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NUTRIENT INTAKE, NUTRIENT TARGETS AND FEED APPLICATION TO
PROMOTE OPTIMAL PRODUCTION AND FEED EFFICIENCY IN THE SEA
URCHIN Lytechinus variegatus (ECHINODERMATA: ECHINOIDEA)
by
LAURA E. HEFLIN
STEPHEN A. WATTS, COMMITTEE CHAIR
LOUIS D’ABRAMO
JAMES MCCLINTOCK
DANIEL SMITH
DANIEL WARNER
A DISSERTATION
Submitted to the graduate faculty of The University of Alabama at Birmingham
in partial fulfillment of the requirements of the degree of
Doctorate of Philosophy
Birmingham, AL
2015
NUTRIENT INTAKE, NUTRIENT TARGETS AND FEED APPLICATION TO
PROMOTE OPTIMAL PRODUCTION AND FEED EFFICIENCY IN THE SEA
URCHIN Lytechinus variegatus (ECHINODERMATA: ECHINOIDEA)
LAURA E. HEFLIN
BIOLOGY
ABSTRACT
Important considerations for the development of sea urchin aquaculture will
include understanding nutrition and feed management. These reported investigations
contribute to knowledge of dietary protein and carbohydrate requirements in Lytechinus
variegatus and feed management strategies. The first evaluation of economic feasibility
of sea urchin aquaculture as related to feed costs is presented and the geometric
framework (GF) to assess nutrient intake targets is first applied to cultured aquatic
organisms to yield recommendations for nutrient balancing in formulated urchin feed.
Data- derived models for dietary protein and carbohydrate intake predict
increased rates of growth and production among urchins fed diets containing 18% dietary
carbohydrate levels as compared to urchins fed diets containing 12% dietary
carbohydrate as the level of dietary protein increases up to ca. 30%. The energetic cost of
dry matter tissue production suggested more energetic cost (decreased energy efficiency)
is required to increase gonad production relative to somatic growth. For both 18 and 12%
levels of dietary carbohydrate, cost per gram of wet weight gain was predicted to be
maximized at dietary protein levels of 25- 35% or lower, regardless of feed ingredient
costs. For urchins fed a standard maintenance feed, increased gonad dry matter
production and gonad index were observed among individuals fed at least once per day,
regardless of ration size.
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GF results indicated that adults (120 g) maintained an average dietary protein
intake of ca. 0.047- 0.061 g day-1 but did not regulate carbohydrate intake. Juvenile sea
urchins demonstrated flexible intake target ranges for dietary protein and carbohydrate.
When single diets did not allow the realization of intake targets, urchins maintained weak
homeostatic regulation, utilizing ‘fixed proportion’ strategy to maintain a constant ratio
of error between protein and carbohydrate intake. Among juveniles provided diets in
combination, urchins consumed more of the most balanced (equi- proportioned) diet to
regulate protein and carbohydrate within diffuse target ranges. Regardless of whether
juveniles were fed single diets or diet combinations, the protein intake target was
prioritized over that of carbohydrate.
Keywords: sea urchin, protein, carbohydrate, nutrition, feed management, nutritional
geometry
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DEDICATION
To my husband, J. Wayne Morgan, and my sister, Anna M. Heflin for their
support, patience and encouragement, for never losing faith in me and never letting me
give up. Thank you, Wayne, for the long hours spent in the laboratory helping me with
labor-intensive projects and the time and money invested in my professional
development. Also, to my mentor Dr. Stephen A. Watts for his friendship and support
and for believing in me more than I believe in myself.
