2821
The Journal of Experimental Biology 201, 2821–2831 (1998)
Printed in Great Britain © The Company of Biologists Limited 1998
JEB1615
MORPHOLOGICAL, PHYSIOLOGICAL AND METABOLIC COMPARISONS
BETWEEN RUNNER-LIKE AND SHEET-LIKE INBRED LINES OF A COLONIAL
HYDROID
NEIL W. BLACKSTONE*
Department of Biological Sciences, Northern Illinois University, DeKalb, IL 60115, USA
*e-mail: [email protected]
Accepted 21 July; published on WWW 22 September 1998
Summary
variaton in flow, the redox state of the polyp
Hydractiniid hydroids display a range of morphological
epitheliomuscular cells was measured using the
variation from sheet-like forms (i.e. closely spaced polyps
fluorescence of NAD(P)H. In response to feeding-induced
with high rates of stolon branching) to runner-like forms
changes in gastrovascular flow, runner-like colonies show
(i.e. widely spaced polyps with low rates of stolon
greater redox variation than sheet-like ones, plausibly
branching), thus exemplifying the patterns of heterochrony
corresponding to the greater amounts of flow generated by
found in many colonial animals. A sheet-like and a runnerthe former colonies relative to the latter. Perturbing the
like inbred line of Podocoryne carnea were produced to
investigate this heterochronic variation further. Selection
system with dilute solutions of 2,4-dinitrophenol similarly
on colony morphology at the time of the initiation of
indicates that runner-like colonies contain more
functionally oxidizable NAD(P)H. The correlation between
medusa production resulted in dramatic differences by the
gastrovascular flow and morphological differences
F5 and F6 generations. Compared with colonies of the
supports the hypothesis that the former mediates the
sheet-like inbred line, runner-like colonies exhibited
timing of colony development, perhaps in concert with the
smaller sizes at the initiation of medusa production, more
irregular colony shapes and diminished stolon development
observed variation in the redox state of polyp
relative to polyp development. In addition to these
epitheliomuscular cells.
differences in colony morphology, runner-like colonies also
exhibited larger medusae and a greater amount of
gastrovascular flow to the peripheral stolons. To assess
Key words: clonal, colony, heterochrony, hydroid, inbreeding,
NAD(P)H fluorometry, Podocoryne carnea, redox state, metabolism.
differences in the metabolic capacity underlying this
Introduction
Heterochrony, an evolutionary change in the timing of
development, is a pervasive theme in studies of evolutionary
morphology (e.g. Gould, 1977; Alberch et al. 1979; Bonner,
1982; McKinney, 1988; Wake et al. 1991; McNamara, 1997).
Nevertheless, the factors underlying most morphological
heterochronies remain to be elucidated. Are morphological and
life history heterochronies direct consequences of genetic
heterochronies? Do metabolic and physiological parameters
mediate heterochronic variation in some cases? Can seemingly
unrelated genetic changes produce similar heterochronies by
affecting the same metabolic or physiological properties? Here,
selection and inbreeding experiments (e.g. Green, 1981;
Monteiro et al. 1997a,b; Wilkinson et al. 1998) and
morphological, physiological and metabolic assays of the
resulting inbred lines are used to investigate the mechanistic
basis of heterochrony in colonial hydroids.
Patterns of heterochrony in hydractiniid hydroids have been
well characterized (Blackstone and Buss, 1991; Blackstone,
1992, 1996). Species of Hydractinia and Podocoryne generally
exhibit contrasting suites of morphological and life history
traits (‘sheets’ versus ‘runners’) and in this way exemplify
patterns characteristic of many colonial animals (Buss and
Blackstone, 1991). The morphology of these organisms can be
idealized as comprising feeding and reproductive entities (e.g.
polyps) that are connected to other entities by a fluid-carrying
system (e.g. stolons; see Harper, 1977; Jackson, 1979).
Runner-like forms show widely spaced polyps with little stolon
branching and anastomosis, while sheet-like forms show
closely packed polyps with extensive stolon branching and
anastomosis. These different morphological patterns
correspond to changes in the timing of the production of polyps
and stolon tips relative to rates of stolon growth and colony
maturation: high rates of production yield sheets, while low
rates yield runners. Furthermore, in hydractiniid hydroids,
relative rates of polyp and stolon production show an inverse
correlation with rates of gastrovascular fluid flow to peripheral
2822 N. W. BLACKSTONE
stolon tips. Compared with colonies of Podocoryne carnea,
mature colonies of Hydractinia symbiolongicarpus exhibit a
low rate of flow to peripheral stolons (Blackstone and Buss,
1992; Blackstone, 1996).
