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Title
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Eng
Characterizing Functional Biodiversity Across the Phylum Ctenophora Using Physiological Measurements
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Date
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2018
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Creator
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Eng
Wilson, Telissa M
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Subject
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Eng
Environmental Studies
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extracted text
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CHARACTERIZING FUNCTIONAL BIODIVERSITY ACROSS THE
PHYLUM CTENOPHORA USING PHYSIOLOGICAL
MEASUREMENTS
by
Telissa M. Wilson
A Thesis
Submitted in partial fulfillment
of the requirements for the degree
Master of Environmental Studies
The Evergreen State College
December 2018
© 2018 by Telissa M. Wilson. All rights reserved.
This Thesis for the Master of Environmental Studies Degree
By
Telissa M. Wilson
Has been approved for
The Evergreen State College
by
_________________________________
Erik V. Thuesen, Ph.D.
Member of the Faculty, Zoology
_________________________________
Date
ABSTRACT
Characterizing functional biodiversity across the phylum Ctenophora using
physiological measurements.
Telissa M. Wilson
Ctenophores are marine predators that are well known for their
bioluminescence and the diffraction of light off their many cilia.
Ctenophores are the largest organisms to utilize cilia to power locomotion as
opposed to muscle. Specimens of Ctenophora belonging to a range of habitat
depths and six distinct orders (Beroida, Cydippida, Thalassacalycida,
Platyctenida, Cestida, and Lobata) were collected for this study.
Examinations of their enzyme activities in relation to specimen wet mass,
feeding morphology, minimum depth of occurrence, number of comb plates
and specific ctene row surface area were conducted. Specific topics
addressed within this research include 1) relationships between the muscle
enzyme creatine kinase and ctenophore locomotion, 2) whether members of
different groups within Ctenophora display unique metabolic scaling
patterns, relationships between metabolic scaling patterns and feeding
strategies, and 3) whether ctenophores support the ‘visual interactions
hypothesis’. Results indicate a significant relationship between creatine
kinase activity and comb plate density. The number of comb plates does not
appear to change in correlation with overall body mass, whereas the specific
surface area of ctene rows does show increase, and specific density of comb
plates shows significant decrease. When considered as a whole, members of
Ctenophora show no significant correlation of body mass to the metabolic
enzymes studied. Yet when treated individually, different groups such as
Beroida and Lobata displayed species-specific scaling. Over all members
studied, the order Lobata showed comparatively lower CK and CS activity.
Species-specific scaling is also visible in the creatine kinase scaling patterns
of Beroida vs Lobata. Ctenophores were found to support the visual
interactions hypothesis. This research shows support for the diversity of
biochemical adaptations found throughout the phylum Ctenophora, and
subsequent studies may benefit by treating each group within the phyla
individually.
Table of Contents
LIST OF
FIGURES………………………………………………………………......vi
LIST OF
TABLES…………………………………………………………………..viii
ACKNOWLEGEMENTS…………………………………………..…......ix
CHAPTER ONE:
BACKGROUND……………………………………………..……………..1
Introduction………………………………………………………………….1
Ctenophore Taxonomy…………………………………………....................3
Ctenophore Morphology and Ecology……………………………................4
Functional Biodiversity……………………………………………………...6
Measuring Functional Biodiversity……………………………….….6
Metabolism as an Index of Functional Diversity……………………..8
Enzymatic Measurements as Proxies for Metabolism…………..........9
Comparative Ecophysiological Studies in other Marine Taxa……………..10
Visual Interactions Hypothesis……………………………..........................11
CHAPTER TWO: MANUSCRIPT……………………...........................13
Introduction………………………………………………………………...15
Materials and Methods…………………………………………………......17
Specimen Collection………………………………………………....17
Enzymatic Activity Measurements……………………………….......18
Morphometric Measurements…………………………………….....20
Statistical Analysis…………………………………………………..21
iv
Results……………………………………………………………………...21
Enzymatic Activities………………………………………………....22
Metabolic Scaling………………………………………………........22
Morphometric Characteristics………………………………............23
Enzymatic Activities in Relation to Depth……………………….......24
Discussion………………………………………………………...…….......24
Enzymatic Activity…………………………………………..….........24
Scaling………………………………………………………….........25
Morphometrics…………………………………………………........26
Enzyme Activities in Relation to Depth……………….......................28
Conclusion…………………………………………………….…................28
CHAPTER THREE: CLOSING REMARKS…………………..............30
Conclusion………………………………………………….........................30
Figures………………………………………………………….…..............32
Tables……………………………………………………………….….......41
References.....................................................................................................45
v
List of Figures
MANUSCRIPT:
FIGURES………....…………………………………….............................32
Figure 1. a) Creatine kinase activity as a function of body mass in 27
species of Ctenophora. b) Citrate synthase activity as a function of
body mass (g)......................................................................................32
Figure 2. a) Creatine kinase activity as a function of body mass for beroid,
cydippid, and lobate ctenophores. b) Citrate synthase activity as a
function of body mass for beroid, cydippid, and lobate
ctenophores.........................................................................................34
Figure 3. The relationship between creatine kinase and citrate synthase
metabolic potential provide a method to examine the relative
importance of locomotion (CK) vs overall aerobic metabolism (CS)
for individual species………..............................................................36
Figure 4. a) Citrate synthase activity as a function of body mass viewed by
feeding morphology b) Creatine kinase activity as a function of body
mass viewed by feeding morphology………………..........................37
vi
Figure 5. Ctenophore comb plate densities, total numbers of combs and
ctene row surface areas as a function of total body wet mass………39
Figure 6. Citrate synthase and creatine kinase activities of California
ctenophores compared to those of pelagic fishes from the same
region..................................................................................................40
vii
List of Tables
MANUSCRIPT: TABLES…………………………………………..........41
Table 1. Enzymatic activities of ctenophores collected off California and
Washington…................................................................................................41
Table 2. Scaling relationships of enzymatic activities of ctenophores from
California & Washington……………………………........................43
Table 3. Morphometric characteristics of oceanic ctenophores from
California and Washington……………………………….............…44
viii
Acknowledgments
I would like to extend heartfelt thanks to my advisor Dr. Erik V.
Thuesen. This work would not have been possible without the constant
support, unfailing guidance, gentle prodding, and inspiration that he
consistently provided. I am grateful to have him as a mentor.