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ACKNOWLEDGMENTS
I thank my mentor, Dr. Stephen Watts, for giving me this opportunity and for
guiding me along the path to becoming a sound researcher through patience, advice and
improvement of my critical thinking skills. I thank my dissertation committee members
Dr. Louis D’Abramo, Dr. Daniel Warner, Dr. James McClintock, and Dr. Daniel Smith
for challenging me to view my research and science as a whole from a fresh perspective,
for reviewing manuscripts and for their guidance and support throughout my dissertation
research. I thank Dr. Addison Lawrence and Dr. John Lawrence for manuscript review
and for assisting in my professional development throughout my graduate career. I would
like to thank Dr. Anne Cusic, Alan Whitehead, Rebecca Vance and Raymond Oden for
advising me in developing my teaching skills and for allowing me the opportunity to
mature as an educator. I would like to thank Dr. Robert Makowsky for his tireless
assistance with statistics and for assisting me in improving my statistical knowledge
throughout both my Masters and PhD work. I would like to thank Dr. Victoria Gibbs for
her mentorship and guidance. I thank R. Jeffery Barry, J. Christopher Taylor and Michael
Williams for tireless, often unrewarded, technical assistance throughout the course of my
dissertation research. I thank Lacey Dennis, Samara Hunter, Marlee Hayes, Yuan Yuan
and Adele Fowler for their friendship and moral support as well as technical assistance. I
appreciate the assistance and support of all other past and present members of the Watts
lab including Dr. Mickie Powell, Dr. Warren Jones, Dr. Julie Price, Dorothy Mosley,
Kate Kohlenberg, Lindsay White, Susan Sewell, Courtney Duncan, Pryia Patel, Amanda
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Monroe, Sharon Cai, Jasmine Nelson, Matthew Snead, Shara Legg, Jonathan Sahawneh,
John Bradford, Jamiko Rose, Ryan Miller, Steve Padgett, Kim Trawick, Jessica Etling,
Christina Richardson, and Dr. Anthony Sicardi. Thank you to the Department of Biology,
UAB Graduate School and NOAA Sea Grant for financial support during my graduate
training.
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TABLE OF CONTENTS
Page
ABSTRACT...................................................................................................................… ii
DEDICATION................................................................................................................... iv
ACKNOWLEDGMENTS...................................................................................................v
LIST OF TABLES............................................................................................................. ix
LIST OF FIGURES........................................................................................................... xi
INTRODUCTION...............................................................................................................1
EFFECT OF LEVELS OF DIETARY PROTEIN AND CARBOHYDRATE ON THE
CULTURE OF JUVENILE SEA URCHIN Lytechinus variegatus AND ECONOMIC
CONSIDERATIONS FOR DIETARY FORMULATION.............................................. 31
FEEDING TIME, FREQUENCY AND RATION AFFECTS GROWTH AND ENERGY
ALLOCATION IN YOUNG ADULTS OF THE SEA URCHIN Lytechinus
variegatus...........................................................................................................................85
BALANCING MACRONUTRIENT INTAKE IN CULTURED Lytechinus
variegatus.....................................................................................................................…124
GENERALIST FEEDING STRATEGIES UTILIZED BY JUVENILES OF THE SEA
URCHIN Lytechinus variegatus.................................................................................…155
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Page
CONCLUSIONS.........................................................................................................…209
GENERAL LIST OF REFERENCES................................................................................215
APPENDIX A: INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE
APPROVAL……………………………….......................................................................222
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LIST OF TABLES
Table
Page
EFFECT OF LEVELS OF DIETARY PROTEIN AND CARBOHYDRATE ON THE
CULTURE OF JUVENILE SEA URCHIN Lytechinus variegatus AND ECONOMIC
CONSIDERATIONS FOR DIETARY FORMULATION
1. Calculated nutrient levels on an “as fed” basis for the base
experimental diet……………………………………………………………...….43
2. Calculated protein and carbohydrate levels (as were fed), total energy, protein:
energy, and protein: carbohydrate caloric ratios in each of
the ten diets tested………………………………………………………………..44