Experimental studies of heterochrony allow the betweenspecies pattern in these hydroids to be mimicked by
manipulation of colonies of a single species (Blackstone and
Buss, 1992, 1993; Blackstone 1997a, 1998; for analogous
studies of other organisms, see for example Stebbins and
Basile, 1986; Meyer, 1987; Müller, 1991). Putatively, these
hydroids incur substantial energetic costs in circulating the
gastrovascular fluid throughout the colony. The application of
dilute solutions of 2,4-dinitrophenol to colonies of Podocoryne
carnea results in a condition of ‘loose-coupling’ of oxidative
phosphorylation, a decrease in the amount of ATP available
for generating gastrovascular flow and a consequent
diminution of the rate of flow to peripheral stolons. Correlated
with this diminished flow are changes that parallel patterns of
heterochrony: the rates of production of polyps and stolon tips
increase relative to rates of stolon growth and colony
maturation. Alternatively, gastrovascular flow can be
diminished by increasing the frequency with which a colony is
fed (e.g. from three to six times per week), possibly because a
higher rate of feeding increases the resistance of the stolon
tissues to fluid flow or the absorption of fluid by these tissues,
or both (Blackstone, 1997a; Van Winkle and Blackstone,
1997). Similar to treatment with uncouplers, feeding
manipulation results in changes which parallel patterns of
heterochrony; again, the rates of production of polyps and
stolon tips increase relative to rates of stolon growth and
colony maturation (see also Braverman, 1974). While
increased feeding produces a surfeit of nutrients and seems in
many ways the opposite of the energy-poor state produced by
uncoupling, its effects on colony physiology (e.g. flow rate) are
similar. In combination, the between-species data (Blackstone
and Buss, 1992; Blackstone, 1992, 1996) and the experimental
manipulations (Blackstone and Buss, 1992, 1993; Blackstone
1997a, 1998) suggest that flow rate is the principal
physiological mechanism underlying heterochronic changes in
these hydroid colonies.
Nevertheless, when the rate of gastrovascular flow in P.
carnea is constant, the rate of polyp and stolon tip initiation
can be increased by further shifting the cellular redox state in
the direction of oxidation (Blackstone, 1997a, 1998). In fact,
uncoupling of oxidative phosphorylation generally triggers
metabolic activation and a shift of the redox state in the
direction of oxidation (Heytler 1981; Hajnóczky et al. 1995),
and these effects are observed in hydroids (Tardent, 1962;
Blackstone and Buss, 1992, 1993; Blackstone, 1997a, 1998).
Uncouplers function as proton ionophores, diminishing protonmotive force, raising the level of ADP, and thus strongly
activating oxidative phosphorylation and shifting the
mitochondrial matrix redox state in the direction of oxidation.
In parallel, feeding triggers strong contractions of the polyp
musculature (Wagner et al. 1998; Dudgeon et al. 1998), and
the increased metabolic demands of these epitheliomuscular
cells are probably responsible for the observed increase in
oxygen uptake and the shift of the redox state in the direction
of oxidation subsequent to feeding (Blackstone, 1997a). Thus,
the experimental data are consistent with a direct effect of
cellular redox state on the rate of polyp and stolon tip initiation,
such that relative oxidation favors a high rate of initiation,
while relative reduction leads to a low rate of initiation. Such
a pattern is, in fact, concordant with the extensive work of an
earlier generation of hydroid biologists (for reviews, see Child,
1941; Tardent, 1963; Rose, 1970).
To investigate the mechanistic basis of heterochrony further
in these hydroids, a sheet-like and a runner-like inbred line of
Podocoryne carnea were produced by selecting colony
morphology at the time of the initiation of medusa production.
The resulting inbred lines were evaluated at the F5 and F6
generations for differences in colony and medusa morphology,
gastrovascular flow physiology and polyp epitheliomuscular
cell redox state.
Materials and methods
Study species
Mature colonies of Podocoryne carnea (Sars) release freeswimming medusae that must feed prior to producing gametes,
and the swimming planula larvae subsequently colonize hermit
crab shells or other surfaces (Edwards, 1972). Colony
development in P. carnea begins with the metamorphosis of
the planula larva into a primary polyp. Stolons extend from the
primary polyp. The stolons encase fluid-filled canals that are
continuous with the gastrovascular cavity of the polyp. In cross
section, stolons consist of a fluid-filled lumen encased by
endoderm, ectoderm and a rigid, acellular perisarc.
Gastrovascular fluid circulates in the lumen of the stolons and
carries food and possibly other metabolites from the feeding
polyp to other parts of the colony; contractions of the muscular
polyp largely propel the gastrovascular fluid (Schierwater et al.
1992; Buss and Vaisnys, 1993; Dudgeon and Buss, 1996; Van
Winkle and Blackstone, 1997). Colony development from a
primary polyp can be mimicked by surgically explanting 1–2
polyps from a colony onto a new surface. In both cases, P.
carnea develops by lineal extension of the stolons, initiation
of new stolonal tips by branching, formation of connecting
stolons by anastomosis and iteration of feeding polyps
(gastrozooids) on the stolons, forming a loose network of
polyps and stolons typical of many runner-like forms. Once the
available surface is covered, P. carnea colonies increase polyp
and stolon tip formation, producing a more closely knit
network of stolons and ultimately initiating the sexual
(medusoid) phase of the life cycle as reproductive polyps
(gonozooids) develop.
In colonies of various sizes, polyp epitheliomuscular cell
contractions commence upon feeding and continue actively for
less than 24 h; waste material is subsequently regurgitated, and
the polyp becomes relatively quiescent until the next feeding
(Wagner et al. 1998; Dudgeon et al. 1998). In response to
feeding, colony oxygen uptake increases and the redox state of
Inbred lines in a colonial hydroid 2823
the epitheliomuscular cells shifts in the direction of oxidation;
when polyps become quiescent, the redox state of these cells
shifts in the direction of reduction (Blackstone, 1997a). Polyp
and stolon tip initiation and stolon growth may occur
principally when polyps are quiescent and not actively
contracting (Blackstone, 1996; N. W. Blackstone, unpublished
data).