Thank you also to Sappitah Ahmath, Tiffany Bachtel, and Jojo
Froehlich who helped invaluably by performing numerous enzyme assays
with me in the lab. Thank you to Steve Haddock and the Western Flyer crew
at Monterey Bay Aquarium Research Institute for allowing us to participate
in research cruises to collect specimens. Special thanks to Kaile Adney and
the staff at the Science Support Center for their help in acquiring necessary
research supplies. I also could not have made it this far without the support
of my colleagues from the WSDA Plant Pathology Lab. I am grateful for
their support and flexibility.
Thank you to the Evergreen State College Foundation and National
Science Foundation awards DEB-1542673 and DEB-1542679 for funding
this research.
Lastly, I would like to thank my husband Gabriel Harder. I am forever
grateful for his support and help. It was no small feat to hold up our
ix
household as a single parent while I was away on long research cruises and
working through weekends. Thank you also to my parents Julie Martin,
Caleb Martin, Brad Wilson, and Marty Wilson, who supported me
throughout the entire process and allowed me to work long hours and late
nights, while providing care for my young daughter and cooking us meals.
All of you helped to raise her while I was away, and the comfort of knowing
she was in loving hands helped me to accept the time I couldn’t spend with
her.
x
CHAPTER ONE: BACKGROUND
Introduction
The planet’s five oceans hold more ecological space than all the
terrestrial biomes put together. The phylum Ctenophora makes up a small
group of macrozooplankton with a large ecological role throughout the
world’s oceans, both in surface waters, midwater, and bathypelagic realms.
All orders are pelagic, with the exception of Platyctenida which is composed
of benthic ctenophores that creep across substrate or attach to something on
the sea floor. Ctenophores are gelatinous predators that range in size from
the tiny Minictena luteola, with a 1.5 mm diameter, to the massive Cestum
veneris, measuring up to 3 meters long. These predators are important
players throughout the oceanic ecosystem, and sometimes large increases
called ‘blooms’ can cause extensive damage to typical ecosystem
functioning, including economically important fisheries (Mills, 2001). As
our planet’s climate changes, and these changes are felt in the ocean through
increased acidity, temperature, salinity and hypoxia, it is expected that such
changes can have profound effects on marine invertebrates and their food
webs (Mills, 1995; Mills, 2001; Thuesen et al., 2005). Research has
suggested that biodiversity is critical to ecosystem health, and that human
1
driven climate change and other anthropogenic effects are generating
negative impacts on ocean biodiversity (Worm & Lotze, 2016; Hooper et al.,
2005). There is a real need to characterize the biodiversity in the world’s
oceans before these negative impacts result in irrecoverable damage. The
objective of this research is to address gaps in our understanding of
ctenophore ecophysiology and functional biodiversity.
Until recently, most ctenophore research has focused on surface
dwelling specimens due to the difficulties involved with observing and
capturing species from the depths and also the fragility of the deeper
specimens (Mills, 1995; Haddock, 2004; Robison, 2004). Improvements in
SCUBA collection methods (Haddock & Heine, 2005) and remotely
operated vehicles (ROVs) have increased our ability to study these
organisms. Despite these advances, most current publications are focused on
the same few species of ctenophores, mostly because they are readily
available, physically robust, and because of the attention they have garnered
from impacts to economically important fisheries. Additionally, many
species have yet to be described, and almost nothing is known about their
physiology and functional biodiversity. This study will utilize physiological
measurements to elucidate characteristics of functional biodiversity found
2
throughout the phylum. This study is the first of its kind to characterize
functional biodiversity across multiple orders of Ctenophora.
Ctenophore Taxonomy
The phylum Ctenophora is comprised of eight presently recognized
orders; Platyctenida, Cestida, Beroida, Cydippida, Lobata, Cambojiida,
Cryptolobiferida, and Thalassocalycida. Currently there are over 150
described species, and it is estimated that this only accounts for half of the
extant species, with several still undescribed and many more undiscovered
(Appeltans et al., 2012; Mills, 1998). The current ctenophore family tree is
constructed mainly from physiological and morphological evidence of
homology, with new versions based on gene regions and transcriptome data
being published yearly. Many researchers have questioned the traditionally
assigned orders in Ctenophora (Harbison, 1985; Mills, 1998; Ryan et al.,
2013; Podar et al., 2001; Moroz et al., 2014; Borowiec et al., 2015), and
there has been a large debate regarding the phylogenetic placement of the
phylum Ctenophora as an early branching metazoan lineage (Nosenko et al.,
2013; Dunn et al., 2008; Wallberg et al., 2004; Ryan et al., 2013; Moroz et
al., 2014). A few studies have posited that Porifera is the sister lineage to
metazoan, and not ctenophores as suggested by others (Simion et al., 2017;
Borchiellini et al., 2001; Jekely et al., 2015). Novel methods are being
3
utilized to clarify the uncertainties in this deep phylogenetic problem, and
more and more researchers are finding support for Ctenophora as the sister
lineage to all of Metazoa (Shen & Rokas, 2017; Whelen et al., 2017).
Piecing together early metazoan evolution will become easier with
sequencing of additional Ctenophora and Porifera genomes.
Ctenophore Morphology and Ecology
Ctenophores come in a large array of diverse body structures, from
large sac-like beroids to flat belt shaped cestids. All ctenophores are
characterized by a body plan involving rotational symmetry and numerous
specialized morphological and physiological features unique to the phylum
(Dunn et al., 2015). Ctenophores are unique in Metazoa in that they are the
only organisms known to have colloblasts (adhesive cells used in prey
capture) and they are the largest organism to utilize cilia for locomotion.
Most ctenophores have eight rows of ciliated comb plates which extend from
the aboral end of the organism up the sides towards the oral end. Some
species, such as Cestum veneris have severely reduced comb rows (Harbison
et al., 1978). A few genera also utilize cilia on appendages called auricles,
which are used in feeding behavior (Haddock, 2007). Ctenophores are
known to possess the enzyme creatine kinase, yet it is unknown whether or
not this enzyme plays a role in ciliary locomotion in these organisms.
4
Ctenophores lack advanced muscle tissues, but are known to have very large
smooth muscle cells, and one genus, Euplokamis, has striated muscle tissue
(Tamm & Tamm, 1989; Mackie & Mills, 1992). Another aspect of this
research is to investigate the potential role of the muscle enzyme creatine
kinase in ctenophore locomotion.
