Research Summary
Metabolic scaling refers to the
change in physiological process in proportion to body size. The measures of
metabolic rate integrate different functions performed by animals. For colonial
animals, their metabolic allometry has been hypothesized to be different from unitary
animals. For instance, unitary organisms can be modeled a sphere with volume
increasing more rapidly than surface area. Colonial organisms can be modeled as
a cylinder with r >> h (r = radius, h = height).Consequently, in the
later, surface area increases in proportion to volume. Thus, they might exhibit
isometric scaling of metabolism. The animals can increase their total biomass
beyond the environmental constraints operating on their modules. The research
proposal includes a background discussion of metabolic scaling law as well as
colony integration. The proposed work utilized two colonial species that differ
in colony integration. Sympodium is a
“distributed” colony, using cilia to propel its gastrovascular fluid and
obtaining food from symbiotic dinoflagellates. Hydractinia is an “integrated” colony, consuming prey with large
centrally located polyps and using muscular contractions to pump food-rich
gastrovascular throughout the colony periphery. Measures of oxygen metabolism
versus size will test the effects of integration and geometry.
Introduction
The constraints in physiological
rates of animals as evident in metabolic allometry describe a disproportional
relationship between body size and basal metabolic rate. The relationship is
described using a power functionY = YoMb .
Metabolic allometry occurs when the scaling coefficient, b, is not equal to 1,
and the mass-specific metabolic rate changes as the body size increases. The
empirically measured value of scaling coefficient approaches 0.75 in different animals,
and many theories help to explain the dominance of ¾ scaling across different
sizes. The different theories predict a scaling exponent of 0.75, or 0.67, or
0.86 depending on the assumptions applied for two-dimensional organisms like
colonial animals. There is an alternative view of metabolic scaling that
proposes a range of scaling exponents predicted using surface area, and mass
(Hulbert, 2014).
Colonial animals are expected to
deviate from negative allometry because their structures exhibit modular
iteration. Their shape can be modeled as a cylinder with r >> h, Thus,
isometric scaling where the scaling exponent is 1 of the colony metabolic rate
and mass is predicted as an emergent property of modularity. The colony
metabolic rate is a linear function of the number of component modules. For
colonial organisms, the metabolic turnover rates like growth and reproduction
converge towards isometric scaling (Hartikainen et.al., 2014).
Size can be easily manipulated in
colonial organisms. The body size is an influential variable measured to
understand the inter-specific variability in the metabolic requirements.
Metabolism refers to the rate at which an organism exchanges and transforms
resources from the environment. The main issue on the study of body size and
metabolism has always been how metabolic rate changes with the size of the
organism. The changes could have a basis on a broad category of measures that
include length, surface area, and mass. The change in the size of an organism
is the change in scale. The metabolic scaling theory has interrelated concepts
and empirical observations that help to create links between different levels
of the organism in biology and ecology. The theory attempts to provide a
unified theory to understand metabolism as a driving pattern and process in
biology from the cellular level to the biosphere (White & Kearney,
2014).
Background
Geometry:
The metabolic scaling law is an
extension of Kleiber’s law that posits that the metabolic rate of organisms is
the basic biological rate that explains many of the observed patterns in
ecology. Metabolic scaling theory attempts to offer a unified theory for the
importance of metabolism in driving the pattern and process in biology (Agutter
& Tuszynski, 2011). Allometry is a technique previously used to analyze the
relative growth and showed basal metabolic rates to vary with the 0.73-0.74
power of the body mass (Glazier, 2014). It usually follows a power function: Y = YoMb where
Y= a measure of metabolic rate, Y0= normalization constant, M= body
mass or some other size measure and b = allometric scaling coefficient (the
slope after log-log transformation).
Y = YoMb
Kleiber suggested the use of mass ¾ due
to its easy calculation with a slide rule. However, there have been other
scaling exponents ranging from 0.80 to 0.97 and the scaling of mammalian metabolic
rate during growth displaying multi-phasic allometric relationships with
scaling components more than ¾. The relative tissue size is crucial in the
determination of metabolic rate.
The
3/4 exponent is found only in resting metabolic rate. Higher levels of
metabolic activity produce higher exponents. Many researchers accept the
reality of allometric scaling but contend with the basal metabolic rate being
0.75 (Farrell-Gray & Gotelli, 2005). The skeptics claim that the true value
is 0.66 or 0.67 since the principal determinant of metabolic scaling is a
surface-to-mass ratio.
Allometric
metabolic scaling implies that bigger animals have a different metabolic rate
per unit mass as compared to smaller organisms. The metabolic rate of animals
gets measured as the rate at which an animal uses oxygen or produces carbon
dioxide. When oxygen is present, gas exchange is not a problem for small
animals due to their high surface-to-volume ratio.
