oneeighth inch from the floor of the ring. When the well
is full and covered with a cover slip, the sperm canl
be seen with a low pow er microscope and the number
per unit area of membrane counted.
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1320
PLANT PHYSIOLOGY
Table I. The Ch'rmotactic Response to Sirenin of the
Male Gantetes of Allomyces macrogyinus and
A. arbuscula
The number of gametes attached to 0.47 mm2 of
mnembrane after 60 minutes is reported. Each figure is
the average of 3 separate determinations.
Species
Male gametes
Expt.
1.
.1.
mc(leroyynits
arbuscula
1
113
4
on
membrane
Expf.
2
235
25
initting 3 assays to be made at
tinme. Each male
gametangium of A. miacrogymus yielded an average
of 48 gametes so that the colncentration used in each
assay was 40,000 per ml. The imiale ganietangia of
.4. arbuscula yielded ani average of 24 gamietes
making the concentration in the assay 20,000 per ml.
At the end of the 3-hour discharge period the assay
apparatus was put in place containing a concentration of sirenini found to be highly chemotactic
againist mnale strain M-4 (3). The results are
shown in table 1.
The response of the male ganmetes of A. mi acrogynus is vigorous. In actual numbers it is of the
same order of mlagnitude previously observed for
M-4 male strain gametes to the same concentration
of sirenin. In contrast, the male gametes of 4.
arbutscula are poorly reactive. The results show
that sireniin produced by a hybrid female strain is
highly effective with at least one of the parent
species. The response of the 4. arbtusclda gametes
is far less thani can be accounted for by the fact
thlat their concentration is half that of the A4. ntacrogynus gametes. Thus, if strain M-4 male gametes
at 40,000 and 20,000 per ml are assayed against the
same concentration of siren,in the lower concentration
of gametes gives a reading equal to 60 % of the
higher concentration whereas the response of A.
arbuscula to sirenin (table T) is only 4 to 11 %
of that of A. miacrogynuiis.
The poor response of A. arbuscula male ganietes
can be explained at present in 2 possible ways.
First is the assunmption that each species has its
own characteristic sirenin. This would mean that
the F-1 female strain produces a sirenin that is the
a
similar to that of the A. mnacrogynus parent.
Alternatively, it can be assumed that both species
produce the same sirenin but that the male gametes
of A. arbuscula are much less sensitive to sirenin.
Experiments of the type described are difficult
to do. The small number of replicates that can be
assembled in any one experiment and the relatively
low concentration of gametes that can be attained
are serious impediments to critical work. Efforts
are now in progress to produce sirenin with the
wild-type species and to obtain male gametes on a
niass scale from the hermaphroditic parent species
for assay purposes. If several technical problems
can be solved, it will be possible to find out if there
same or
are species-specific sirenins.
Acknowledgments
The technical assistance of Mrs. Vroni Waltoni is
Financial support w as provided by the National Science Foundation under grant
GB-1 107.
gratefully acknowledged.
Literature Cited
1. EMIERSON, R. 1941. Ani experimental studx of the
life cycles and taxonomy of Allomntces. Lloydia
4: 77-144.
2. EMERSON, R. AND C. M. WILSON. 1954. Interspecific hybrids and the cytogenetics and cytotaxonomy of Euallomyces.
Mycologia 56: 393-434.
3. MACHLIS, L. 1958. Evidence for a sexual hormone in Allomyces. Physiol. Plantarum 11: 18192.
4.
MACHLIS, L. 1958. A procedure for the purifica-
tion of sirenin. Nature 181: 1790-91.
5. MACHLIs, L. 1968. Zoospore chemotaxis in the
watermold A41lomnyces. Phvsiol. Plantarum. In
press.
6. MACHLIs, L., W. H. NUTTING, AND H. RAPOPORT.
1968. The structure of sirenin. J. Am. Chem. Soc.
90: 1674-76.
7. MACuLIs, L., Wi. H. NUTTING, M. W. WILLIAMIS.
AND H. RAPOPORT. 1966. Productioni, isolatioln and
characterization of sirenin. J. Am. Chem. Soc.
5: 2147-52.
8. SPARROW, F. K.. JR. 1960. Aquatic Phycomycetes.
Second Edition. University of Michigan Press.
1187 p.
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Copyright © 1968 American Society of Plant Biologists. All rights reserved.