BULLETIN OF MARINE SCIENCE. 41(1): 59-67.1987
REPRODUCTIVE BIOLOGY AND DEVELOPMENT OF
THEMISTE LAGENIFORMIS, A
PARTHENOGENIC SIPUNCULAN
John F. Pilger
ABSTRACT
The reproductive biology ofthe sipunculan Themiste lageniformis from Aorida is reported.
The populations have temporally stable biased sex ratios with females outnumbering males
24: I. Measurements of coelomic oocytes and records of spawns define an annual cycle of
reproduction. Small oocytes begin to appear in the coelom in early spring and reach maximum
size by July. Spawning extends from July through January but reaches its greatest frequency
during August and September. Themiste lageniformis is unique among the Sipuncula in that
females produce eggs which spontaneously activate and develop into normal larvae. Multiple
hypotheses are considered as explanations of this phenomenon, but only facultative parthenogenesis is supported. Themiste lageniformis is the only sipunculan species reported to
use this reproductive mode.
Reproductive periods are known in several species of the Sipuncula (see Rice,
1975, review). Most of these studies are based on spawning data alone and many
did not document the process in the species through a full year. Gametogenic
cycles, on the other hand, have been studied in Golfingia vulgaris (Gonse, 1956a;
1956b), Phascolosoma agassizi; (Towle and Giese, 1967; Rice, 1967; 1975), Themiste petricola (Amor, 1977), Golfingia vulgaris (Gonse, 1956a; 1956b), G. pugettensis (Rice, 1975), and G. minuta (Gibbs, 1975). These studies provide a more
complete understanding of reproductive periodicity in the Sipuncula.
The subject of this study is Themiste lageniformis Baird, 1868 (=Dendrostoma
signifer Selenka and de Man, 1883), a species that is distributed in the IndoPacific, India, Africa, Hawaii, and the southeastern United States (Florida). The
annual cycle of gametogenesis and spawning was investigated as part of a larger
study of reproduction in the species and is reported here. T. lageniformis is unique
among the Sipuncula in that it reproduces by parthenogenesis (Pilger, 1978). The
evidence that supports parthenogenesis as the reproductive mode is presented
and hypotheses relating to sex determination and cytogenetic aspects of part henogenesis in the species are proposed.
MATERIALS AND METHODS
Monthly collections of T. lageniformis were made from September 1977 through March 1979 at
Boot Toe Point (27°28.4'N, 80ol8.3'W) near the Fort Pierce Inlet to the Indian River. Additional
specimens were obtained from occasional collections at Bessie Cove (27°II.I'N, 80 9.7'W) near the
Saint Lucie Inlet and a single collection from Missouri Key (24°40.6'N, 81 14.3'W) in the Aorida
Keys. At Boot Toe Point and at Bessie Cove the animals live between oysters (Crassostreae virginica),
which form clusters on intertidal sand bars and mangrove roots. At Missouri Key, the worms live in
burrows which they form in coral rubble. The specimens were maintained in the laboratory isolated
in individual compartments of plastic boxes. The sea water was changed twice a week. Daily records
of spawning were kept during the 19-month study-period. Spawnings by the ·same individual on
separate days were recorded as separate events.
The sex of each individual was determined either by observing coelomic oocytes through the semitransparent body wall or, ifeggs were not seen, by removing a small sample of the coelomic fluid with
a syringe and needle and examining it microscopically for the presence of gametes. In instances where
no coelomic gametes were present, the sex of the individual could not be determined and was recorded
as "unknown."
0
0
59
60
BULLETIN OF MARINE SCIENCE, VOL. 41, NO. I, 1987
Table I. Percentage of males and females of Themiste lageniformis from three Florida locations
during 1977-1979
% Identifiable
Location
Boot Toe Point
(Ft. Pierce Inlet)
Bessie Cove
(St. Lucie Inlet)
Missouri Key
Males
Number of
undetermined sex
Total number
96.7
3.3
9
411
95.9
100
4.1
0
5
0
79
5
Females
The progression of oogenesis was monitored by measuring the diameter of coelomic oocytes. A
sample of coelomic fluid was removed with a syringe and needle from each of 10 females selected at
random from the monthly collection. An ocular micrometer was used to measure the diameter of 50
oocytes in each female.
Observations of normal embryonic development were made on laboratory cultures maintained in
IIS-mm diameter glass finger bowls with approximately 200 ml of sea water. The water was changed
every other day. The cultures were maintained at a temperature of 24-26°C and observed regularly
with stereo and compound light microscopes.