The relationship between metabolic
rate and body mass has been well-studied in biology due to several factors. This
relationship among species is interpreted as a primary constraint by which
ecological processes, from individuals to ecosystems, aregoverned. The second
aspect is that there is still considerable debate over the exact value and
mechanistic basis of exponent b (Glazier, 2005). The basic principle for
whole-organism metabolic allometry is that the volume, hence the body mass, of
cells converting energy increases faster than the total effective surface area
across which energy and material resources get exchanged with the external and
internal environment as animals continue to grow.
The mechanistic
theories have different assumptions concerning the flow and partitioning of
assimilated energy into and through an animal. The West, Brown, and Enquist
(WBE) model, for instance, makes the assumption that the entire animal
metabolic rate gets limited by the internal transport of resources through a
volume filing, hierarchical, fractal-like, pathway. The Dynamic Energy Budget
(DEB) model, on the other hand, is based on surface area to volume
relationships that determine the uptake ad use of food and oxygen. The
Metabolic-Levels Boundary (MLB) model makes the assumption that the energy
utilization and power generation for an activity are limited by volume and
scale isometrically with mass while fluxes of metabolic resources, wastes, and
heat get limited by surface area and scale allometrically with mass as
two-thirds.
The form of an
organism has a close linkage to its function, and the relationship is important
in the organisms whose individual form is highly flexible. The modular
organisms consist of repeated building blocks which may be reflected in the
ability of a colony to reallocate priority of resource transport among its
units. For the social insect colonies as well as individual animals, the rate
of biological processes scales with the body size.
Metabolic
integration:
The extent to
which parts of a colonial organism are physically or physiologically integrated
influences the pattern of metabolic scaling. When modules are physiologically
similar and independent, the entire colony metabolic rate is expected to be the
product of the metabolic rate per module and the total number of modules and
hence scale isometrically.
Modules are not
typically co-dependent as tissue and organs are in unitary organisms; however,
the more the modules become physiologically dependent on each other, the more
the relationship scale allometrically similar to unitary organisms.
Resource-transport systems are a significant element of colonial integration
that is critical in understanding variation in metabolic scaling. Various
benefits emerge from the internal metabolic integration and include the sharing
of resources throughout the organism, enhance an expectation for integration to
take place in modular animals under several conditions despite the costs. The
evolution of transport systems is a central component in the evolution of
multicellularity in animals (Parrin et.al., 2010).
The power of metabolic allometry in ecology
derives from the diversity of life exhibiting a non-linear scaling pattern in
which the metabolic rates are not proportional to mass (Agutter & Wheatley,
2004). One theory postulates that the supply of energy is a significant physiological
constraint and another postulate that behavior regulates the demand for energy.
There is the likelihood that the increase in colony size reduces the proportion
of individual active engagement for scaling at a colony level. Some colonies
exhibit a hypometria allometry in which exponent < 1(Agutter & Wheatley,
2004).
Colonies can live in different
states comprised of loose, highly integrated, or a transition to a fully
integrated individual. The highly integrated colonies have a high likelihood of
exhibiting standard metabolic scaling (with exponent = ¾) as the case in the
fully integrated individuals when measured at resting metabolic rate(Siblyet.al.,
2012).
Testing
theories:
In modular
animals, the expectations and consequences for metabolic isometry are precise;
however, there are many reasons to expect varying scaling exponents that
deviate from isometry. Various scaling exponents have emerged for modular
animals, together with a more detailed explanation of the biology of modular
organisms. The representation of modular growth has always been the perpetual
addition of identical units. However,
when modules within colonies vary in size and shape, modules still vary as a
result of differences in age, ontogeny, reproductive state, nutritional state,
and position. The degree of increase of respiration of an entire colony
together with the number of modules added will, hence, be dependent on the
particular arrangement, integration and functional responsibility of individual
modules influencing the uptake and utilization of energy. The differences among
genotypes, growth forms, and species in the aspect of variation that takes
place among modules and during the lifetime of an individual is likely a great
source of information concerning the limitation or enhancing of variation
surface area, volume ratio of the transport system in metabolic scaling. This
variability is sometimes biologically critical rather than being noise and
offers a great chance to understand the causes and impacts of metabolic
scaling. The interest of metabolic scaling in modular animals also can be attributed
to the aspects of these organisms that allow differentiation among theories,
since it is possible to artificially manipulate body mass, size and shape. The
predictions of b for competing for mechanistic theories are similar, and it is
necessary to use manipulative experiments of size and oxygen metabolism to
determine the cause and consequence relationships between body mass and
metabolic rate (White & Kearney, 2014).
