Environmental split between germ cell parasitism and somatic cell synergism in chimeras of a colonial urochordate
Israel Oceanographic and Limnological Research, National Institute of Oceanography, Tel Shikmona, POB 8030, Haifa 31080, Israel
* Author for correspondence (e-mail: buki{at}ocean.org.il)
Accepted 12 July 2004
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Summary |
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Key words: Botryllus, chimerism, fusion-rejection, green-beard allelism, stem cell
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Introduction |
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Colonies and subclones grow by lateral expansion on the substrate. When
different colonies meet their peripheral ampullae under natural or laboratory
conditions, they may anastomose to form a vascular parabiont (cytomictical
chimera; Rinkevich and Weissman,
1987a). This occurs if they share one or both alleles on a highly
polymorphic haplotype (Oka and Watanabe,
1957
; Sabbadin,
1982
; Scofield et al.,
1982
), the fusibility/histocompatibility (Fu/HC;
Weissman et al., 1990
) locus.
Thereafter, during a period of a few days to several months, massive apoptosis
and phagocytosis processes, called `colony resorption', morphologically
eliminate all zooids originating from one of the genotypes within a chimera
(Rinkevich and Weissman,
1987a
; Weissman et al.,
1990
; Rinkevich et al.,
1993
). This phenomenon appears to be genetically controlled by
multi-level hierarchical organization of the Fu/HC and additional
histocompatibility alleles (Rinkevich et
al., 1993
).
It has been suggested that the development of such a complex, genetically
based, selfnonself recognition system emerged as an adaptation for
weakening the threat of cell lineage competition and parasitism
(Buss, 1982;
Grosberg and Quinn, 1986
), a
possible scenario resulting from natural fusions in the wild
(Ben-Shlomo et al., 2001
).
However, by using polymorphic microsatellite markers, recent studies
(Pancer et al., 1995
;
Stoner et al., 1999
) have
demonstrated that, even after complete morphological resorption of one partner
in an anastomosed entity, the blood, the soma and the germ cells of the
`winner' partner are, in many cases, chimeric, pointing to cell lineage
parasitism (Pancer et al.,
1995
; Stoner and Weissman,
1996
; Magor et al.,
1999
; Stoner et al.,
1999
). Moreover, it is also common to find outcomes where the
whole mass of gonads, as well as the soma, is derived from the resorbed
genotype. The germ cell parasitism events are reproducible, show hierarchical
patterns and are sexually inherited, as shown by breeding experiments
(Stoner et al., 1999
). Somatic
cell parasitism, on the other hand, while reproducible under controlled
mariculture conditions and characterized by hierarchical organization, does
not reveal the trait of sexual inheritance through a pedigree
(Stoner et al., 1999
).
Therefore, gametic and somatic competitive routes, although reproducible,
appear to be unlinked (Stoner et al.,
1999
; Magor et al.,
1999
).
Botryllus chimeras are common in nature
(Ben-Shlomo et al., 2001).
Several studies (Buss, 1982
;
Grosberg and Quinn, 1986
;
Rinkevich and Weissman, 1992
;
Rinkevich and Shapira, 1999
;
Rinkevich, 2002
) were set up
to define the evolutionary significance of natural chimeras by evaluating the
fitness costs and benefits of chimerism as compared with the state of
genetically homogeneous entities or by analysing bi-vs-multipartner
chimeras. No definite benefit was recorded in the kingdom Animalia for any
case of chimerism. Conversely, scientific interest focused on the possible
threat of germ cell parasitism in chimeras, which is particularly relevant for
organisms where germ cell sequestration remained undetermined until late in
ontogeny or when their full attainment along the life span of the organism was
not achieved (Buss, 1982
,
1983
). No such analysis has
been devised for a case where germ cell parasitism and somatic cell parasitism
split. In the present study, we test the hypothesis that directionality of
somatic cell parasitism in Botryllus chimeras is a plastic trait. We
found that, under adverse environmental conditions, the chimera entity
continuously responds to natural selection forces by exhibiting different
phenotypic combinations of its genetic components. This leads to two
conflicting types of interactions that are simultaneously exhibited by the two
genotypes: germ cell parasitism vs somatic cell synergism.
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Materials and methods |
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Tissue processing
Tissue samples were taken from zooids deprived of gonads. A longitudinal
incision was made between the atrial and branchial siphons with a fine needle.
Both edges of the incision were retracted with fine forceps, and zooids were
lifted and removed with a fine needle and forceps. In sexually matured
colonies, the male gonads were carefully removed with this needle, washed
several times with sterile (0.22 µm) seawater and each was placed in a
small Petri dish submerged in a small drop of seawater. The tip of a sharp
needle was used to release the sperm, which were collected using a Pasteur
pipette. Blood was collected by cutting (with industrial metal blades) the
peripheral ampullae of colonies growing on glass slides that were wiped
beforehand with soft towels and placed under a dissecting microscope. Released
blood cells and haemolymph were collected using pulled glass
micropipettes.