Culture conditions
Colonies of P. carnea were collected from the Yale Peabody
Museum Field Station in Connecticut. A male and a female
colony were identified, and clonal replicates of these colonies
were propagated by explanting 1–2 polyps and connecting
stolons onto glass microscope slides. Generally, colonies (i.e.
polyp stages) were grown on glass slides or coverslips
suspended in floating racks and in 120 l aquaria containing
Reef Crystals artificial sea water (salinity 35 ‰) with
temperature control to 20.5±0.5 °C, undergravel filtration and
50 % water changes weekly. Ammonia, nitrites and nitrates
were maintained below detectable levels (Aquarium Systems
test kits). Colonies were fed to repletion with brine shrimp
nauplii 3 days per week. Analysis has shown that, under these
culture conditions, ‘random’ statistical effects (e.g. time
effects, tank effects, rack effects; see Sokal and Rohlf, 1995)
are negligible (Blackstone and Buss, 1991).
All mating experiments were carried out using water from a
tank that was completely free of any hydroids. Medusae were
isolated from mature colonies contained overnight in finger
bowls in an incubator at 20.5±0.2 °C. Medusae were cultured
in finger bowls under similar conditions with daily feedings of
brine shrimp followed by water changes. Under these
conditions, medusae matured and produced gametes in 3–5
days. Subsequent to mating and larval maturation (3 days),
competent larvae were induced to metamorphose by means of
ionic imbalance (53 mmol l−1 CsCl solution, see Blackstone
and Buss, 1991), and metamorphosing larvae were deposited
on 12 mm diameter glass coverslips. Small coverslips were
used in the mating experiments to hasten maturation. In later
assays of morphology and physiology using clonal replicates,
15 mm coverslips were used in part to allow more precise
assays and in part to assess whether colony morphology
remained consistent on a larger surface. On both sizes of
coverslip, colonies were effectively confined to one side of the
coverslip by cutting back encrusting stolons from the reverse
side on a daily basis.
Production of inbred lines
Medusae were isolated from the pair of field-collected
colonies. Male and female medusae were cultured together,
and embryos were isolated after 5 days. Larvae were matured
and metamorphosed onto 12 mm coverslips. F1 colonies were
grown on one side of these coverslips until medusa buds
appeared on gonozooids. Colonies were then imaged, and the
areas of the empty (i.e. unencrusted) coverslip and of the
individual polyps were measured (see below). These
measurements were then used as a guide in selecting two sets
of parents, one set sheet-like, the other set runner-like. These
parents and their offspring defined the two inbred lines.
Subsequent generations of each line were then propagated in a
similar manner. At each generation for the sheet-like line,
extreme sheet-like parents were selected, while for the runnerlike line, extreme runner-like parents were selected. Often,
however, these choices of parents were tempered by
deficiencies in medusa production. Many extreme sheet-like
colonies grew asexually with little or no medusa production,
whereas some extreme runner-like colonies had very few
gonozooids and thus produced too few medusae to be useful
for breeding. The most extreme phenotypes could not therefore
be used for breeding (Fig. 1). In later generations, inbreeding
depression became apparent largely in terms of the viability of
the gametes. Thus, only one F6 colony of the sheet-like line
was successfully produced (Fig. 1). Despite these difficulties,
the inbred lines differed dramatically in colony morphology
(Fig. 2). For the assays conducted, four runner-like F6
colonies, one sheet-like F6 colony and three sheet-like F5
colonies were used.
Morphological comparisons of colonies and medusae
Three clonal replicates of each of the four sheet-like and four
runner-like colonies were explanted onto 15 mm coverslips.
Generally, colonies covered the surface at the same rate and
initiation of medusa production occurred at the same
chronological time in both sheet- and runner-like lines (16±1
days after explanting; mean ± S.E.M.). At the time that medusa
buds became visible on the gonozooids of a replicate, that
replicate was measured using image analysis technology (e.g.
Marcus et al. 1996). Briefly, a high-resolution MTI CCD-72
camera attached to a macro lens was used to project each
colony onto a color monitor interfaced with a PC-compatible
microcomputer (Pentium, 90 MHz CPU, 32 Mbytes RAM)
equipped with an overlay frame grabber board (640×480 pixels
with 12-bit depth per pixel). Using OPTIMAS software,
background-subtracted images of the colonies were acquired
using illumination appropriate to produce three distinct
luminance thresholds: the polyps (lightest), the stolons
(intermediate) and the empty coverslip (darkest). Using these
thresholds, the software identified and measured the areas of
the empty (i.e. unencrusted) coverslip and of the individual
polyps (Blackstone and Buss, 1992). Classification macros
were used to identify and exclude coverslip areas outside the
edge of the colony. The total colony area and perimeter were
also measured. Data files were analyzed using PC-SAS
software.
Inbred lines were compared using nested univariate
(ANOVA) and multivariate analysis of variance (MANOVA)
(clonal replicates nested within colonies, colonies nested
within inbred lines) for size (total area), for ‘size-free’ shape
(perimeter divided by the square root of area, see Blackstone
and Buss, 1991) and for the relationship between the total area
of polyps and the total area of empty, unencrusted coverslip
enclosed within the colony. Both polyp area and empty,
unencrusted inner area were expressed as a fraction of the total
2824 N. W. BLACKSTONE
0.9
F1
0.8
0.7
0.6
0.6
0.5
r
0.4
r
0.4
r
0.3
r
0.3
0.2
0.2
s
0.1
0.1
s
0
s s
0
0
0.1 0.2 0.3
0.4
0.5 0.6
0.9
F3
0.8
Inner area/total area
F2
0.8
0.7
0.5
Fig. 1. Bivariate scatterplots of the amount of stolon
development (inversely correlated to inner area/total
colony area) and the amount of polyp development
(polyp area/total area) at the initiation of medusa
production for all representatives of five generations
of inbreeding that survived (50–95 % per
generation). Inner area is the total area of empty
unencrusted coverslip enclosed within the colony.