RESULTS
Sex Ratio. - The percentage of males and females and the number of "unknowns"
at each of the collection sites is shown in Table 1. At each location the females
far outnumbered the males. At Boot Toe Point and at Bessie Cove where substantial populations of T. lageniformis are present, the populations consist of
approximately 96% females and 4% males. Each of the five specimens collected
at Missouri Key were determined to be female but the small sample size leaves
the precise estimation of the sex ratio of that population in question. Little variation was present in the percentages of males and females at Boot Toe Point over
the 19-month study-period (Fig. 1). The percentage of females in the samples
ranged from 92% to 100%.
On the basis of their external morphology, males of T. lageniformis cannot be
distinguished from females. To test for a size-based sexual dimorphism, the wetbody weight of males and females collected from Boot Toe Point was determined
and plotted (Fig. 2) and the distributions compared. A Kolmogorov-Smirnov test
for goodness of fit (Sokal and Rohlf, 1969) was applied to the data and the result
indicated that the two distributions were not significantly different (P > 0.05).
Oogenesis. - The gonads ofsipunculans are small bits of tissue situated at the base
of the retractor muscles. Only the earliest stages of gametogenesis take place in
the gonad, however, since gametes soon are released to float freely within the
coelomic fluid. In oogenesis, only the growth phase occurs in the coelom. When
oocyte growth is complete, the nephrostome "recognizes" and selectively removes
these oocytes from the coelom and stores them in the sac-like nephridium until
they are released during spawning.
The size-percent frequency distributions of coelomic oocytes in specimens collected from September 1977 through March 1979 is shown in Figure 3. The
smallest coelomic oocytes are about 10 ILm in diameter and the largest reach
nearly 200 ILm. The shapes of the size-percent frequency distributions varied
considerably with time. During March 1978 and February and March 1979 the
distributions were distinctly unimodal. The modal classes for these months were
35 ILm, 35 ILm, and 53 ILm respectively. From April through August 1978 the
PILGER: SIPUNCULAN
100
~
~
S
~
e-e
e
e-----e_/
"'--/
e
61
REPRODUCTION
e
"'-e
__
,
/
e
"e-e-e-e
"'-e
90
u
"...
OJ
()
OJ
0..
80
SON
D
1977
J
F
M
AM
J
J
1978
A
SON
D
J
F
1979
Figure I. Variation in percentage of females of T. lageniformis at Boot Toe Point from September
1977 through February 1979. No obsel"Vations were made during December 1977.
distribution became increasingly bimodal, indicating that the small oocytes were
growing into larger size classes. Because the percent frequency of small oocytes
was not reduced significantly during this period, one can assume that coelomic
oocyte production kept pace with the growth of small oocytes into the larger size
classes. The percent frequency of the model class representing the larger sized
oocytes was greater than the percent frequency for the smaller oocytes during July
and August. September, October, and November 1978 were characterized by a
gradual reduction in the percent frequency of both modal classes. During both
December through March periods there was a progressive increase in the percentage of small coelomic oocytes while large oocytes were absent or in the minority.
Spawning. - When spawned, the oocytes of T. lageniformis had an adhesive coating which made them readily stick to any solid surface. This property diminished
steadily and was no longer effective when the larvae began to crawl during the
second day of development.
The record of spawning frequency is depicted in Figure 4. Repetitive spawnings
by single individuals occurred many times during the study period. One particularly fecund female spawned five times during a 6-day period. Spawning begins
in mid-July and continues through January or possibly February. One individual
spawned during the May-June 1978 observation periods and none spawned between that period and the mid-July period. The greatest frequency of spawning
occurred during August-September when a total of 66 spawns were recorded,
representing nearly 40% of the study group. The frequency of spawns tapered off
slowly from December, 1977 through February, 1978 but ended more abruptly
at the end of the next season.
Development. - In early observations of T. lageniformis from Florida, Rice (pers.
comm.) noticed that laboratory cultures of adults would quickly come to contain
Themiste larvae. When the adults were maintained in isolated compartments
larvae still appeared. These observations led to the hypothesis that reproduction
in Themiste lageniformis occurs through parthenogenesis.