3. Parameter estimates, tests of significance and goodness of fit for various measures
of Lytechinus variegatus growth models……………………………………...…48
FEEDING TIME, FREQUENCY AND RATION AFFECTS GROWTH AND ENERGY
ALLOCATION IN YOUNG ADULTS OF THE SEA URCHIN Lytechinus variegatus
1. Feeding treatments…………………………………………………………….…92
2. Calculated nutrient levels on an “as fed” basis for the base
experimental diet………………………………………………………………....94
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Table
Page
BALANCING MACRONUTRIENT INTAKE IN CULTURED Lytechinus variegatus
1. Calculated fish meal and wheat starch levels (as fed), agar, synthetic seawater,
salt and percent moisture of diets………………………………………………133
2. Pairwise diet combinations (4 treatments) offered to urchins (n=5 per
treatment)………………………………………………………………………136
GENERALIST FEEDING STRATEGIES UTILIZED BY JUVENILES OF THE SEA
URCHIN Lytechinus variegatus
1. Calculated nutrient levels on an “as fed” basis for the base
experimental diet..................................................................................................166
2. Calculated protein (% dry matter, lipid from animal sources removed),
carbohydrate (% dry matter) and lipid (% dry matter, including lipid from animal
sources) levels in each of the five feeds tested and non-nutritive ingredients and
food concentrations of moist diets prepared……………………………………168
x
LIST OF FIGURES
Figure
Page
INTRODCTION
1. Hypothetical protein- lipid nutrient space with 2 foods…………………………14
EFFECT OF LEVELS OF DIETARY PROTEIN AND CARBOHYDRATE ON THE
CULTURE OF JUVENILE SEA URCHIN Lytechinus variegatus AND ECONOMIC
CONSIDERATIONS FOR DIETARY FORMULATION
1. Schematic of recirculating system……………………………………………….41
2. Percent wet weight gain………………………………………………………….51
3. Total wet weight gain…………………………………………….………………52
4. Gonad dry matter production…………………………………………………….53
5. Dry gonad index………………………………………………………………….54
6. Gut dry matter production………………………………………………………..56
7. Dry gut index…………………………………………………………………….57
8. Lantern dry matter production…………………………………………………...58
9. Test dry matter production……………………………………………………….59
10. Food conversion ratio (FCR).……………………………………………………62
11. Production efficiency (PE)……………………………………………………….63
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Figure
Page
12. Protein efficiency ratio (PER)……………………………………………………64
13. Production energy efficiency (PEE)……………………………………………..65
14. Economic analysis of sea urchin diets…………………………………………...67
FEEDING TIME, FREQUENCY AND RATION AFFECTS GROWTH AND ENERGY
ALLOCATION IN YOUNG ADULTS OF THE SEA URCHIN Lytechinus variegatus
1. Schematic of recirculating system…………………………………………….…93
2. Total wet weight gain (g, ±SEM) by treatment...…………………………….….98
3. Total dry matter production (g, ±SEM) by treatment……...…………………….99
4. Gonad dry matter production (g, ±SEM) by treatment…...…………………….100
5. Gonad index (%, ±SEM) by treatment..………………………………………..101
6. 62 day gut dry matter production (g, ±SEM) by treatment...…………………...102
7. 62 day gut index (%, ±SEM) by treatment.......………………………………...103
8. Test dry matter production (g, ±SEM) by treatment.…………………………...104
9. Lantern dry matter production (g, ±SEM) by treatment...……………………...105
10. Lantern: test index (%, ±SEM) by treatment...…………………………………106
11. Dry matter production efficiency (%, ±SEM) by treatment...………………….108
12. Feed conversion ratio by treatment (±SEM)……………………………………109
BALANCING MACRONUTRIENT INTAKE IN CULTURED Lytechinus variegatus
1. Average daily diet intake (g, ±SEM)...…………………………………………138
2. Percent daily protein and carbohydrate intake by diet combination……………140
3. Average daily macronutrient intake (mg, ±SEM)………………………………141
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Figure
Page
GENERALIST FEEDING STRATEGIES UTILIZED BY JUVENILES OF THE SEA
URCHIN Lytechinus variegatus
1. Schematic of recirculating system……………………………………………...164
2. No choice diets and pairwise diet combinations..………………………………171
3. Total mean diet intake (g, as fed, includes feed plus water and agar binding
matrix, ±SEM)…………………………………………..……………………...175
4. Total mean macronutrient intake points (g, ±SEM)……………………………176
5. Average total mean macronutrient intake points (g, ±SEM) among urchins fed
diets with 8 and 12% food concentration in exp. 1……………………………..177
6. Total mean dry feed intake (g, dry matter, no water or agar binding matrix,
±SEM) among exp. 1, 8 and 12% food concentrations………………………...178
7. Total mean protein (dark bars) and carbohydrate (pale bars) intake (g, ±SEM)
among urchins in exp. 1, 8 and 12% food concentrations…………...…………179
8. Total mean protein to non-protein energy intake (cal, ±SEM) among urchins in
exp.1.....................................................................................................................180