All colonies were grown and measured on 12 mm
coverslips (see text). Parent colonies for the next
generation are indicated (r, parent of runner line; s,
parent of sheet line); unfortunately, the most
extreme forms often have low fertility. By F5,
representatives of the line selected to be runner-like
(circles) show little polyp and stolon development,
while representatives of the line selected to be sheetlike (squares) show extensive polyp and stolon
development. For these lines, bivariate distributions
show highly significant differences for F3
(MANOVA, F=20.3, d.f.=2,35, PⰆ0.001), F4
(F=17.2, d.f.=2,34, PⰆ0.001) and F5 (F=43,
d.f.=2,31, PⰆ0.001). The total numbers of colonies
grown to maturity (runners and sheets, respectively,
for each generation after the first) were: F1=21,
F2=12 and 11, F3=23 and 15, F4=17 and 20, F5=13
and 21, F6=8 and 1. The asterisks mark colonies
used in experiments.
0.9
0
0.7
0.6
0.6
0.5
0.5
0.3
0.2
s
s
0
0.1
0.2
0.3
0.4
F5
0.8
0
0.6
0.4
0.5
0.4
0.3
0.3
0.2
0.2
0.1
s
0
0.1
area (note that the total area of stolons can be calculated as 1
minus this combined fraction, although this third variable was
not used in the analyses). Polyp area is clearly a measure of
polyp development; empty, unencrusted inner area is largely a
measure of stolon branching and anastomosis (i.e. as these
aspects of stolon development increase, inner area decreases).
While polyps can shield empty inner area from observation and
measurement, in practice this is a minor source of error because
stolon development is generally most extensive at the base of
the polyps. This is particularly true at the time of the initiation
of the sexual (medusoid) phase of the life cycle and at
subsequent times. Thus, at the time in development when
morphology was measured, polyp area and unencrusted inner
area behave as largely independent measures of two different
aspects of colony development (Fig. 2; see further discussion
0.2
0.3
* s*
0.4
0.1 0.2
s
0.3
0.4
0.5
0.6
F6
0.8
0.7
r
s
0.9
0.6
0
r
0
0.7
r
0.6
r
0.1
0.5 0.6
0.9
0.5
0.4 0.5
0.3
0.2
0.1
0
0.3
F4
0.4
r
r
0.2
0.8
0.7
0.4
0.1
0.9
*
**
*
0.1
*
0
0 0.1
0.5 0.6
Polyp area/total area
*
0.2
0.3
0.4
0.5
0.6
in Blackstone, 1996). While some heterogeneity of variances
was apparent in some of the measures used, generally all of
these data approximately meet the assumptions of parametric
statistics. Both natural logarithm and arcsine transformations
provided a poorer fit to these assumptions.
Subsequent to imaging, all colonies were grown until
medusae began to be released. All three replicates of one
runner-like colony failed to release a single medusa despite
repeated isolations; with the exception of this colony, the
initiation of medusa release occurred at the same
chronological time in both lines (21.9±0.6 days after
explanting for sheet-like lines and 22.1±0.6 days after
explanting for runner-like lines; means ± S.E.M.). Each colony
was isolated nightly until a total of five medusae had been
obtained. The morning after its release, each medusa was
Inbred lines in a colonial hydroid 2825
Fig. 2. Background-subtracted
images of F6 representatives of
inbred lines growing on 15 mm
diameter glass coverslips at the
initiation of medusa production
(A, runner-like; B, sheet-like).
Both images were taken 14 days
after explanting.
imaged while swimming horizontally. Images were taken
when the medusa was relaxed, not contracted. The length of
the bell (excluding the base of the tentacles) and the
maximum width were recorded for each medusa. As a
consequence of the failure of one runner-like colony to
release any medusae, the nested analysis of variance
(medusae nested within replicates, replicates nested within
colonies, colonies nested within inbred lines) was highly
unbalanced and could not be used effectively (see Sokal and
Rohlf, 1995). Differences among the medusae from the sheetlike and runner-like inbred lines were therefore compared
graphically and using a simple one-way multivariate analysis
of variance of logarithme-transformed data.
Video microscopic measurements of peripheral
gastrovascular flow
At the time that colonies covered one side of the coverslips
(2–2.5 weeks after explanting and immediately prior to the
time that medusa buds became visible on the gonozooids),
gastrovascular flow to three peripheral stolons was measured
in each replicate for all four colonies of both inbred lines.
Gastrovascular flow reaches a maximum 2–8 h after feeding
(Schierwater et al. 1992; Wagner et al. 1998; Dudgeon et al.
1998); all these studies were carried out 3–5 h after feeding.