There are three main feeding strategies that have been observed in
ctenophores, although several variations on the three themes exist (Haddock,
2007). The three most common feeding strategies use either tentacles, lobes,
or an engulfing mechanism. Most members assigned to Cydippida feed
entirely via the use of two tentacles. Also, the cydippid larval stage present
in all ctenophores (except the Beroids) typically use tentacles for feeding.
Members that feed using tentacles are typically sit-and-wait predators. They
hang out in the water column with their tentacles fully extended and wait for
prey. When contact with prey is made, the numerous sticky colloblasts
adhere to the prey and the tentacles rapidly retract, pulling the prey close to
the organism’s body, and then the animal rotates to bring its mouth to the
prey. Ctenophores eat copepods, other zooplankton, and even other
ctenophores. While tentacles are the main feeding mechanism for some
ctenophores, others utilize different strategies but still have reduced tentacles
present (Haddock, 2007).
5
The lobate feeders are harder to generalize. Lobates are characterized
by small to large oral lobes, presence of auricles, and reduced tentacles near
the mouth. Lobate feeders swim towards prey, passively or actively
(depending on the species), and when contact is made, the lobe(s) bring the
prey towards the mouth (Harbison et al., 1978; Haddock, 2007). In addition
to feeding, some lobate ctenophores also use their lobes to propel themselves
through the water (Haddock, 2007).
Species that feed solely by engulfing belong to the order Beroida.
Their body is shaped like a large sack, with an oral opening on one end.
Engulfers swim through the water with their mouths open, and are thought to
use chemical signals to attract and/or sense prey (Haddock, 2007). As
mentioned previously, members of Beroida do not have a cydippid larval
stage, and so are restricted to this one form of feeding throughout life.
Measuring Functional Biodiversity
Measuring functional biodiversity in the oceans is an important step to
predicting how environmental perturbations may affect marine ecosystems.
Biodiversity is critical to ecosystem functioning, and there is a large gap in
our knowledge regarding the functional biodiversity within the phylum
Ctenophora. Biodiversity is considered the “number and composition of the
6
genotypes, species, functional types, and landscape units in a given system”
(Diaz & Cabido, 2001). Functional diversity is the total sum of functional
traits that organisms possess that play a role in how they interact with and/or
affect their environment and ecosystem. Functional traits can be
measurements based on morphology, physiology, ecology, behavior,
phenology, energetics, or almost any other relevant categorical or
quantitative functional trait. There are no unanimously accepted functional
traits that are approved for use in a functional diversity study, and the
definition of functional diversity across multiple disciplines and publications
is often ambiguously defined.
Analysis of functional diversity has become increasingly prevalent in
studies aimed at predicting ecosystem impacts from declines in biodiversity.
Functional traits are now considered to be superior measures to use over
previous methods which assessed biodiversity using species richness and
species abundance (Schleuter et al., 2010; Diaz & Cabido, 2001; Petchey et
al., 2006). There are numerous studies that lay down various methods and
indices for analyzing functional diversity, yet, as with selecting traits there is
still no agreed-upon one proper way to go about it (Petchey et al., 2006;
Rosado et al., 2013). Several researchers do agree that using multiple traits is
7
a better approach than just using a single functional trait (Lefcheck et al.,
2015; Villeger et al., 2008).
Metabolism as an index of Functional diversity
Since the introduction of the term ‘functional diversity’, there has
been an explosion of different indices used by researchers to better
understand species’ ecological roles. In the marine literature, physiological
traits such as the rate of metabolism have been used extensively to
understand species’ ecological niches, phenotypic adaptations, and
biodiversity (Thuesen et al., 1998a &1998b; Seibel & Childress, 2000;
Thuesen & Childress, 1994). In the case of many marine studies on
zooplankton, the chosen trait (metabolism) is studied comparatively with
information regarding habitat depth, body size, taxonomic groupings, and
any available life history knowledge (Barnett et al., 2007; Pomerleau et al.,
2015; Thuesen & Childress, 1994; Thuesen et al., 1998a; Seibel et al., 1997).
Measurements of metabolism used in this context can provide a window into
the functional diversity of difficult to observe marine taxa. Metabolism is
traditionally measured using oxygen consumption data, and preferably on
specimens in a resting state. As the organism respires it uses up the available
oxygen, resulting in consumption rates per unit time. Oxygen consumption
rates provide useful information regarding metabolism in robust ctenophore
8
species. Unfortunately, many ctenophores cannot withstand the experimental
procedures and disintegrate completely, sometimes even before successful
transfer to a respiration chamber.
Enzymatic measurements as proxies for metabolism
The difficulties working with fragile specimens and the restrictions
involved with assaying living specimens prompted researchers to evaluate
alternative methods for characterizing metabolism. Preliminary research by
King and Packard (1975) showed significant correlation between electron
transport chain activity and respiration in several members of zooplankton
(King & Packard, 1975). Subsequent research on marine fishes investigated
the use of individual aerobic and anaerobic metabolic enzyme activities, and
also found correlation with respiration rates (Childress & Somero, 1990;
Torres & Somero, 1988). The same key metabolic enzymes were assayed
and found to be good indicators of metabolism in pelagic chaetognaths,
nemerteans, and annelids (Thuesen & Childress, 1993a; Thuesen &
Childress, 1993b). Of the enzymes studied, citrate synthase (CS), pyruvate
kinase (PK), and lactate dehydrogenase (LDH) were all found to be good
indicators of metabolic potential. CS plays a key role in the Kreb’s cycle and
is critical in aerobic metabolism. The latter enzymes, PK and LDH, are
considered anaerobic metabolic proxies, as they are enzymes that mostly
9
contribute to glycolysis, although PK has minor roles in aerobic metabolism
as well. In addition to providing valuable ecophysiological information
about hard to study, fragile organisms, these metabolic proxies have
successfully been used in models to predict ecological relationships and
bloom dynamics (Dahlhoff, 2004; Purcell, 2009). Researchers have also
shown that when combined with accurate population estimates, metabolic
proxies can be used to estimate oceanic carbon flux inputs (Childress &
Thuesen, 1992).