9. Average total mean protein to non-protein energy intake (cal, ±SEM) among
urchins fed diets with 8 and 12% food concentrations in exp. 1……………….181
10. Total mean macronutrient intake points for urchins in exp. 2………………….183
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Figure
Page
11. Total mean intake (g, as fed, includes feed plus water and agar binding matrix,
±SEM) of protein and carbohydrate diets within the respective diet combinations
among urchins in exp. 2….…………………………………………………..…184
12. Total mean diet intake (g, as fed, includes feed plus water and agar binding
matrix, ±SEM) among urchins in exp. 2………………………………………..185
13. Total mean dry feed intake (g, dry matter, no water or agar binding matrix,
±SEM)………………………………………………………………………..…186
14. Actual total mean protein (A) and carbohydrate (B) intake among urchins in exp.
2 vs. hypothetical intake expected if urchins had eaten equivalent amounts of each
diet within a diet combination………………………………………………….187
15. Combined total mean protein + total carbohydrate intake (±SEM)……………188
16. Total mean lipid intake (g, ±SEM) among urchins in exp. 2………………...…188
17. Total mean energy intake (cal, ±SEM) among urchins in exp. 2……………....189
18. Total mean protein to non-protein energy intake (cal, ±SEM) among urchins in
exp. 2……………………………………………………………………………190
19. Total mean macronutrient intake points for urchins in exp. 1 and exp. 2...……191
20. Total mean protein to non-protein energy intake (cal, ±SEM) among urchins in
exp. 1, 8% food concentrations and exp. 2……………………………………..192
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1
CHAPTER 1
by
LAURA E. HEFLIN
INTRODUCTION
Sea urchins are a major component of marine subtidal communities and are
classified as ecosystem engineers because they alter both structural habitats and food
resources (Jones et al., 1997, Rogers-Bennett, 2013). Among sea urchin species that
inhabit kelp forests, their grazing controls algal and kelp biomass (thereby causing a shift
in the availability of particular foodstuffs) and creates habitats where microorganisms
may shelter (Rogers- Bennett, 2013). Urchins are also an important prey item for many
species including sea otters, crabs, fish, eels and birds. Over the last several decades, sea
urchin populations have declined in many areas of the world. Waning populations are
largely attributed to commercial over-harvesting in an attempt to supply an increasing
demand for sea urchin roe (Sloan, 1985; Keesing and Hall, 1998; Lesser and Walker,
1998; Andrews et al., 2003; Robinson, 2004). Various measures have been considered to
protect and/or replenish natural urchin populations. However, urchin stocks are not
expected to recover quickly and ongoing efforts are unlikely to result in population
increases that will be sufficiently large enough to supply the commercial market for sea
urchin roe while avoiding decimation of existing populations.
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Despite decreases in the urchin fisheries, the popularity of urchin roe (in Asian
cultures it is usually prepared as sushi or sashimi) continues to grow. Sea urchins are
harvested and eaten worldwide. Japan imports and consumes the majority (around 80%)
of urchin roe (Keesing and Hall, 1998; Andrews et al., 2003; FAO, 2010; NOAA, 2010).
Due to intensified fishing effort, total global harvest of sea urchins increased by
approximately 109,000 metric tons from 1950 to 1995 (NOAA, 2003). Since then,
worldwide landings of sea urchins have trended downward (FAO, 2012; NOAA, 2013;
Rahman et al., 2014). That is largely attributed to a combined decrease in landings in
several major fisheries around the world, including those of Chile, the US, Japan,
Canada, and Mexico (NOAA, 2003; FAO, 2012).
The decline of urchin stocks and simultaneous increase in the market for roe has
generated noteworthy interest in the development of large scale sea urchin aquaculture
(Lesser and Walker, 1998; Keesing and Hall, 1998; Andrews et al, 2003; Robinson,
2004; Kelly, 2005; Cook and Kelly, 2007; Eddy et al., 2010; Humphries et al., 2012;
Rahman et al., 2014). A consistent supply of marketable cultured sea urchins would serve
to supplement urchin harvests from the wild (helping to prevent further depletion of
natural populations) while also satisfying the market for roe. Research is currently being
conducted to develop sea urchin culture techniques but many challenges remain,
noteably, nutrition and those factors which affect nutrient utilization and feed intake.
Sea urchins occupy a wide range of ecological niches throughout the world.
Consequently, nutrient requirements for each species of interest are expected to vary
somewhat among species and will need to be independently evaluated. The species of
interest used in my dissertational research is Lytechinus variegatus. While L. variegatus
3
is not a species that is historically fished (with the exception of several island populations
in the Caribbean), it has high potential for aquaculture (Lawrence and Bazhin, 1998).
Adult L. variegatus are easily induced to spawn in captivity and resultant larvae reach
metamorphosis in 14 to 21 days (McEdward and Herrera, 1999; Buitrago et al., 2005). As
a ruderal species, it reaches maturity quickly, growing to about 45 mm in diameter within
a year (Moore et al., 1963; Beddingfield, 1997) and may attain maximum roe production
at that time (Greenway, 1977). Recently, food experts and several nationally-ranked chefs
in the U.S., have turned their attention to L. variegatus as a possible sustainable source of
urchin roe. Roe of L. variegatus has been prepared and served in upper tier restaurants in
both Alabama and South Carolina. Chefs describe the taste of cultured L. variegatus roe
as sweet and clean with a pleasant umami flavor and have expressed interest in its
inclusion as a permanent menu item. Despite the potential market for roe, techniques are
lacking to establish successful echinoculture of this or any other urchin species. Of
critical importance is the determination of dietary nutrient requirements combined with
the formulation of a least cost practical feed.