The colony was placed in a flow-through chamber with a no.1
coverslip base (Warner Instruments). The chamber reservoir
was maintained at 20.5±0.1 °C (Neslab RTE-100D). The
temperature of the in-flowing sea water was further adjusted
with a thermoelectric device to maintain a constant chamber
temperature (20.5±0.3 °C; chamber temperature was
monitored using a YSI cuvette thermometer with a flexible
probe). Colonies were viewed on an inverted light
microscope (Zeiss Axiovert 135) with a 40× Plan-Neofluar
objective in differential interference contrast (DIC). Using
the MTI CCD camera, three primary stolon tips from
each colony were video-taped (at 30 frames s−1) for 10 min
each.
Gastrovascular flow must be reversed in each distal ‘deadend’ tip. Stolon tips fill as fluid enters; the velocity of the fluid
then decreases to zero. Tips then empty, and the fluid velocity
again decreases to zero. In the region of the stolon
immediately behind the tip, the difference between the width
of the stolon lumen when it is at a maximum (and fluid
velocity is zero) and when it is at a minimum (and velocity is
again zero) provides a measure of the rate of gastrovascular
flow if this difference is measured over time. These width
measurements are taken at the base of the lumen. With the
image analysis system connected to the video recorder, the
width of the stolon lumen was measured at a point 250 µm
behind the tip itself. In this region of the stolon, gastrovascular
fluid velocity falls to zero as the lumen width approaches its
maximum and minimum (thus, velocity itself need not be
measured). Lumen width was measured when the stolon was
full and when it was empty for three consecutive, but nonoverlapping, cycles. For each cycle, the net change in the
lumen width, i.e. the difference between the maximum and
minimum lumen widths, was calculated. Perisarc-to-perisarc
total stolon width (which is invariant throughout the
contraction cycle) and the period (in seconds) of each cycle
were also measured. The interpretation of these measures in
terms of the volumetric rate of gastrovascular flow is
discussed in detail in Blackstone (1996).
Statistical analysis thus focused on the three measured
outcomes: the net change in lumen width per cycle,
contraction cycle period and stolon width. These measures can
be combined into a biologically meaningful measure of
gastrovascular flow rate for each stolon contraction cycle: net
change in lumen width divided by cycle period and stolon
width (meters of lumen width expansion and contraction per
total meters of stolon width per second). Biologically, this rate
measure illuminates the ‘rate of supply’ of food to the tissues
of the stolon tip. Both this rate measure and the individual
flow parameters generally meet the assumptions of parametric
statistics (see Sokal and Rohlf, 1995). To compare inbred
lines, a nested analysis of variance was used with cycles
nested within stolons, stolons nested within replicates,
replicates nested within colonies and colonies nested within
inbred lines.
2826 N. W. BLACKSTONE
Assays of cellular redox state using fluorescent microscopic
measures of NAD(P)H
The characteristic fluorescence of NADH and NADPH
compared with the oxidized forms of these molecules has been
used extensively to measure cellular redox state (for a review,
see Chance, 1991). Currently, this technique is widely used
(e.g. Pralong et al. 1992, 1994; Heineman and Balaban, 1993;
Hajnóczky et al. 1995; Rohács et al. 1997). NAD(P)H
fluorescence includes both mitochondrial and cytosolic
compartments. Under physiological conditions, these
compartments are in a slowly equilibrated steady state, and the
redox states show corresponding behavior when subject to
perturbation (Scholz et al. 1969; Hajnóczky et al. 1995).
Localized measures of NAD(P)H fluorescence were obtained
using the Zeiss Axiovert 135 and ultraviolet light (excitation at
365 nm, barrier filter at 420 nm). Brief exposures were used,
since hydroids are sensitive to ultraviolet light. A colony was
contained in the flow-through chamber at 20.5±0.3 °C as
described above. Images were recorded on film (10 s exposure,
ASA 160 balanced for tungsten filaments), digitized and
quantified with densitometry in OPTIMAS (brighter values
relative to the dark background signal greater reduction). In
such images, stolons appear dark, except for a weak signal from
the chitinous perisarc (stolons lack the muscular fibers
characteristic of polyp epitheliomuscular cells; Schierwater et
al. 1992), while polyps show a much stronger signal. Because
polyps are highly contractile in vivo, only the base can be used
in precise between-polyp comparisons. In cross-sectional
images of the base of a living polyp, the fluorescence of the
base of the polyp epitheliomuscular cell fibers or myonemes can
be clearly identified (Fig. 3). These fibers form a longitudinal
network in a polyp, and their contractions drive the
gastrovascular flow. Both the number of fibers fluorescing
visibly above the dark background and the relative luminance
of the fibers can be calculated (Fig. 3). Since all the fibers of a
polyp are part of the same epitheliomuscular cell network, it is
not clear that these individual fibers can be considered as being
statistically independent. Therefore, the relative luminances of
all visible fibers of a polyp were averaged, and this mean value
was used in statistical comparisons.
Because these measures of NAD(P)H fluorescence were
somewhat time-consuming, only one sheet-like and one
runner-like colony were used. Nine clonal replicates of each
colony were explanted onto 15 mm round coverslips and grown
for 1 week. These replicates were divided into three groups of
three replicates each. Three replicates were assayed 3–5 h after
feeding, three replicates were assayed after being starved for
27–29 h, and three replicates were assayed after being starved
for 27–29 h and treated with 30 µmol l−1 2,4-dinitrophenol in
sea water for this period (for detailed protocols and discussions
of uncoupler treatments, see Blackstone and Buss, 1992, 1993;
Blackstone, 1997a, 1998). For each replicate of each treatment,
three polyps were imaged and measured. A mixed-model
analysis of variance (ANOVA) was used to detect betweentreatment effects for each colony (polyps nested within
replicates, replicates nested within treatments). Since the
Base of polyp
cross section
Polyp
Stolon
width
p3
p1 c2c1 p2
p4
Stolon
Fig. 3. Schematic diagram of a cross section of a polyp base, as
viewed in fluorescence with an inverted microscope, showing the
luminance of the base of the epitheliomuscular cell fibers against the
dark background. Fibers fluorescing visibly against the background
can be counted (here N=11), and luminance relative to the background
can be measured using densitometry. Relative luminance was defined
as the ratio of gray level measures from the center of each fiber (c1
and c2) to gray level measures at the periphery of each fiber (p1, p2,
p3 and p4), i.e. relative luminance = 2(c1+c2)/(p1+p2+p3+p4).