Comparative Ecophysiology studies in other marine invertebrates
Physiological characteristics such as oxygen consumption rates and
key metabolic enzyme activities have been successfully used to elucidate
ecophysiological traits and aspects of functional biodiversity in other marine
organisms. For instance, studies of medusae and chaetognaths have shown
that species that live or migrate within the Oxygen Minimum Zone (OMZ)
tend to be anaerobically poised and shallower species displayed higher
aerobic metabolic potentials (Thuesen & Childress, 1994; Thuesen &
Childress, 1993b). Comparison of oxygen consumption rates and metabolic
enzyme activities in some organisms has shown support for the use of
certain enzymes as indicators of metabolic potential (Thuesen & Childress,
1993b). Enzymatic activities of different species of copepods were able to
10
reveal distinct groups that were associated with aspects of their morphology
and ecology (Thuesen et al., 1998b). All of the ecophysiological
comparative studies mentioned above show that Chaetognatha, Medusae,
and Copepoda are composed of physiologically distinct groups of species,
which suggests that they occupy different functional niches in their
environment.
Visual Interactions Hypothesis
Several studies have shown that organisms with image-forming eyes
have metabolic rates that decrease with increasing depth, in amounts that are
higher than expected once factors such as pressure and temperature are
accounted for (Childress & Mickel, 1985; Seibel et al., 1997; Seibel &
Drazen, 2007; Torres et al., 1994; Torres & Somero, 1988). Interestingly,
organisms such as medusa, copepods, and chaetognaths, which do not
contain image-forming eyes have shown little correlation between metabolic
activity and increased depth (Childress, 1995; Thuesen & Childress, 1994;
Thuesen & Childress, 1993; Thuesen et al., 1998b). The ‘visual interaction
hypothesis’ posits that visually orientating animals involved in predator-prey
interactions have higher metabolic rates in surface waters than the metabolic
rates of animals living in the deep, dark ocean (Childress & Somero, 1979;
Childress & Mickel, 1985). Reduction of light with depth reduces the
11
evolutionary pressure for burst swimming involved in such predator-prey
interactions and results in lower metabolic rates. There is currently no data
published on ctenophores as a group, with respect to the visual interactions
hypothesis. The physiological measurements taken in this study provide a
means to test the validity of the visual interactions hypothesis for the first
time in this abundant group of non-visual marine predators.
12
CHAPTER TWO: MANUSCRIPT
Formatted and prepared for: PLOS ONE
This manuscript has been prepared for submission to a peer-reviewed
journal. It is presented here as a preliminary draft to fulfill graduation
requirements for The Evergreen State College Master of Environmental
Studies program.
13
Ctenophore Ecophysiology
Ecophysiological characteristics of temperate ctenophore
species utilizing physical, morphometric, and enzymatic
measurements
Wilson, T.M. 1*, Thuesen, E.V. 1, Haddock, S.D.H.2
1
The Evergreen State College, Olympia, Washington 98505;
2
Monterey Bay Aquarium Research Institute, Moss Landing, California
95039.
*To whom correspondence should be addressed. Email:
wiltel16@evergreen.edu
-list of key words: Ctenophore, ctene plate, ctene row, morphometric,
metabolic scaling, cilia, creatine kinase.
14
Introduction
Ctenophores are gelatinous macrozooplankton found throughout the
world’s oceans. While most are planktonic, about 20 percent are actually
benthic (Mills, 1998). Originally thought to be a sister clade of Cnidarians,
recent research has placed them much closer to Porifera, as the most basal
multicellular organisms (Dunn et al., 2008; Ryan et al., 2013; Moroz et al.,
2014). Ctenophores inhabit all depth ranges of the ocean from upper
epipelagic zones to 6700 meters. Ctenophore locomotion is powered by
eight rows of ciliated combs, making them the largest organisms to utilize
cilia for movement. Because of their fragility and habitat, ctenophores are
very difficult to collect and study, and as a result, there is a large lack of
information regarding their physiology, ecology, and evolution (Haddock,
2004). Ctenophores typically feed by one of three mechanisms: tentacles,
feeding lobes, or engulfing (Haddock, 2007). Because of their important role
as predators of many commercial and noncommercial fish and shellfish
larvae, they merit further research into many aspects of their biology and
ecology (Shiganova, 1998). Furthermore, through biochemical analysis and
reliable population predictions, their role in oceanic carbon cycling can be
estimated (Childress & Thuesen, 1992). The taxonomy and evolutionary
history of Ctenophora is still largely disorganized, with many species
15
remaining undescribed, and many more species currently clumped into
families to which they may not belong (Appeltans et al., 2012; Haddock,
2004; Mills, 1998). The aim of the current project is to utilize physiological
measurements to help profile the functional diversity within Ctenophora.
Previous studies have shown support for the use of metabolic
enzymes in estimating overall metabolism. Muscle enzymes such as lactate
dehydrogenase, creatine kinase, malate dehydrogenase, citrate synthase, as
well as electron transport system components have been used to estimate
metabolism in fish and zooplankton (King & Packard, 1975; Childress &
Somero, 1979; Somero & Childress, 1990; Thuesen & Childress, 1993a;
Thuesen & Childress, 1993b). All of the organisms for which previous
researchers have used enzymatic activities as metabolic proxies utilize
muscle driven locomotion. Ctenophores are unique in that they rely upon a
non-muscle associated locomotion system (Tamm & Tamm, 1989), and we
are uncertain if such proxies will work for ctenophores.
Although lacking a true mesodermal layer, ctenophores do possess
the largest known smooth muscle cells, and some forms even have striated
muscle cells (Mackie et al., 1988; Martindale, 2005). Recent genome
sequencing results have shown that the ctenophore Mnemiopsis leidyi
possesses the gene for the ATP/ADP regulating enzyme creatine kinase
16
(Blast:http://research.nhgri.nih.gov/mnemiopsis/ontologies/kegg/
hsa00330.shtml). Typically, in other eukaryotic organisms, isoforms of
creatine kinase are expressed in tissues that require large amounts of ATP,
such as muscle tissue, cardiac tissue, and mitochondria (Wallimann et al.,
1992). In addition to buffering intracellular ATP levels, CK also plays a role
in regulating pH and buffering ADP concentrations (Wallimann et al., 1992).
In the current study, due to the unique pairing of muscle and ciliary systems
present in some members of Ctenophora (Tamm & Tamm, 1989), we
hypothesized that if CK provides the necessary energy for powering cilia,
then CK activity will scale allometrically with morphological measurements
of ctenes and ctene rows.
Material and Methods
Specimen collection
Ctenophore specimens were collected during research cruises of the R.V.