In an aquaculture facility, feed costs can account for approximately half of total
variable cost expenditures (Meyers, 1994). Consequently, development of least-cost
formulated feeds is of paramount importance for environmentally and economically
sustainable sea-urchin aquaculture. Formulated feeds are preferred over natural foods in
that they (1) are more nutritionally complete and more consistent in nutrient content; (2)
can be formulated to promote optimal growth and production in a commercial facility;
and (3) allow the addition of feed additives to enhance growth, health and roe production
and quality (pers comm. Watts, 2014). A commercial sea urchin feed should satisfy
4
requirements for essential macronutrients, lipids, protein, and carbohydrates, and
micronutrients, vitamins and minerals. Various studies have investigated the nutritional
requirements of sea urchins for both macro and micronutrients yet specific requirements
remain largely undetermined (Watts et al., 2010) for all life stages. Various studies have
investigated nutritional requirements in L. variegatus (reviewed by Watts et al., 2013b)
and have suggested nutrient ranges. While general ranges have been recommended,
knowledge of essential levels for most nutrients is lacking for L. variegatus. Each
nutrient must be provided at a level that is adequate to support growth, tissue repair,
reproduction, metabolic function and overall good health.
Study of the nutritional content of plants and animals consumed by wild sea
urchins can be helpful in the formulation of a prepared diet. In the wild, sea urchins are
opportunistic omnivores and their dietary intake varies among habitats and seasonally as
the relative availability of food items changes (De Ridder and Lawrence, 1982; Lawrence
et al., 2013). L. variegatus is reported to consume a wide variety of plants and animals
including, but not limited to, algae, sea grasses, crustaceans, gastropods, sponges and
polychaetes (De Ridder and Lawrence, 1982; Watts et al. 2013a).
Generally, the most expensive component of a formulated feed is protein. Among
most eukaryotes, protein is a critical macronutrient that allows for the maintenance of
proper physiological function. In sea urchins, protein has a crucial role in reproduction
and larval development (de Jong-Westman et al., 1995), growth, repair and maintenance
of body tissues, and many other biological functions (Watts et al 2013b). Urchins that
consume diets containing comparatively higher dietary protein levels are reported to
become quickly satiated (Frantzis and Gremare, 1992; Fernandez and Boudouresque,
5
1998; McBride et al., 1998; Meidel and Scheilbling, 1999; Fernandez and Boudouresque,
2000; B.W. Hammer et al., 2004; Daggett et al., 2005; H.S. Hammer et al., 2012), grow
faster (Fernandez, 1997; Cook et al. 1998; Fernandez and Boudouresque, 1998;
Fernandez and Pergent ,1998; Meidel and Scheibling, 1999; Akiyama, 2001; Hammer et
al., 2004; Taylor, 2006), and produce more roe (g/100g, de Jong-Westman et al., 1995;
Fernandez, 1997; Barker et al., 1998; Cook et al., 1998; Meidel and Scheibling, 1999;
Schlosser et al., 2005; Pearce et al., 2002a; B.W. Hammer et al., 2004; Chang et al.,
2005; Marsh et al. 2013; Woods et al., 2008; H.S. Hammer et al., 2012; Heflin et al.,
2012). However, under culture conditions, the inclusion of excess dietary protein can be
problematic due to the increased production of nitrogenous waste that contributes
substantially to poor water quality. Excessive protein (Pearce et al., 2002a; Woods et al.,
2008) or perhaps specific amino acids (Hoshikawa, 1998; Murata et al., 2001; Pearce et
al. 2002a, 2002b; Robinson et al., 2002; Senaratna et al., 2005; Woods et al. 2008) may
also contribute a bitter flavor characteristic to roe, which is highly undesirable in the food
industry. Therefore, it is important to meet but not exceed dietary protein requirements
when formulating a sea urchin diet.
Exact dietary protein requirements for sea urchins are predicted to vary among
species and within conspecifics due to feeding strategies (carnivory, ominvory,
herbivory), life stage, reproductive stage, and exposure to changes in abiotic conditions.