For microsatellite analyses, samples were placed, separately, into 1.5 ml
vials containing 240 µl lysis buffer
(Graham, 1978), homogenized and
extracted with 240 µl phenol:chloroform:isoamylalcohol (25:24:1). DNA
samples were precipitated in ethanol, resuspended in water and used for
microsatellite analyses, as described previously
(Pancer et al., 1995
;
Stoner et al., 1999
). In the
present study, all tissue samples were typed along with microsatellite PB-41
(Stoner and Weissman, 1996
;
Stoner et al., 1997
). In the
amplified fragment length polymorphism (AFLP) analyses, for each genotype,
512 specific loci (resolved bands on the sequencing gel) were first
assigned as described previously
(Rinkevich et al., 1998
).
Since AFLP loci can differ by as much as 10-fold in their ability to be
amplified from low concentrations
(Rinkevich et al., 1998
), a
semi-quantitative evaluation was established as follows: appearance of <20%
of genotypic specific bands revealed only traces of the corresponding
genotype's DNA.
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Results and discussion |
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In the first Fu/HC compatible combination, two partners (A and B) were
categorized with the Fu/HC unlinked microsatellite locus PB-41 as: partner A =
heterozygous alleles 173 bp and 178 bp; partner B = homozygous allele 176 bp
(Fig. 1A). Six chimeras made of
subclones from partners A and B were randomly split into three temperature
regimen (15°C, 20°C, 25°C) groups. Chimeras were monitored for up
to 10 months and were sampled (blood, gonads and total zooid tissue) four
times during this period (Table
1; Fig. 1). In two
cases (chimeras 1, 6; Table 1),
one of the partners was morphologically resorbed within the first two months
after chimeral establishment, and in four cases (chimeras 25), stable
chimeras (Rinkevich and Weissman,
1987b) were established 810 months after allogeneic
fusions. In a single case (chimera 6; Table
1), a chimeral death
(Rinkevich and Weissman,
1987b
) was recorded. In chimeras that had been maintained for 8
and 10 months after fusion at 15°C and 20°C temperature regimen, the
zooidal soma consisted, in most cases, of both partners. On the other hand, in
the 25°C treatment, genotype B disappeared from the soma of 2-month-old
chimeras but appeared in the male gonads. Some of the outcomes for blood cells
were consistent with the sperm analyses
(Table 1), pointing to the
existence of circulating germ cells in the blood
(Sabbadin and Zaniolo, 1979
).
Male gonads in chimeras that had been grown at 15°C and 20°C revealed
only the A genotype. This outcome was further confirmed by self-crosses made
with both of the 20°C chimeras and one of the 15°C chimeras (chimera
2; Table 1) at age 810
months. These crosses were made to test the extent to which the microsatellite
analysis of sperm was a good predictor of progeny production by gametes from
both testes and ovaries. Fifteen progenies were collected and tested; all
revealed the A genotype only (Fig.
1B depicts 12 offspring).
|
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The other three sets of Botryllus chimerical combinations were
sampled 1.52 months after chimeral establishment
(Table 2;
Fig. 1C). Using AFLP
fingerprinting profiles (Rinkevich et al.,
1998), it was possible to elucidate, independently, the
sensitivity of each specific AFLP locus in a mixture of genotypes and to
define cases where only traces of the genotypic DNA of interest are found
(Table 2). In two of the three
genotypic combinations (CD and GH), the fingerprints of the
chimeric soma differed between those grown in ambient temperature (20°C)
and most of those grown in the two extreme maintenance conditions (15°C,
25°C; Table 2;
Fig. 1C). In chimeric
combination CD, both genotypes appeared in the two 20°C chimeras
while only genotype D was recorded in the soma of two of the chimeras grown
under extreme temperatures (chimeras 1 and 5) and only traces were recorded in
the third chimera (chimera 2). In zooidal soma analyses of combination
GH, genotype H (but not G) was expressed only in 20°C chimeras, as
opposed to the chimeras grown at extreme temperatures, where both genotypes
appeared simultaneously. Sperm identity was consistent and unchanged at all
temperature regimens and in all three chimeral combinations (12 chimeras).
Only in combination EF did soma fingerprints appear to be the same at
all temperatures.
|
Thus, this study shows that the somatic constituent of chimeric
Botryllus entities is a plastic event. The competitive (parasitic)
relationships of interacting genotypes, under variable environmental
conditions (seawater temperature was used here as an example of an
environmental parameter), are frequently altered. This outcome contradicts the
state of germ cell parasitism, where almost no alterations were recorded
(Tables 1,
2;
Stoner et al., 1999) and where
directionality was probably dictated genetically by primitive germ cell
lineage hierarchy (Stoner et al.,
1999
).