Hypothesis
More integrated colonies have
metabolic scaling exponents of 0.75 in the resting state and less integrated
colonies have a higher exponent and no clear resting state.
Study species and
culture conditions:
Colonies are explanted as two-polyp
fragments and grown on 12 mm diameter and cover glassto larger and smaller
sizes. Colonies grow to reach the edge of the cover glass nearly, for the
larger sizes. Some colonies grow as small part of the edge of the cover glass,
for the smaller sizes, and the assays are done 3 h or 24 h after feeding. Colonies
of Hydractinia symbiolongicarpus and Sympodium sp. are used. The growth of
the colonies was limited to one side of the coverslip.
Sympodium sp.
can be either blue or green and grows in reefs and shallow lagoons of the
ocean. They are a fast-growing, sheet-like species that colonize or encrust the
rocks and shells on which they grow. They are widely available due to fast
growth and adaptability to the aquarium environment. They get most of their
nutrients through symbiotic algae (zooxanthellae) contained within the coral.
They can also do well when they receive nutrients from phyto-plankton filtered
from the water currents but are not directly fed. Their gastrovascular fluid is driven by
ciliary action (Harmata et.al., 2013). Sympodium sp. is cultured at 27° C, dKH
9, pH 8.2, specific gravity 1.026.
Hydractinia
symbiolongicarpus are among the many hydractinia species that are widely
known. Hydractinia feed on smaller
invertebrates found in the shallow mud, but in laboratory environments, they
feed on brine shrimp. They consist of a network of gastrovascular canals in a
plate of tissue called the stolonal mat.
Large polyps in the center of the colony feed and then, using
myo-epithelial contractions, pump gastrovascular fluid to the growing
peripheral zone of the colony.Hydractinia
is cultured at 20.5° C, dKH 9, pH 8.2, specific gravity 1.021.
Materials
and Methods
Study
Species and Culture conditions for Hydractinia symbiolongicarpus and sympodium
sp.
All the experiments will be carried
out using single clone of each of the hydractinia
symbiolongicarpus and sympodium sp.
H. symbiolongicarpus are sheet-like
due to the formation of a stolonal mat in the initial stages of development and
produce varying amounts of peripheral stolons.
The colonies will be cultured at
~20.5 °C on 15-mm round glass cover slips suspended in floating racks in an aquarium
having Reef Crystals artificial sea water with 32% salinity and under gravel
filtrations illuminated for 12 hours per day. The water will be changed on a
weekly basis and colonies fed to repletion three times a week with 3-day-old
brine shrimp. The replicate colonies will be produced by explanting 1-3 polyps
surgically and connecting the tissue from a source colony to the cover slips.
Explants will be held in place using nylon threads fastened to the cover slip
by aquarium glue. After the explants are attached to the cover slips, they
begin to grow into small colonies and the threads and glue removed. The cover
slips will be cleaned twice weekly on the non-feeding days by use of a
paint-brush. Additionally, colony growth will be restricted to one side of the
cover slip by scrapping the stolons from the reverse side with a razor blade.
The experiments will be carried out at ~20.5 °C in culture aquariums except
when kept in glass finger bowls in incubators (Harmata & Blackstone, 2011).
The general imaging
protocols
For the experiments to be conducted,
the images will be acquired and analyzed using Image-Pro Plus software.
Whole-colony images will be acquired with a Hamamatsu Ocra-100 cooled CCD
camera attached to a macro lens. For the images of mitochondrion-rich cells and
that of gastro vascular cavity, the camera will be attached to a Zeiss Axiovert
135 inverted microscope. The colonies will be imaged in seawater in disposable
chambers with frequent water changes for temperature maintenance. All the
statistical analysis will be performed using PC-SAS software.
The experiment will also be
performed on the colonies of sympodium sp.
which will be grown and studied in the lab. They are symbiotic-containing
octocorals that belong to the Holaxonia-Alcyoniina clade of octocorals. Sympodium sp. belongs to the family
Xeniidae. In regard to thermal and light perturbation, the symbionants leave
the tissue and accumulate in the lumen of the gastrovascular system. The
sensitivity of sympodium sp. to
thermal perturbation is relative but not as high as that of Sarcothelia sp.
The control and treated colonies
will be explanted from the same mother colony for each of the experiments. The
experimental colonies will be grown on 12 mm and 15 mm round cover glass and
cultured using the standard methods. The conditions for culturing include; 27
°C, 1.026 specific gravity, 8.2 pH, 400 ppm calcium, 1200 ppm magnesium, 0 ppm
nitrate and 12 h dark, 8 h illumination at 30 µmol photons m-2 s-1, 4 h illumination
at 110 μmol photons m-2 s-1. Only fresh materials will be used for the
experiment and the colonies will be grown on the cover glass 1 to 2 weeks
before use.