Previous studies suggest that 20% dietary protein may be adequate for growth and
production among several species (Akiyama et al., 2001; Pearce et al., 2002a, 2002b;
B.W. Hammer et al., 2004; H.S. Hammer et al., 2006; Heflin, 2010; Eddy et al., 2012).
Juvenile Pseudocentrotus depressus fed a purified diet exhibited highest test growth at
6
dietary protein levels between 20-50% and greatest feed efficiency at protein levels
between 20-40% (Akiyama et al., 2001), indicating that 20% protein may be adequate for
juveniles of this species. Similarly, both adult and juvenile Strongylocentrotus
droebachiensis are thought to require approximately 20% dietary protein (Pearce et al.,
2002a). Other species, including L. variegatus may sometimes (perhaps depending on life
stage) have higher requirements for protein: Heflin et al. (2012) reported a 0.5 g increase
in wet weight gain over nine weeks among adult L. variegatus for every 1% increase in
dietary protein up to 36%. Hammer et al. (2012) observed low feed conversion ratio
(FCR), increased dry matter production, and increased production efficiency at 31%
dietary protein, as compared to lower protein levels, in small adult L. variegatus.
However, a previous study suggests that 20% dietary protein may be adequate for growth
and production among large adults (Hammer et al., 2006). Among juvenile L. variegatus,
a minimum of 21% dietary protein is suggested for growth and survival (Hammer et al.,
2004).
In addition to dietary protein, sea urchins require a source of dietary energy.
Although lipids are required by sea urchins (Gibbs et al., 2009; Hammer et al., 2010;
Gibbs et al., 2015) and are high in energy, urchins do not appear to have the ability to
process large quantities of lipids readily. Few lipases are found within the urchin gut
(Lawrence, 1995; Lawrence et al., 2013) and the predicted low oxygen content of their
tissues may confound oxidation as well. Amino acids derived from dietary proteins can
also be catabolized for cellular energy. Sea urchins receiving adequate protein but low
(perhaps inadequate) dietary energy exhibit decreased growth and production, suggesting
that dietary protein is catabolized to meet energy needs when dietary lipid or
7
carbohydrate levels are insufficient (Schlosser et al., 2005; Hammer et al., 2006).
However, the process is inefficient (Marsh et al., 2013), economically expensive and
detrimental to the culture environment. Carbohydrates are more economical and appear to
be easily processed by sea urchins. Many carbohydrases have been categorized within the
urchin gut (Lawrence et al., 2013), and carbohydrates appear to be the primary energy
source for herbivores and omnivores, including sea urchins (Marsh et al., 2013).
Energy requirements of sea urchins are expected to be comparative low relative to
other organisms. They manifest a low rate of respiration (Lawrence and Lane, 1982), are
poikilotherms and are sessile. A suitable sea urchin feed would presumably have a high
protein: energy ratio. Among juvenile L. variegatus, individuals fed formulated diets
containing 104-112 mg protein kcal-1 consumed more feed and had higher growth rates
than urchins fed a formulated diet containing 82 mg protein kcal-1. Hammer et al. (2006)
saw similar results in adult L. variegatus fed isocaloric feeds with different protein:
energy ratios. Heflin et al. (2012) observed higher wet weight gain (despite equivalent
feed intake) for adult L. variegatus fed diets with high protein: energy ratios as compared
to those fed low protein: energy ratios (diets ranged from 39- 95 mg P kcal-1).
The results of these cited studies collectively indicate that the level of one nutrient
relative to another (e.g. protein, carbohydrate and lipid) must be considered when
preparing formulated diets. Many nutrients are known to interact extensively with other
nutrients, causing different responses by the organism under investigation. The results of
some studies also suggest that if essential nutrients are not balanced within a diet, animals
may compensate by over or under- consuming a specific nutrient (Raubenheimer and
Simpson, 1993, 2003; Simpson and Raubenheimer, 1993, 2012), possibly resulting in
8
sub-optimal outcomes. Improperly balanced nutrient ratios in feeds may affect both feed
consumption and feed utilization. To date, the previous studies examining the
relationship between dietary nutrients have not provided a clear understanding of the
interaction between dietary protein and carbohydrate. This information will be important
in the formulation of a diet that will provide sufficient protein for optimal growth and
production, while minimizing its use as an energy source.
For organisms raised in culture, determining optimal diet composition is only the
first of several steps in optimizing nutrient intake and availability. Researchers must also
identify and control factors that may inhibit efficient utilization of nutrients. In a culture
facility, feeding regimes are usually chosen based upon convenience (Tacon, 1995).