Natural chimerism is a common phenomenon, not only in the
Botryllus system. Chimeras from a variety of protists, plants and
animals are documented in nature (Buss,
1982). Several studies (Buss,
1982
; Rinkevich and Weissman,
1987b
,
1992
;
Rinkevich and Shapira, 1999
)
have dealt with the evolutionary significance of chimerism by evaluating
fitness costs and benefits of the chimeral entity, relative to the state of
genetically homogeneous individuals. Of all the different suggested classes of
benefits for chimerism (none has been recorded in organisms more developed
than primitive slime molds and algae;
Rinkevich and Shapira, 1999
),
one has focused on the possible synergistic impact of both partners' genetic
constituents on the chimeric fitness
(Rinkevich and Weissman,
1987a
; Rinkevich and Shapira,
1999
). It was suggested (Buss,
1982
) that since a chimera has a greater store of genetic
variability, and hence a wider range of effective physiological qualities and
characteristics, this organismic state may tolerate a greater range of
environmental variation than the organismic state of genetically homogeneous
entity.
Within chimeras of Botryllus schlosseri, parasitic germ lines
hitchhike and pass throughout successive generations without being visible to
natural selection forces (Pancer et al.,
1995; Stoner et al.,
1999
; Rinkevich,
2002
,
in press
). Hitchhiking onto
the soma of positively selected genotypes provides the parasitic forms with
the inevitable advantage of establishing new progenies. However, this may
eventually turn into a Pyrrhic victory (Rinkevich,
2002
,
in press
) by causing possible
development of super-parasitic entities, specialized in allogeneic invasion
and germ cell parasitism. Three evolutionary selected mechanisms
(diversification of fusibility allele repertoire, the establishment of
multichimeric entities and the induction of programmed life spans) reduce
opportunities for parasitic forms to hitchhike to a high frequency with
selected genotypes and may shape more benign germ cell parasitic forms that
share overlapping future expectations with their hosts
(Rinkevich, 2002
). These
benign forms are expected to contribute cells for somatic functions, forming
entities with fitnesses that depend on the combined genomic fitness of the
partners, as seen in the present study. It should also be taken into
consideration that, while intraspecific interactions within chimeras go on,
larger botryllid chimeras successfully control feeding substrates. This should
effectively prevent colonization of that surface area by other competitive
species and/or may increase interspecific competitive abilities
(Rinkevich and Shapira,
1999
).
The apparent synergism for fine-tuning a plastic combination of the genetic
components in Botryllus chimerical soma (to better fit adverse
environmental conditions) may represent a typical form of a green-beard
(Dawkins, 1976) allelism on
historecognition elements. All components of a green-beard effect
(Dawkins, 1976
;
Haig, 1996
;
Keller and Ross, 1998
;
Riley and Gordon, 1999
;
Queller et al., 2003
)
a detectable phenotypic feature (the Fu/HC allele), the ability to
recognize this feature and the ability to respond [in the Botryllus
system: three sets of different responses are expressed towards genotypes
possessing or not possessing this feature: fusion and rejection
(Oka and Watanabe, 1957
;
Sabbadin, 1982
;
Scofield et al., 1982
),
allogeneic resorption (Rinkevich and
Weissman, 1992
; Rinkevich et
al., 1993
), somatic and germ cell parasitism
(Rinkevich and Weissman,
1987a
; Pancer et al.,
1995
; Stoner and Weissman,
1996
; Magor et al.,
1999
; Stoner et al.,
1999
; Rinkevich,
2002
)] are present in Botryllus chimeras and are
mediated by a single green-beard allorecognition haplotype.
In the wild, any single Botryllus population at any specific time
represents unprecedented extensive polymorphism of Fu/HC alleles (up to
several hundred; Rinkevich et al.,
1995), a phenomenon that may reduce the chances of forming natural
chimeras. However, preferential gregarious settlement of Fu/HC compatible
oozooids in nature (Grosberg and Quinn,
1986
) results in compatible allogeneic contacts and high
percentages of Botryllus chimeras
(Ben-Shlomo et al., 2001
). In
the case of a natural chimera (i.e. carrying AB vs AC
allorecognition alleles), the shared selfish allorecognition `A' allele will
always attain its 50% share in the germ line, regardless of any germ line
hierarchical combination (i.e. AB or AC colony is the winner).
At the same time, chimeric fitness is synergistically fine-tuned by all its
genetic constituents to fit changes in environmental conditions (such as
seawater temperature). This cooperation on the somatic level clearly benefits
the chimera as compared with genetically homogenous Botryllus
colonies. In the Botryllus system, therefore, a single
allorecognition green-beard allelism directly assesses allelic kinship and
simultaneously modifies two `conflicting' responses within a whole chimeric
entity (germ cell parasitism vs somatic cell cooperation). It not
only `ensures' its proportional transmission to subsequent generations but
also enhances fitness of the entity (the vehicle; sensu
Dawkins, 1976
) that houses the
germ line. The genotype that wins the soma is, thus, neither sporadic nor
random, and the synergistic expression of somatic constituents is dictated by
selfish genetic elements working through green-beard effects. It is also
interesting to note that the green-beard gene in the red fire ant system
(Keller and Ross, 1998
) is also
hallmarked by widespread polymorphism.
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Acknowledgments |
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