Western Flyer off central California utilizing SCUBA and ROV collection
methods during May 17-23, 2014, September 16-22, 2014, December 13-18,
2016, and July 24-31, 2018. Specimens were collected by ROV Doc Ricketts
using detritus samplers, or suction samplers for the hardier species (Haddock
et al., 2017). When possible, all specimens used in this study were
17
photographed live on board the ship. Specimens free of parasites and with
cleared gut contents were frozen in liquid N2 at sea, shipped on dry ice to
Olympia, Washington, and later transferred to -80 °C freezer for storage. All
specimens were analyzed within 6 months of capture. Pleurobrachia bachei
(A. Agassiz, 1860) specimens were collected utilizing 1-L “jelly-catchers” in
southern Puget Sound at Oakland Bay Marina, in Shelton, Washington in
July 2014, and in Budd Inlet, Olympia, Washington in July 2016.
Enzymatic activity measurements
Rates of citrate synthase (CS) and creatine kinase (CK) of 26 different
species of ctenophores sampled from a range of depths were assayed. CS
was chosen as a predictor of overall specimen metabolism, based on its
ability to correlate well with overall metabolic rate, resistance to freezing,
and applicability to fragile specimens (Thuesen & Childress, 1994). CK
activity was chosen due to its potential role in ciliary locomotion. Both
enzymes were assayed in duplicate for each specimen.
Frozen specimens were weighed on a Metler Toledo analytical balance, and
homogenized in 10 mmol TRIS/HCL buffer using handheld glass
homogenizers (15 ml or 40 ml) on ice, or when specimen size required, a
Waring commercial blender. Homogenate was centrifuged ten minutes at
18
4°C, 6600 g. All activity was measured within one hour of homogenization
using a Hewett- Packard diode array spectrophotometer with a temperature
controlled cuvette held at 20°C. All biochemical reagents for enzymatic
assays were obtained from Sigma Aldrich, with the exception of NADH
(AcrosOrganics).
The enzymatic activity of creatine kinase was assayed utilizing a coupled
enzymatic reaction involving the secondary enzymes, hexokinase and
glucose-6-phosphodehydrogenase (Dawson, 1970; Szasz et al., 1976). In this
assay, CK dephosphorylates phosphocreatine, resulting in the production of
ATP. The ATP is further reacted by hexokinase to phosphorylate glucose,
which then is reacted with G6-PDH to produce NADPH. The production of
NADPH is followed spectrophotometrically at 340 nm, and is directly
proportional to CK activity. The final cuvette reaction volume of 1 ml
contained 100 mM Imidizole Buffer (pH 7.1), 10 mM MgCl2, 20 mM
glucose, 1.8 mM ADP, 3.0 mM phosphocreatine, 1.3 mM NADP, 1600
Units L-1 G-6-PDH, and 2800 Units L-1 hexokinase. The activity was
measured for up to six minutes, excluding a lag phase of approximately 100
seconds. Efficacy of the assay conditions were confirmed using tissue from
store bought cod filets (results not shown) (Somero & Childress, 1990).
19
Due to differences of pH presented in CK assay protocols (Dawson &
Eppenberger, 1970; Szasz et al., 1976; Somero & Childress, 1990), a range
of pH conditions were tested to determine the optimum pH for CK activity
measurements in ctenophore tissue homogenates. Specimens of P. bachei
were assayed in triplicate with solutions at pH of 6.5, 6.7, 6.9, 7.1, 7.3, and
7.5 (results not shown). A pH of 7.1 was chosen as the optimum pH for CK
activity measurement in ctenophores.
The enzymatic activity of citrate synthase was measured as an increase of
absorbance at 412 nm via its reaction with the substrate 5, 5-Dithiobis (2nitrobenzoic acid) (DTNB). The final cuvette reaction volume of 1 mL
contained 50 mM imidazole/HCL buffer (pH 7.8 at 20°C), 0.5 mM
oxaloacetate, 0.1 mM acetyl-CoA, 0.1 mM DTNB, and 1.5 mM MgCl2
(Childress & Somero, 1979). CS activity was measured for up to six
minutes, with addition of oxaloacetate after background was detected, and
any background rate was subtracted from the latter rate. All activities are
given in units (µmols of substrate converted to product per minute) g-1.
Morphometric measurements
Morphometric measurements from 13 different species were analyzed.
Morphometric analyses were carried out on a subset of the total specimens
20
(N=24). All specimens were photographed live with reference scale bars
onboard ship, with the intent to capture width, height, length, and area of
ctene rows. A combination of live footage stills (shot prior to capture by
cameras on the ROV Doc Ricketts) and photos of live specimens onboard
ship were used to calculate total ctene plate numbers. At least four of the
eight ctene rows were included in the ctene plate counts per specimen. Due
to the size and shape of each specimen, it was impossible to obtain counts
from all eight rows from any one photo, and means of visible rows were
used. All CS and CK measurements were analyzed for relationships between
enzymatic activities and morphometric characteristics.
Statistical analysis
Power regressions were carried out on log-transformed measurements to
improve linearity, utilizing KaleidaGraph, version 4.5.2 statistical software.
A confidence level of 95% was applied when interpreting correlations.
Results
A total of 125 specimens of 26 species were used in this study. Of the total,
117 specimens were from California and eight were from the Puget Sound in
Washington.
21
Enzymatic activities
Enzymatic activities were successfully measured on 125 different specimens
from 26 different species, belonging to six different orders, Lobata,
Cydippida, Beroida, Platyctenida, Thalassocalycida, and Cestida (Table 1).
Thirteen of the species are new to science and were given operational names.
Haeckelia beehleri displayed the highest CS activity, whereas Kiyohimea
usagi displayed the lowest, 0.1920 units g-1 and 0.0030 units g-1, respectively
(Table 1). Beroe gracilis displayed the highest CK activity, whereas
Lampocteis sp. B displayed the lowest, 0.6408 units g-1 and 0.0115 units g-1
respectively (Table 1).
Metabolic scaling
Over the 26 species sampled, CS and CK activities did not significantly
correlate to body mass when species were viewed individually (p> 0.05, Fig.