However the provision of feed (frequency, time of day and amount per feeding event)
will affect biomass production and cost of production for many organisms. Feed
provision that is inconsistent with the eating habits of the organism being cultured may
remain uneaten in the water for a period of time. Prolonged exposure to water results in
nutrient leaching and overall degradation. The nutrient loss and water fouling that occur
increase production costs. To maximize return on investment in feed, it will be important
for sea urchin culturists to be aware of intrinsic feeding strategies of wild sea urchin
populations and to feed in a manner that optimizes, as practically as possible, feed intake
among urchins held in culture.
Meal timing often has an influence on growth and feed efficiency in both aquatic
and terrestrial organisms. Many urchin species, including L. variegatus are reported to
follow diel (usually nocturnal) feeding rhythms (Ebling et al., 1966; Lewis, 1964; Ogden
et al. 1973; Lawrence and Hughes-Games, 1972; Dance, 1987, Lison de Loma et al.,
9
1999; Boudouresque and Verlaque, 2001; Vaἳtilingon et al., 2003; Watts et al., 2013).
These patterns appear to be in response to predator-prey interactions (Dance, 1987;
Barnes and Crook, 2001; Boudouresque and Verlaque, 2001) and possible effects of
feeding patterns on feed utilization and efficiency are not reported. However, diel feeding
rhythms are correlated with hormonal fluctuations among other aquatic species and are
reported to affect feed utilization and efficiency (Spieler, 1977; Delahunty et al., 1978;
Sundararaj et al., 1982; Noeske and Spieler, 1984; Perez et al., 1988; Azzaydi et al.,
2000; Bolliet et al., 2001). It seems reasonable to expect that sea urchins may also be
subject to circadian cues and therefore, may benefit from feeding at a specific time of
day.
Most aquatic organisms need to be fed once to several times each day. However,
if an intermittent feeding regime would be effective for farmed sea urchins, feeding every
other day or even less often would allow culturists to reduce labor costs and thus increase
the opportunity for economic viability. Sea urchins are somewhat unusual in that they
will feed intermittently and may periodically reduce consumption for one or several days.
Among wild sea urchin populations, differences in frequency of feeding are often
attributed to availability of food. Some species may have frequent access to food and may
graze continually (Watts et al., 2013). Others may only have periodic access to food due
to the habitat in which they live (Dix, 1970). There is conflicting information about the
ultimate effect of intermittent feeding on urchins held in culture (McCarron et al., 2009)
but reduced roe production is reported in several urchin species, including L. variegatus
(Minor and Scheibling, 1997; Lawrence et al., 2003; McCarron et al., 2009; Zhao et al.,
10
2013), indicating that a substantial intermittent feeding regime will most likely be
unsuitable for sea urchins held in culture.
Historical nutritional studies seek to establish nutrient requirements and feed
management strategies among sea urchins by offering individuals one of several feeds
which differ in the concentration of one or two nutrients and measuring outcomes defined
as survival, growth, production and feed intake among treatments. These studies lay the
ground work for future nutritional research and are indispensable in that they provide
valuable information about nutrient requirements and enable researchers to make
recommendations about feed formulations, dietary intake levels and dietary supplements
(Simpson and Raubenheimer, 2012). However, due to the complexity of nutrition and
nutrient utilization, considerable labor and time are required to gain detailed knowledge
about a species using traditional nutritional research methodologies. Consequently, there
are only a few species for which we have extensive knowledge of nutritional
requirements (Simpson and Raubenheimer, 2012) in conjunction with key factors that
motivate organisms to regulate dietary intake are not entirely understood.
Alternate experimental designs common to nutritional ecology may allow
aquaculture researchers to explain intake strategies and measure growth outcomes in
cultured organism by observation of self-regulated feed intake behavior and ecosystem
(culture system) interactions. Optimal foraging theory (OFT) proposes that animals will
self-regulate feed intake to optimize net energy acquisition, thus increasing fitness
(Emlen, 1966; Pianka, 1966). Although all organisms must fulfill energy requirements,
OFT does not consider requirements for specific macro and micronutrients and is
therefore, most likely too narrow to fully explain feed intake choices among organisms.
11
Also, within the confines of most culture systems (excluding pond culture), foraging
would not be a significantly energy consumptive process, thus experimental approaches
using this theoretical approach would not be informative.