Peripheral measures were taken outside the area of fiber luminance to
measure the local background luminance surrounding each fiber.
runner-like and sheet-like colony were tested at different times,
direct statistical comparisons between runners and sheets were
not made, but the effects of the treatments on the two colonies
were compared graphically.
Results
Morphological comparisons between colonies and medusae
Colonies inbred for sheet-like morphologies initiate medusa
production at larger total areas than runner-like colonies
(Fig. 4A; using the colonies-within-lines effect as the error
term, F=14.9, d.f.=1,6, P<0.01). At the initiation of medusa
production, sheet-like colonies also exhibit more regular colony
shapes, that is, smaller perimeter/area0.5 values (Fig. 4B; using
the colonies-within-lines effect as the error term, F=15.0,
d.f.=1,6, P<0.01). Sheet-like colonies also show a significant
difference from runner-like colonies in the relationship between
polyp area and unencrusted inner area (Fig. 4C; using the
colonies-within-lines effect as the error term in a MANOVA,
F=15.5, d.f.=2,5, P<0.01). For the most part, sheet-like colonies
show greater stolon branching and anastomosis and thus
considerably less unencrusted inner area enclosed within the
colony. Note that three runner-like replicates cluster fairly close
to the sheet-like colonies (Fig. 4C). These three replicates are
all from colony 4 in Fig. 4A,B. Examination of the images
suggests that this colony grows in a manner similar to other
runner-like colonies; however, it initiates medusa production at
such small sizes that anastomoses between stolons have not yet
formed and thus little or no unencrusted area is enclosed within
the stolons. This produces not only small total areas at the
initiation of medusa production (Fig. 4A) and small inner
area/total area ratios (Fig. 4C) but also relatively large polyp
area/total area ratios (Fig. 4C). In general, some differences are
Inbred lines in a colonial hydroid 2827
180
160
140
120
100
80
60
40
20
0
A
1
2
3
4
Colony
30
B
25
Shape
20
Video microscopic measures of peripheral gastrovascular
flow
Prior to the initiation of medusa production, sheet-like
colonies exhibit a significantly smaller gastrovascular flow rate
than runner-like colonies (Fig. 6; using the colonies-withinlines effect as the error term, F=41.1, d.f.=1,6, P<0.001). Since
flow rate is a composite of three measured flow parameters (net
change in lumen width per cycle, contraction cycle period and
stolon width), it is useful to examine the between-inbred line
difference in these variables individually. Stolon width and
contraction cycle period show weak and non-significant
differences (Fig. 7B,C; F=4.6, d.f.=1,6, P>0.05, and F=3.9,
d.f.=1,6, P>0.05, respectively, for each variable using the
colonies-within-lines effect as the error term). The net change
in lumen width during each cycle, however, shows a large and
statistically significant difference (Fig. 7A; F=29, d.f.=1,6,
P<0.002, again using the colonies-within-lines effect as the
error term). Thus, the between-line difference in flow rate
derives primarily from large differences in the amount that the
lumen opens and closes with each contraction cycle in response
to the polyp-driven gastrovascular flow, rather than from
differences in stolon width or contraction cycle period.
15
Assays of cellular redox state using fluorescent microscopic
measures of NAD(P)H
Replicates of both the runner-like and the sheet-like colony
exhibit similar responses 3–5 h after feeding, after more than
24 h of starvation and after more than 24 h of starvation
combined with treatment with 30 µmol l−1 2,4-dinitrophenol
(Fig. 8). Both feeding and treatment with the uncoupler
dinitrophenol shift the cellular redox state in the direction of
oxidation (fewer fibers are visible against the dark background
and those that are visible exhibit lower values of relative
10
5
0
1
2
3
4
Colony
0.7
Inner area/total area
MANOVA of log-transformed data, F=381, d.f.=2,102,
PⰆ0.001).
C
0.6
0.5
0.4
1.2
0.3
0.2
0.1
0.04
1
0.08
0.12
0.16
Polyp area/total area
0.2
Fig. 4. Morphological comparisons of three clonal replicates of four
colonies inbred for runner-like morphologies (open columns and
circles) and four colonies inbred for sheet-like morphologies (filled
columns and squares) grown on 15 mm coverslips and measured at
the initiation of medusa production. Colonies are paired arbitrarily,
and means and standard errors are shown in comparisons of total area
(A) and perimeter/area0.5 or ‘size-free’ shape (B). Standard errors
provide a measure of between-replicate, within-colony variation. In
C, a bivariate scatterplot compares the amount of stolon development
(inversely correlated to inner area/total colony area) with the amount
of polyp development (polyp area/total area). Inner area is the total
area of empty unencrusted coverslip enclosed within the colony.