Based on these observations, simple isolation experiments were conducted and
histological examinations made to test the hypothesis that parthenogenesis is
taking place and to test the alternative hypotheses of hermaphroditism or sperm
storage by females. The results of these studies showed that isolated females of
T. lageniformis from Florida always spawn eggswhich begin to cleave and develop
without apparent fertilization. It did not matter whether the female had been
associated with other worms in its oyster cluster or not. Morphologically, females
do not have special modifications of their reproductive systems which might
62
BULLETIN OF MARINE SCIENCE, VOL. 41, NO. I, 1987
0.40
0·30
FEMALES n=346
MALES
n==16
0.30
0.40
6
•
.1
I
.2
•
.3
•
.4
WEIGHT
I
.
.5
I
.6
•
.7
•
.8
(g)
Figure 2. Relative frequency of wet-body weight of males and females collected from Boot Toe Point.
The two distributions are not significantly different (Kolmogorov-Smimov test, P > 0.05).
facilitate sperm storage and internal fertilization. Finally, careful examination of
the coelomic fluid revealed no evidence of sequential or simultaneous hermaphroditism.
Early development of these partheno-produced embryos is typical of many
sipunculans. The larval stage differs from other sipunculans in that the trochophore
stage is absent and a crawling, lecithotrophic pelagosphera is produced by the end
ofthe second day. These larvae are very active and frequently extend and retract
their introverts. Although swimming begins when they are about 3 days old they
never totally give up crawling and they gradually revert back to that mode of
travel. When they are approximately 14 days old the larvae can no longer swim.
This is due to morphological changes such as their increase in size and the loss
of the metatroch. Although some individuals rapidly glide along the bottom of
the culture dish, the terminal organ soon begins to produce adhesives which will
anchor the larva to the bottom, to other larvae, or to debris.
Metamorphosis is a gradual process whereby the larva assumes the adult characters. Although some of the most dramatic changes take place in the head, little
is known about the mechanisms which cause these changes. When the larva is 7
days old hooks become evident on the introvert and a pair of tentacle buds begin
to form dorsal to the mouth. Further development includes the elaboration of
the tentacular crown and the change of the mouth opening from its ventral position
to a terminal location within the tentacular ring.
DISCUSSION
Biased sex ratios are not uncommon in the Sipuncula. The species which show
this phenomenon include Golfingia minuta (Keferstein, 1863), G. vulgaris (Keferstein, 1863), G. elongata (Keferstein, 1863; Claparede, 1863), G. pugettensis
63
PILGER: SIPUNCULAN REPRODUCfION
10%
200
180
160
E
.3
L
"
"
~
E
•
'"
~
>-
c
u
0
c
..
0
Sept.
Oct.
Nov.
Dec.
~
Jan.
~
Feb.
Mar.
1977
Apr.
May
June
July
Aug.
1978
200
'~D
~
L
"
"
E
'"
~
">-
c
u
0
c
~
Sept.
Oct.
Nov.
1978
Dec.
Jan.
Feb.
Mar.
1979
Figure 3. Size-percent frequency distributions of coelomic oocytes in specimens collected at Boot
Toe Point from September 1977 through March 1979. Scale bar represents 10%. • No measurements
made.
(Cole, 1952), and Themiste lageniformis (Awati and Pradhan, 1936; Pilger, 1978).
In all of these species it is the females which outnumber the males. Golfingia
minuta has since been determined to be a protandrous hermaphrodite (Akesson,
1958) and it is assumed that Keferstein's (1863) observation was based on a
population which was at a point in the cycle when the female condition was
dominant.
Studies by Awati and Pradhan (1936) of Themiste lageniformis from India
revealed a sex ratio of 60 females to I male. They did not investigate this phenomenon further but speculated that it may be due to protandry, fertilization by
minute males, or parthenoglusis (sic). Williams' (1977) study of development of
Themiste lageniformis from Hawaii did not report sex ratios.
The annual cycle of oogenesis in T. lagentformis is similar to the cycles reported
for Golfingia min uta (Gibbs, 1975), G. vulgaris (Gonse, 1956a; 1956b), and G.
pugettensis (Rice, 1975), Phascolosoma agassizii (Rice, 1975; Towle and Giese,
1967) and Themiste petricola (Amor, 1977) in that small oocytes are present in
the coelomic fluid throughout the year and that the oocyte size frequency distributions become bimodal as oogenesis progresses. The only other known sipunculan reproductive cycle is that of Phascolosoma arcuatum (Green, 1975). In that
64
BULLETIN OF MARINE SCIENCE, VOL. 41, NO. I, 1987
70
'"
~
60
'0
...•
>
:;; 50
...•"
g
.-<
•....