Measures of oxygen
uptake rate and data analysis
The rate of oxygen uptake is assayed
in colonies using Strathkelvin Instruments oxygen meter. The oxygen uptake
measurement is taken in a 3-minute span for over 30 minutes in the dark. Slopes
of linear regression versus time is used to calculate metabolic rate (mg O2 L-1
min-1), which then serves as the outcome for regressions using size as the
predictor. Colonies of the two species
will be tested on feeding and non-feeding days.
It is expected that this will affect metabolic rate in Hydractinia, but
not in Sympodium, since the latter are not fed. To gain further insight into
the metabolic state of these species, experiments on oxygen metabolism and mass
will examine starved colonies (minimum metabolic rate) and uncoupled colonies
(maximum metabolic rate).
Measurement of oxygen
metabolism
Oxygen generation can be helpful in
the determination of the health of colonies and the assessment of whether photo
systems are functioning correctly. The net oxygen consumption is observed in
the dark due to respiration whereas the net oxygen production takes place in
exposure to light due to photosynthesis. The use of an inhibitor, DCMU, to
block photo system II makes oxygen metabolism in the light to resemble that in
the dark (Netherton, Scheer, Morrison, Parrin
& Blackstone, 2014).
Strathkelvin 1302 electrode and
781-oxygen meter in a glass chamber will be used to measure dissolved oxygen. A
Neslab RTE-100D recirculating chiller operating at 27º C will help to maintain
a constant temperature. The colonies of sympodium
sp. will be grown on 12 mm diameter round cover glass to fit in the 13 mm
diameter glass chamber. A magnet will be attached at the back of another 12 mm
cover glass for stirring having a drop of silicone grease to the cover glass on
which the colony resides. The oxygen uptake readings will be recorded for 21
minutes in total in the dark and 21 minutes in the light in 1 mL. seawater with
12.5 μL dimethyl sulfoxide (DMSO). The process will then be repeated in 1 mL.
Sea water with 12.5 μL from a stock solution of 0.008 mol L-1 DCMU
(3-(3,4-dichlorophenyl)-1,1-dimethylurea) dissolved in DMSO to a final
concentration of 100 μmol L-1. Five colonies will be used to measure oxygen
metabolism. For each colony, the slope of oxygen concentration versus time will
be calculated for the untreated colony in the light as well as in the dark and
for the same colony treated with DCMU in the light and the dark. The slopes
will be averaged and pair comparison t-test performed. The mean slope in light
for the control experiment will be compared to the average slope in the light
for the treated colonies and the average slop in the dark for the controls compared
to that in the dark of the treated colonies.
Protein assay
The size of small, experimental
colonies size can be measured by total protein (Agutter & Wheatley,
2004). The relationship between total
protein and mass can be determined for larger colonies and extrapolated to
small sizes. Bradford protein assay is an analytical procedure used to measure
the concentration of protein in a solution. The assay relies on the observation
that the absorbance maximum for an acidic solution of Coomassie Brilliant Blue
G-250 changes from 465 nm to 595 nm when protein binding occurs. The
hydrophobic and ionic interactions cause stabilization of the anionic dye shown
by a visible color change (Ku et.al., 2013). Total protein compares absorbance
of sample to a standard to determine total protein and hence size.
CBTM Protein
Assay
An improved Coomassie Dye based
protein assay is based on the Bradford Protein Assay. The assay is appropriate
for the simple and fast estimation of the protein concentration. The assay has
a basis on a single Coomassie dye based reagent. The binding of the protein to
the dye results to a change in color from brown to blue. The change in the
color is proportional to the protein concentration and protein estimation is
performed using little protein. The CB
Protein Assay uses a simple protocol and ready to use reagents that do not
require pre-filtering or dilution. The procedure involves simple mixing of the
protein solution with CB Protein Dye and reading the optical density. The
protein-dye complexes attain a stable end point within 5 minutes, and the CB protein
Assay method is compatible with the reducing agents and various laboratory
agents. The assay has a traditional bovine serum albumin (BSA) protein standard
or non-animal protein standard. DMSO at a concentration of 10 % is compatible
with the CB protein assay, hence will be appropriate for the protein assay
experiment involving the colonial animals. The method is highly sensitive, has
a flexible protocol, is ready to use the assay reagents without any
preparation, and has a long shelf life up to 12 months of stability.
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