1 and Fig. 2). CS and CK activities for specimens belonging to the order
Lobata were comparatively lower than activities displayed by orders Beroida
and Cydippida (Fig. 2 and Fig. 3). CK activities showed a decrease with
body mass for the orders Beroida, Cydippida, and Lobata (y=0.128x^(0.289); R=0.74, p<0.0012; y=0.0623x^(-0.109); R=0.17, p>0.05 and
y=0.0394x^(-0.0855); R=0.34, p>0.05 respectively, Fig. 2a, Table 2). CK
22
activities of engulfer, tentacle, and lobe feeders showed a significant
decrease with body mass (g) (y=0.128x^(-0.289); R=0.749, p<0.0012,
y=0.0867x^(-0.207); R=0.478, p<0.059, and y=0.0413x^(-0.111); R=0.449,
p<0.0087 respectively, see Fig. 4.b and Table 2). CS activity did not
correlate significantly with body mass when viewed by feeding strategy
(p>0.05, Fig. 4.a). CS activity of engulfers displayed an inverse scaling
relationship comparative to that of tentacle and lobe feeders (Fig. 2.b).
Morphometric characteristics
An undescribed Nephaloctena (sp. A) displayed the highest comb plate
density, 1528.3 (comb plates g-1), whereas Beroe abyssicola had the lowest,
27.9 (comb plates g-1) (Table 3). Comb plate density showed a significant
decrease with body mass (y = 190.52x-0.87; R = 0.96, p < 0.0001; Fig. 5),
while comb row area (mm2) showed a significant increase with body mass (y
= 4.64x0.52; R = 0.63, p < 0.01; Fig. 5). Surface area of comb plates (mm2)
and CK activity were not correlated (p<0.05, figures not shown). CK and CS
activities did not correlate with comb plate density or number of combs
(p<0.05, figures not shown). The total number of combs did not significantly
correlate with body mass (Fig. 5).
23
Enzymatic activities in relation to depth
The minimum depth of occurrence (MDO) was considered to be the depth
below which 95% of the population of each species lives (Childress, 1995).
This was calculated for each species using the MBARI Video Annotation
and Reference System database (VARS, Schlining & Jacobsen Stout, 2006)
for ROV observations over twenty-eight years and by personal
communication for undescribed species (Table 1). CS and CK activities
showed no significant decline with minimum depth of occurrence in
ctenophores. CS activity of pelagic fishes from the same region significantly
declined with depth (Childress & Thuesen, 1995) (y=10.355x-0.56; R =
0.77, p < 0.001 Fig. 6).
Discussion
Enzymatic activity
Enzyme activities yielded some interesting characteristics when specimens
were grouped by the three orders with the most abundant number of
specimens, Beroida, Lobata, and Cydippida. Lobata displayed the lowest CK
and CS activities, and may represent a group of ctenophores that are less
active feeders. This has been supported by observations that some members
of Lobata are indeed very slow foragers (Matsumoto & Harbison, 1993).
24
Ctenophores that feed using the engulfing strategy displayed positive scaling
with CS activity, whereas all other feeding types for CS activities displayed
negative scaling trends. Lobe and tentacle feeding ctenophores are typically
considered filter feeders and sit and wait predators, respectively, while the
Beroids that comprise the ‘engulfers’ feeding type actively swim towards
prey (Matsumoto & Harbison, 1993). The positive scaling trend observed for
CS activity of engulfers may suggest that Beroids rely mostly upon
anaerobic metabolism during feeding interactions. However, current studies
suggest that traditionally held monophyletic orders such as Lobata and
Cydippida may in fact be polyphyletic. One study, which sampled 26
species, from 5 orders, compared 18s ribosomal RNA sequences and found
that traditional assigned orders based on morphological data need to be
reassigned (Podar et al., 2001) and that several species are more closely
related to members assigned to different orders. It may be worth revisiting
the enzymatic measurements as a function of body mass when grouped by
order once ctenophore phylogenetics are resolved.
Scaling
Overall, ctenophores displayed higher CK activities than CS activities.
Creatine kinase in muscle tissue provides adenosine triphosphate (ATP) for
muscle contraction. Similarly, our results suggest that CK is responsible for
25
providing ATP to drive cilia, similar to its role in muscle tissue. The high
number of giant mitochondria located in the polster cells associated with the
comb rows (Horridge, 1964) suggests that ctenophores are very aerobic
despite their low CS activity. In contrast to studies of CS activity in other
gelatinous zooplankton, members of this study did not display the common
allometric scaling relationships of decreasing activity with increasing body
mass. Overall, ctenophores displayed variability among the same species and
between species for the enzymatic activities studied. Although the
ctenophores studied here possessed variable scaling relationships, when
viewed collectively they did tend to have an overall negative allometric
scaling relationship of CK activity. Our results suggest that creatine kinase
plays an important role in ctenophore metabolism.
Morphometrics
This study is the first of its kind investigating the possible role of CK
between ctenophore locomotion and ctenophore cilia. Morphometric results
were obtained for a total of 13 different species, representative of five
different orders. Our results indicate high metabolic diversity among species
within the same given order, including members of Beroida despite high
morphological similarities within the order. Our results do not suggest a
relationship between ctene characteristics and CK activity. Arginine kinase
26
is an ancient phosphagen kinase, present in many invertebrates, which may
be worth investigating for its potential role in supplying ATP for ciliary
locomotion. Previous research has suggested that arginine kinase may
supply the needed energy in the form of ATP to drive ciliary motion in the
single celled organism Paramecium caudatum (Noguchi et al., 2001).
Adenylate kinase is another potential candidate worth investigating for
supplying ATP in ctenophore cilia. In addition to investigating other
phosphagen kinases, the ctenophore Velamen parallelum warrants further
study. This species had the lowest number of ctenes and among the highest
CK activity. Due to the ribbon-like nature of this species, photographing and
counting of ctenes was difficult and unfortunately, this species was not
included in our morphometric analysis. Velamen parallelum is known to
escape predation using a rapid undulating muscle driven motion (Stretch,
1982), which may account for its high CK activity despite low ctene counts.
Future morphometric studies of CK activity in this organism could be
interesting.
Both Beroida and Cydippida have relatively developed muscle tissue for
ctenophores, but this tissue is known to be associated with the mouth and the
tentacular sheath, respectively (Tamm & Tamm, 1989; Mackie et al., 1988).
This observation may support our finding that ctene morphometrics did not
27
positively scale with CK activity in these groups. The significant scaling
found between CK activity and mass when viewed by feeding strategy also
support this observation.
Enzymatic activities in relation to depth
Ctenophores, which lack image-forming eyes, support the visual interactions
hypothesis. Despite enzymatic activities that range over two orders of
magnitude for both pelagic fishes and ctenophores, there were no significant
effects of habitat depth on enzymatic activities of ctenophores. Although the
visual interactions hypothesis has been challenged (Childress et al., 2008),
overwhelming evidence supports this evolutionary hypothesis that animals
involved in visual predator-prey relationships in surface waters have much
higher metabolic rates than animals living in the perpetually dark, deep sea.