40
~
30
'"
0.
'"
•••
20
i"
10
o
z
SON
0
1977
J
F
M
AM
J
1978
J
A
SO
NO
J
F
MA
1979
Figure 4. Spawning observations oflaboratory maintained specimens from September 1977 through
April 1979. Data for each interval represent observations on 100 individuals collected at the beginning
of each interval. * No observations during interval.
species, oocytes were completely absent from the coelomic fluid during March
and April. Size frequencies were not computed for p, arcuatum so a comparison
of modal characteristics cannot be made.
The distinctly bimodal character of the oocyte size frequency distributions of
T. lageniformis is very interesting when one considers that the modes do not
march, that is, they do not progress through the size classes from small to large.
The small modal class ranges from 22 /-tm to 53 /-tm (mean = 37 /-tm) while the
large modal class ranges from I 19 /-tm to 167 /-tm (mean = 140 /-tm). Modes are
never present in the 53 /-tm to 119 /-tm size range. Because small oocytes are present
in the coelomic fluid throughout the year (i.e., continuous production) and there
is no evidence for differential loss of oocytes, the frequency of each oocyte size
class may be interpreted as being proportional to the amount of time an oocyte
spends in a particular class. The modes, therefore, may represent size ranges in
which the rate of oocyte growth is much slower than the flanking size ranges. The
slow periods may represent a time when a complex biosynthetic activity such as
vitellogenesis is taking place. A biochemical study of oogenesis is necessary to
test this hypothesis.
Two sipunculan species are known to reproduce asexually. Sipunculus robustus
produces new individuals by transverse fission and lateral budding (Rajulu and
Krishnan, 1969; Rajulu, 1975) while asexual reproduction in Aspidosiphon brocki
involves only transverse fission (Rice, 1970). Although gonads are present in both
species, sexual reproduction has not been observed in either of them.
The present study examined reproduction in the sipunculan Themiste lagenijormis. Females of this species from the central east coast of Florida were found
to produce eggs which developed spontaneously after spawning. Parthenogenesis
is the best explanation for this phenomenon since other explanations, such as
sperm storage and hermaphroditism with selffertilization, were not supported by
the results.
This represents the first report of parthenogenesis in the Sipuncula. Interestingly,
however, it is not the first time that it has been suggested for this species. Awati
and Pradhan (1935; 1936) studied populations of T. lageniformis from India and
documented an extremely biased sex ratio where females outnumbered males 60
to 1. Three possible explanations were given: (1) protandry, (2) fertilization by
PILGER: SIPUNCULAN
REPRODUCTION
65
minute males, and (3) facultative parthenoglusis (sic). They did not note whether
or not eggs spawned by isolated females would develop spontaneously. In light
of the present study, however, facultative parthenogenesis is the explanation of
choice. I am not aware of any observations of T. lageniformis from India since
the work of Awati and Pradhan.
While parthenogenesis is the probable reproductive mode in the Indian population of T. lageniformis, the situation in Hawaii is much clearer. Specimens
collected during November 1984 near Coconut Island, Hawaii and maintained
in isolated compartments in the laboratory spawned eggs which activated and
developed spontaneously just as the F10rida specimens had done (c. L. Hunter,
pers. comm.).
Parthenogenesis is an uncommon reproductive mode among phyla closely related to the Sipuncula. No members of the Echiura, for instance, have been
reported to use this method of reproduction (Gould-Somero, 1975). Among the
polychactous annelids, Gibson (1977) could not find males in populations of
cirratulids from South Africa, although females contained oocytes that were ready
for spawning. Based on these observations, he concluded that parthenogenesis
did occur. Two species of marine enchytraeid oligochaetes are known to reproduce
parthenogenetically (Lasserre, 1975). In these, sperm is required to initiate egg
activation but male and female pronuclei don't always fuse (Christensen, 1960).
Such cases of pseudo fertilization are not examples of pure parthenogenesis because
sperm are necessary for egg activation and development. They are, however,
parthenogenetic in the cytogenetic sense, since male chromosomes do not fuse
with female chromosomes and they are not incorporated into embryonic cells.