Width (mm)
Total area (mm2)
apparent between the inner area and polyp area measures of the
experimental colonies (Fig. 4C) and the same colonies as they
were originally grown (Fig. 1). Differences in substratum size
(12 mm versus 15 mm coverslips) and in initial polyp size
(small primary polyp versus fully grown polyp explant)
probably produce this discrepancy.
In general, medusae from sheet-like colonies are distinctly
smaller than those from runner-like colonies (Fig. 5;
0.8
0.6
0.4
0.4
0.6
0.8
1
Length (mm)
1.2
Fig. 5. Bivariate scatterplot of the length and width of medusae from
runner-like colonies (circles, N=45) and sheet-like colonies (squares,
N=60).
Lumen width change (µm)
2828 N. W. BLACKSTONE
3
2
1
1
2
3
4
Colony
Fig. 6. Means and standard errors of flow rates (meters of lumen
expansion and contraction per total meters of stolon width per
103 seconds) for three stolons per replicate and three replicates per
colony of each of the four runner-like (open columns) and four sheetlike (filled columns) colonies, which are paired arbitrarily. Standard
errors provide a measure of between-replicate, within-colony
variation.
luminance), while starvation for more than 24 h shifts the
cellular redox state in the direction of reduction (more fibers
are visible against the dark background and these fibers exhibit
higher values of relative luminance). For replicates of the
runner-like colony, both the number of fibers fluorescing
(Fig. 8A) and the relative luminance of these fibers (Fig. 8C)
show statistically significant between-treatment differences
(F=5.9, d.f.=2,6, P<0.05, and F=6.3, d.f.=2,6, P<0.05,
respectively, for each variable using the replicates-withintreatments effect as the error term). For replicates of the sheetlike colony, only the relative luminance of these fibers
(Fig. 8D) shows statistically significant between-treatment
differences (F=9.6, d.f.=2,6, P<0.05, using the replicateswithin-treatments effect as the error term), while the number
of fibers fluorescing (Fig. 8B) shows a non-significant effect
(F=2.7, d.f.=2,6, P>0.05, using the replicates-withintreatments effect as the error term).
Discussion
While the synthesis between development and evolution can
be framed in strictly genetic terms (e.g. Pennisi and Roush,
1997), such an approach is probably an oversimplification.
Genes are but one aspect of developmental mechanisms
(Nijhout, 1990), and an understanding of the metabolic and
physiological aspects of these mechanisms will be crucial to
any definitive synthesis (e.g. Sinervo and Basolo, 1996; Zera
et al. 1998). In this context, selection and inbreeding
experiments (e.g. Green, 1981; Monteiro et al. 1997a,b;
Wilkinson et al. 1998) can provide useful insights, particularly
when combined with an experimental approach. In colonial
animals, selection and inbreeding experiments have generally
not been used; such organisms can nevertheless be examined
using these methods (Mokady and Buss, 1996).
In the case of P. carnea, experimental manipulations have
suggested a relationship between heterochronic variation and
20
18
16
14
12
10
8
6
4
2
0
120
Contraction period (s)
0
A
1
2
3
4
1
2
3
4
1
2
3
4
B
100
80
60
40
20
0
50
Stolon width (µm)
Flow rate (ms-1)
4
C
40
30
20
10
0
Colony
Fig. 7. Means and standard errors for the three flow parameters used
to calculate the rate measures in Fig. 6 for three stolons per replicate
and three replicates per colony of each of the four runner-like (open
columns) and four sheet-like (filled columns) colonies, which are
paired arbitrarily.
gastrovascular flow physiology. Selection and inbreeding of
heterochronic variants of P. carnea support this hypothesis.
Colonies selected on the basis of morphology and subsequently
inbred produced distinct lines of runner- and sheet-like
morphologies. Representatives of these inbred lines showed
large differences in the rate of gastrovascular flow to peripheral
stolon tips. Examination of the flow parameters suggests that
this difference in flow rate derives primarily from the amount
that the stolon lumen opens and closes with each contraction
cycle. A strikingly similar pattern of flow parameter
differences is obtained by treating colonies with uncouplers of
oxidative phosphorylation such as dinitrophenol, in contrast to
feeding manipulations, for instance (Blackstone, 1997a). The
oscillations of the stolon lumen are in response to the
gastrovascular flow, which is largely driven by contractions of
the polyps. Polyps of runner-like colonies may thus have a
greater capacity for large and sustained contractions than
Inbred lines in a colonial hydroid 2829
polyps of sheet-like colonies. Alternatively, polyps of sheetlike colonies may be supplying greater numbers of stolons with
flow, thus diminishing the amount of flow to each stolon.
If polyps of runner-like colonies have a greater capacity for
large and sustained contractions than polyps of sheet-like
colonies, differences in metabolic capacity should be apparent
between these inbred lines. Polyps of P. carnea initiate
contractions in response to feeding and continue these
contractions for less than 24 h (Wagner et al. 1998; Dudgeon
et al. 1998). At wavelengths suitable for detecting NAD(P)H,
fluorescent microscopic measurements of both the visible
number of polyp epitheliomuscular cell fibers and their relative
luminance indicate that the cellular redox state oscillates
dramatically in a runner-like colony in response to these
feeding-related contractions. At 3–5 h after feeding, polyps are
contracting maximally, and the few muscle fibers visible
against the dark background exhibit a low relative luminance.