Conclusion
Members of Ctenophora covered in this study clearly display unique patterns
of biochemical diversity. The significant functional groups that emerged in
this study suggest that ctenophores have evolved different physiological
strategies to adapt to their environment. Because of such physiological
diversity, future studies should take care not to lump different species
together when analyzing metabolic characteristics, but should take
morphological, functional, and taxonomical differences into consideration
28
during analyses. While the role between CK and cilia is still unclear in
ctenophores, our study suggests CK plays a vital role in ctenophore
metabolism. It would be interesting to see if CK activity has potential for use
as a metabolic indicator for the phylum. Lastly, a more complete knowledge
of ctenophore physiology is crucial to establishing a baseline for their role in
oceanic ecosystems, predicting and understanding bloom dynamics, and
perceiving how they may be impacted by both anthropogenic-driven climate
change, and natural environmental fluxes (Childress & Thuesen, 1992;
Mills, 2001).
29
CHAPTER THREE: CLOSING REMARKS
Conclusion
Enzymatic activities of ctenophores sampled for this study suggest that the
group is highly diverse physiologically, as well as morphologically. When
all specimens sampled were evaluated for enzymatic scaling with body
mass, there were no significant trends visible. While there were no
significant scaling patterns between either CK or CS when viewed by
individual specimen wet mass, several significant patterns emerged when
comparing enzymatic measurements within different functional groups. Of
the functional groups investigated, the three most common feeding strategies
were particularly good indicators of functional diversity when viewed by CK
activity. The feeding strategies used in this study are not all inclusive, and
there are many variations that exist within the phylum. Future studies would
benefit by characterizing and including the other feeding strategies which
exist within the phylum. The feeding strategies described by engulfing,
tentacles, and lobate did not cover all of the species used within this current
study.
Morphometric investigation of the role of CK in powering ciliary movement
in ctenophores was inconclusive. This study focused on a subset of the total
30
specimens collected, and future studies may benefit by including more
specimens per species sampled. A single small and a large representative
was chosen for each species used in our morphometric study. In future
studies, it may be worthwhile to investigate specimens that constitute a
larger size range, and also explore activities of different phosphagen kinases.
Additionally, further studies using ctene preps and the biochemicals required
for CK activity could help to elucidate the relationship between ciliary
motion and CK.
Depth related decline in CS and CK activities were not detected for the
species included in this study. The species sampled represent six of the eight
orders currently assigned in the phylum, and suggest that ctenophores show
support for the visual interactions hypothesis. Additionally, it was observed
that the deepest specimen collected and analyzed, an undescribed Tjalfiella
sp., had among the highest CK activity measured. It is worth noting that CK
and CS activities have not been thoroughly tested for their potential to be
suitable metabolic proxies in ctenophores. This is partly due to the
difficulties in obtaining respiration measurements on fragile and rare
species. However, a comparison of oxygen consumption rates and CK
activity in the more robust species of ctenophores may provide useful
insights.
31
Figures
Figure 1. a) Creatine kinase activity as a function of body mass in 27
species of Ctenophora. Ctenophores do not show significant scaling in
creatine kinase activity (units g-1) over all species tested.
32
Figure 1. b) Citrate synthase activity as a function of body mass (g).
Ctenophores do not show significant scaling in either of these enzymes over
all species tested. Refer to Figure 1a for symbol legend.
33
Figure 2. a) Creatine kinase activity as a function of body mass for beroid,
cydippid, and lobate ctenophores. When groups are viewed separately,
significant correlations were found between CK activity and body mass for
members of Beroida (y=0.128x^(-0.289); R=0.74, p<0.0012). Cydippid and
lobate species did not display significant results (y=0.0623x^(-0.109);
R=0.17, p>0.05 and y=0.0394x^(-0.0855); R=0.34, p>0.05 respectively).
34
Figure 2. b) Citrate synthase activity as a function of body mass for beroid,
cydippid, and lobate ctenophores when viewed separately did not yield
significant correlations for CS activity and body wet mass
(y=0.0384x^(0.0856); R=0.11, p>0.05; y=0.0339x^(-0.109); R=0.24,
p>0.05; and y=0.00953x^(-0.126); R=0.24, p>0.05 respectively). When
viewed as a whole, different orders displayed unique scaling patterns, with
the order Beroida exhibiting an inverse scaling relationship.
35
Figure 3. Creatine kinase vs. citrate synthase. Members of Lobata are
represented with circle symbols and circled in blue, all other species are
circled in red.
36
Figure 4. a) Citrate synthase activity as a function of body mass viewed by
feeding morphology. Different feeding groups did not display significant
correlation with body mass (P>0.05), but appear to display unique clustering
by functional group.
37
Figure 4. b) Creatine kinase activity as a function of body mass viewed by
feeding morphology. CK activities of engulfer, tentacle, and lobate feeding
groups significantly scaled with body mass (y=0.128x^(-0.289); R=0.749,
p<0.0012, y=0.0867x^(-0.207); R=0.478, p<0.059, and y=0.0413x^(-0.111);
R=0.449, p<0.0087 respectively).
38
Figure 5. Ctenophore comb plate densities (u), total numbers of combs
(
) and ctene row surface areas () as a function of total body wet mass
for 13 species, two specimens each (when possible). Comb plate densities
are correlated significantly with body mass (y = 190.52x^(-0.87); R = 0.96,
p < 0.0001). Comb row surface areas are also correlated significantly with
body mass (y = 4.64x^(0.52); R = 0.63, p < 0.01). The metabolic enzymes
creatine kinase (CK) ( ) and citrate synthase (CS) (u) show no significant
difference with body size
39
Figure 6. Citrate synthase and creatine kinase activities of Washington and
California ctenophores as a function of minimum depth of occurrence
compared to pelagic fishes from the same region. The slopes of the
regression lines for ctenophores are not significantly different from zero.
The slope of the regression line for CS activities of pelagic fishes is
y=10.355x-0.56; R=0.77, p< 0.001 (Childress & Somero, 1979; Childress &
Thuesen, 1995).