Other animals undergo cyclical parthenogenesis or heterogony in which parthenogenetic generations alternate with sexual generations in a more or less regular
fashion (White, 1973). Such organisms have the advantages of dispersal and
recolonization in their parthenogenetic state and genetic variability in their sexual
state. Cyclical parthenogenesis is not likely in T. lageniformis because, in several
years of observation, non parthenogenetic females have never been discovered.
Parthenogenesis as a mode of reproduction in T. lageniformis raises many
interesting questions that relate to its importance in the animal's life history, to
sex determination of offspring, and to the cytogenetics of the process.
Whether parthenogenesis in T. lageniformis is obligatory or facultative is not
clear. If it is obligatory, then what is the function of males? Males are generally
absent in populations where parthenogenesis is obligatory (White, 1973). If parthenogenesis is facultative, then fertilization should take place ifeggs are spawned
in the presence of sperm. Due to the paucity of males and the temporal problems
involved with having a female spawn in the presence of freshly spawned sperm,
it has not yet been established whether or not the male nucleus enters an oocyte
spawned in these conditions. Nonetheless, the presence of males in the population
leads me to hypothesize that parthenogenesis is facultative and that, due to the
scarcity of males, sexual reproduction is a rare event.
The biased sex ratio may be explained by the mode of reproduction in Themiste
lageniformis. My working hypothesis is that parthenogenesis produces only females (thelytoky) and that sexual reproduction produces both males and females
(deuterotoky). Given the facts that males comprise only 4% ofthe population and
that unfertilized eggs are always capable of activating parthenogenetically, then
fertilization must be considered a relatively rare event. Ifmales are produced only
by this rare event, and females are produced by it in addition to the very common
process of parthenogenesis, a disproportionate sex ratio would result with females
far outnumbering males. This hypothesis can be tested by raising partheno-pro-
66
BULLETIN OF MARINE SCIENCE, VOL. 41, NO.1,
1987
duced and sexually produced larvae to sexual maturity and comparing the sex
ratio in the offspring.
A further approach to the understanding of parthenogenesis in T. lageniformis
is through the cytogenetics of the system. Suomalainen (1950) classified cytogenetic systems of parthenogenetic organisms on the basis of whether the adults are
haploid or diploid. In haploid parthenogenesis, individuals develop from haploid
eggs and are haploid as adults. In diploid parthenogenesis the individuals are
usually diploid or sometimes polyploid. These may be the result of automixis
where haploid eggsdevelop parthenogenetically but the diploid chromosome number is restored by fusion of the egg nucleus with one of the polar body nuclei or
by fusion of two of the sister nuclei in one of the early cleavages. Apomictic
individuals, on the other hand, develop from diploid eggs in which chromosome
reduction has not taken place.
The ideal approach to the study of this aspect of parthenogenesis is to determine
the chromosome numbers of specific stages in the life history of the animal through
squash techniques. Unfortunately, the large amount of yolk in Themiste eggs
obscures the chromosomes such that accurate and precise counts have not been
obtained. An alternative strategy to understanding the cytology is to use cytophotometric techniques on Feulgen stained cells and tissues from various stages
in the life history to obtain relative DNA values. Preliminary results with this
technique indicated that Feulgen-DNA values for parthenogenetic and zygogenic
embryos did not differ significantly from each other. At first glance these results
appear to support diploid parthenogenesis as the operational mechanism since
partheno-produced individuals have the same DNA values as diploid forms.
These results are ambiguous, however, in light of the fact that it is not known if
the male nucleus participates in fertilization or if the oogonia undergo meiosis
prior to spawning.
Although many unanswered questions have arisen from the present study, it is
clear that reproduction in Themiste lageniformis from the central east coast of
Florida is characterized by an annual cycle of oogenesis and spawning and that
parthenogenetic reproduction is used by this and other populations ofthe species.
ACKNOWLEDGMENTS
This research was supported by a Smithsonian Institution postdoctoral fellowship. It is a pleasure
to thank Dr. M. E. Rice for her encouragement, stimulating discussions, and unceasing generosity in
my use of the Smithsonian Marine Station facilities. The support facilities of Agnes Scott College are
gratefully acknowledged.
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PILGER:SJPUNCULAN
REPRODUCTION
67
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---.
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---.
1970. Asexual reproduction in a sipunculan worm. Science 167: 1618-1620.
---.
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agassizii. Physio!. Zoo\. 40: 229-237.
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DATEACCEPTED: October 27, 1986.
ADDRESS: Department of Biology. Agnes Scott College, Decatur, Georgia 30030.