The redox state of these cells is thus probably shifted in the
direction of oxidation because of the heavy metabolic demand
and the consequent high levels of ADP (Chance and
Baltscheffsky, 1958; Chance and Thorell, 1959; Scholz et al.
1969; Hajnóczky et al. 1995). Perturbations of the system
Numbers of fibers
30
25
25
20
20
15
15
10
10
5
5
0
0
1.1
Relative luminance
30
A
C
1.1
1.08
1.08
1.06
1.06
1.04
1.04
1.02
1.02
1
1
Runner-like
B
D
Sheet-like
Fig. 8. Means and standard errors of measures of NAD(P)H
fluorescence for epitheliomuscular cell fibers of three polyps of each
of three colonies for each treatment [3–5 h after feeding (filled
columns), starved for more than 24 h (cross-hatched columns) and
starved for more than 24 h and treated with 30 µmol l−1 dinitrophenol
(open columns)]. Both the number of fibers (A,B) fluorescing visibly
relative to the dark background and the relative luminance (see Fig. 3)
of these fibers (C,D) are shown for runner-like and sheet-like colonies.
using dilute solutions of dinitrophenol support this
interpretation; polyps treated with dinitrophenol exhibit a
similar number of muscle fibers and with similar luminance to
polyps 3–5 h after feeding. In contrast, polyps starved for more
than 24 h are relatively quiescent, and the large number of
muscle fibers visible against the dark background exhibit a
high relative luminance. Cellular redox state is thus probably
shifted in the direction of reduction owing to the low metabolic
demand and the abundance of substrate and ATP.
In the sheet-like colony, similar feeding-related oscillations
in cellular redox state occur, but they appear to be less
dramatic. There is a tendency for polyps 3–5 h after feeding
and polyps treated with dinitrophenol to have fewer visible
muscle fibers than polyps starved for more than 24 h but, in
contrast to the runner-like colony, this trend is not statistically
significant. In terms of relative luminance, the sheet-like
colony exhibits a significant effect in the same direction as in
the runner-like colony. Nevertheless, the relative luminance of
muscle fibers from polyps starved for more than 24 h is not
dramatically greater than that of polyps 3–5 h after feeding and
that of polyps treated with dinitrophenol. These data suggest
that the sheet-like colony contains less functionally oxidizable
NAD(P)H than the runner-like colony, although further assays
should be undertaken to support this interpretation. A lower
level of functionally oxidizable NAD(P)H suggests diminished
‘reducing power’ and thus a diminished capacity to perform
metabolic work (e.g. Harold, 1986). Polyp epitheliomuscular
cells in sheet-like colonies may therefore lack the metabolic
capacity to match the rates of gastrovascular flow found in
runner-like colonies. Nevertheless, at this time, alternative
explanations must also be considered. For instance, the
extensive development of stolons in the sheet-like colony may
require substantial amounts of substrate to be maintained.
Because of this high allocation of substrate into the stolons,
sheet-like polyps may be somewhat depleted of substrate more
than 24 h after feeding, thus shifting the cellular redox state
more in the direction of oxidation.
An unanticipated consequence of the selection on colony
morphology was a strong effect on medusa size. Sheet-like
colonies exhibited considerably smaller medusae than runnerlike colonies. While this may represent a stochastic
consequence of inbreeding, it also may be a genuine ‘trade-off’
between colony and medusa morphology. Additional
inbreeding experiments should be carried out to test this
hypothesis. Possibly, medusa size may be a consequence of
selection on colony morphology. For instance, large polyps
with extensive muscular development may be capable of the
large and sustained contractions that produce high rates of
gastrovascular flow to peripheral stolon tips and result in the
development of runner-like colonies. Thus, selection for
runner-like colonies may produce colonies with large polyps,
and large medusae may typically develop from such large
polyps. Apparent genetic ‘trade-offs’ between colony
morphology and medusa-related life history traits (e.g.
dispersal distance) might in this way derive from such simple
structural consequences (Buss and Blackstone, 1991). To test
2830 N. W. BLACKSTONE
this hypothesis definitively, polyp size must be experimentally
manipulated and the effects on colony and medusa morphology
must then be assessed (see Dudgeon and Buss, 1996; Sinervo
and Basolo, 1996).
The metabolic and physiological differences discussed here
might underlie the heterochronic differences in any or all of
several ways. Changes in the timing of the production of polyps
and stolon tips relative to rates of stolon growth and colony
maturation require different patterns of signaling in runner- and
sheet-like colonies. Different rates of gastrovascular flow in
these colonies may provide hydromechanical signals which
influence colony development (Van Winkle and Blackstone,
1997; Dudgeon et al. 1998). Gradients of morphogens
emanating from polyps (Plickert et al. 1987) may be affected
by cellular redox state (Blackstone, 1997b; Jantzen et al. 1998),
thus also differentially influencing colony development.
Finally, specific characteristics of the feeding-related
oscillations of both gastrovascular flow and polyp redox state
may signal different patterns of colony development. Biological
and biochemical oscillations are ubiquitous (Chance et al.
1973), and amplitude and frequency modulation of such
oscillations may be a common signaling mechanism (Berridge,
1997; Dolmetsch et al. 1997). Since some observations suggest
that colony growth and morphogenesis are periodic, the last
hypothesis may be the most likely.
Helpful comments were provided by L. Buss, B. Chance, S.
Dudgeon and an anonymous reviewer. The National Science
Foundation (IBN-94-07049) provided support.
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