40
Tables
Table 1. Enzymatic activities of ctenophores collected off California and Washington
Order
Family
Genus and species
Cydippida
Haeckeliidae
Haeckelia beehleri
Pleurobrachiidae
Hormiphora californensis
Pleurobrachia bachei
Euplokamidae
Euplokamis dunlapae
Undescribed Cydippida
Cydippida sp. A
Cydippida sp. B
Cydippida sp. C
Cydippida sp. D
Cydippida sp. E
Cydippida sp. N
Cydippida sp. G
Dryodoridae
Dryodora sp. A
Mertensiidae
Wet weight
range (g)
Enzymatic activity (mean ± SE, number of specimens)
CK (units g-1)
CS (units g-1)
MDO (meters)
0.2860-1.1206
0.4320 ± 0.103, 2
0.1920, 1
10
0.4600-3.3950
0.3548-0.6600
0.1067 ± 0.009, 9
0.0555 ± 0.018, 8
0.0458 ± 0.014, 6
0.0602 ± 0.011, 7
20
7.9
0.1291-0.1487
0.5505 ± 0.049, 4
na
423.6
4.0300-7.5000
0.2315-2.2040
0.4150-1.8610
1.14
0.195
0.1260-0.1380
0.6160-2.2880
0.1675 ± 0.002, 2
0.0528 ± 0.012, 3
0.0247 ± 0.002, 2
0.1100, 1
0.1900, 1
0.0695 ± 0.018, 2
0.0991 ± 0.031, 5
0.0400 ± 0.002, 2
0.0353 ± 0.025, 3
0.0086 ±0.004, 2
0.0750, 1
0.0580, 1
0.0505 ± 0.044, 2
0.0459 ± 0.002, 5
na
450
1000
450
200
33
750
0.59
0.0430, 1
0.0500, 1
30
41
Mertensia sp. A
Platyctenida
Tjalfiellidae
Tjalfiella sp. A
Thalassocalycida
Thalassocalycidae
Thalassocalyce inconstans
Lobata
Bathocyroidae
Bathocyroe fosteri
Bolinopsidae
Bolinopsis infundibulum
Eurhamphaeidae
Kiyohimea usagi
0.126-0.138
0.0695 ± 0.013, 2
0.0505 ± 0.0315, 2
na
4.8840-7.8710
0.0977 ± 0.012, 4
0.0350 ± 0.013, 2
2940
0.6600-1.5200
0.087 ± 0.009, 4
0.0448 ± 0.009, 4
50
0.4280-51.2500
0.0398 ± 0.006, 11
0.0086 ± 0.002, 4
506.5
0.4684-50.6212
0.0388 ± 0.004, 12
0.0075 ± 0.001, 11
40
5.4200-31.2200
0.0215 ± 0.004, 2
0.0030 ± 0.002, 2
51.9
0.2970-192.0400
52.66
0.0342 ± 0.007, 5
0.0115, 1
0.0258 ± 0.014, 5
0.0069, 1
650
850
0.2200-27.3400
0.0319 ± 0.003, 7
0.0079 ± 0.001, 7
1200
0.2970-0.8600
0.2300 ± 0.023, 5
0.0672 ± 0.013, 5
10
12.4430-44.1500
0.4898-36.6600
1.1390-97.5600
0.0488-0.3636
0.0637 ± 0.016, 3
0.0476 ± 0.005, 15
0.1672 ± 0.030, 6
0.5285 ± 0.117, 8
0.0410 ± 0.019, 3
0.0555 ± 0.008, 13
0.0606 ± 0.020, 4
na
453
212.1
17.6
200.1
Lampoctenidae
Lampocteis sp. A
Lampocteis sp. B
Undescribed Lobata
Lobata sp. V
Cestida
Cestidae
Velamen parallelum
Beroida
Beroidae
Beroe abyssicola
Beroe cucumis
Beroe forskalii
Beroe gracilis
42
Table 2. Scaling relationships of enzymatic activities of ctenophores from California and Washington.
Significant relationships presented in bold.
Group
Beroida
Cydippida
Lobata
Engulfers
Lobes
Tentacles
Enzymatic activity g-1 wet weight (y) as a function of total body wet weight (M), y=aMb
b (±95% C.I.,n)
Enzyme
A
p
CK
CS
CK
CS
CK
CS
0.13
0.038
0.06
0.033
0.04
0.0095
-0.28, 32
0.085, 20
-0.11, 45
-0.15, 31
-0.09, 31
-0.14, 30
0.0012
0.4383
0.0891
0.3588
0.0621
0.0686
CK
CS
CK
CS
CK
CS
0.12
0.038
0.041
0.0094
0.086
0.035
-0.29, 32
-0.085, 20
-0.11, 32
-0.13, 30
-0.21, 40
-0.16, 30
0.0012
0.4380
0.0088
0.0685
0.0599
0.3530
43
Table 3. Morphometric characteristics of oceanic ctenophores from California and Washington.
Species
Cydippida sp. A
Cydippida sp. N
Cydippida sp. G
Dryodora sp. A
Lobata sp. V
Haeckelia beehleri
Hormiphora
californensis
Pleurobrachia bachei
Thalassocalyce
inconstans
Bathocyroe fosteri
Velamen parallelum
Beroe abyssicola
Beroe cucumis
Beroe forskalii
Morphometric measurements (mean ± SE, number of specimens)
Comb row area
Comb plate density (comb
(mm2)
plates g-1)
Number of combs
228 (± 46.5), 2
32.98, 1
43.4 (± 5), 2
1.27 (± 0.15), 2
1528.3 (± 122.5), 2
201 (± 7), 2
2.85 (± 4.45), 2
173.5 (± 126.25), 2
176 (± 332), 2
3.44, 1
297.8, 1
176, 1
NA
483.7, 1
149 (± 45), 2
2.24, 1
772.73, 1
221, 1
11.23 (± 3.1), 2
NA
331.5 (± 124.5), 2
369.8 (± 51.1), 2
228 (± 20), 2
203 (± 6.5), 2
1.32 (± 0.075), 2
4.85, 1
NA
2.45 (± 0.55), 2
16.2 (± 8.3), 2
59.32 (± 49.05), 2
77.4 (± 22.6), 2
45.25 (± 39.4), 2
NA
27.9 (± 10.26), 2
203.4 (± 126.3), 2
177.02 (± 356.49), 2
70 (± 4), 2
276 (± 24), 2
Lowest Observed
575 (± 355), 2
605 (± 211), 2
489 (± 356.5), 2
44
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