An alternate theory, ecological stoichiometry (ES) is based on the hypothesis that
an organism will flourish when fed a diet with an elemental composition (as opposed to
macro and micronutrient composition) similar to that of its own body tissues
(Raubenheimer et al., 2009). ES often provides a more parsimonious explanation of feed
intake strategies than that of OFT because it considers the combined balance of energy
and nutrient intake and observes its effect on the organism and its interaction within the
ecosystem (Sterner and Elser, 2002). However, ES has shortcomings founded in an
inability to account for functional distinctions that may exist among structurally similar
molecules (Raubenheimer et al., 2009).
A third nutritional framework, the geometric framework (GF), is suggested as a
tool to supplement current research methodologies and expand existing knowledge of
nutritional requirements among aquatic species (Simpson and Raubenheimer, 2001;
Ruohonen et al., 2007). Geometric framework (GF) is an applied nutritional science that
seeks to understand how animals use nutritional choices to adapt to and succeed within
their environment (Raubenheimer and Simpson, 1993; Simpson and Raubenheimer,
2012). Within the GF model, the optimal intake of a particular nutrient is referred to as
the “intake target” and is demonstrated graphically as a discrete point or region in
nutrient space (Raubenheimer and Simpson, 1993; Simpson and Raubenheimer, 1993,
1995, 2012; Fig. 1). The nutrient composition of available foods will determine whether
an organism can reach a particular intake target (Chambers et al., 1995; Simpson and
12
Raubenheimer, 1995; Simpson and Raubenheimer, 2012). A single food will contain a
certain set ratio of nutrients (e.g. protein and carbohydrate). The nutrient content of that
food is represented graphically as a vector projecting from the origin through nutrient
space (Fig. 1) and is referred to as a “nutrient rail” (Raubenheimer and Simpson, 1993;
Simpson and Raubenheimer, 1993, 1995, 2012). The nutritional intake of an animal
feeding on a single food must lie along the trajectory of the nutrient rail created by that
food (Raubenheimer and Simpson, 1993; Simpson and Raubenheimer 1993, 1995, 2012,
Fig. 1). When an individual is restricted to only one food, it may choose to eat more or
less but cannot vary the ratio of dietary nutrients consumed. Conversely, when an
individual has access to two or more complementary foods (and thus, two or more
nutrient rails), it may vary eating patterns whereby the intake target for one or more
nutrients can be achieved as long as the nutrient targets lie between the rails created by
the available foods (Fig. 1, Raubenheimer and Simpson, 1993; Simpson and
Raubenheimer, 1993, 1995, 2012).
Organisms held in culture (excluding those held in outdoor ponds) generally only
have access to one feed and, thus, must consume a ratio of dietary nutrients that lie along
one nutrient rail. In consuming one feed to reach a target for a particular nutrient, an
individual may over or under consume another nutrient (Raubenheimer and Simpson,
1993, 2003; Simpson and Raubenheimer, 1993, 2012), resulting in undesired outcomes.
Alternately, an individual restricted to one food may fail to reach an important nutrient
target by avoiding over or under consumption of another nutrient (Raubenheimer and
Simpson, 1993; Simpson and Raubenheimer 1993, 2012; Hewson-Hughes et al., 2013).
The resulting nutrient deficiency too may have a detrimental effect on survival, growth
13
and reproduction. It will be of paramount importance to identify intake targets (if they
exist) for protein, carbohydrate and lipid among urchins. Failure to provide a feed that
allows organisms to reach individual intake targets without exceeding or falling short of
others can potentially have an adverse effect on organismal health, carcass yield and
reproduction, resulting in loss of profit for commercial culture (Simpson and
Raubenheimer 2001; Ruohonen et al., 2007).
This dissertation represents a series of investigations designed to increase
knowledge of nutrient requirements and feed management techniques and will provide
one of the first evaluations of economic feasibility of sea urchin aquaculture. Part of the
approach includes the first attempt to utilize the GF to assess nutrient intake targets for
aquatic organisms held in culture and accordingly, recommend a condition of nutrient
balancing in formulated feed.
14
9
8
7
IT1
Protein (g)
6
5
Food A
4
3
IT2
IT3
2
Food B
1
0
0
0
2
4
Lipid (g)
6
8
Figure 1. Hypothetical protein- lipid nutrient space with 2 foods. Lines represent nutrient
rails created by 2 hypothetical foods, A and B. Squares (IT1, IT2 and IT3) represent
hypothetical possible nutrient intake targets, all of which can be attained by a
hypothetical animal eating combinations of food A and B (modified from Simpson and
Raubenheimer, 2012).
Chapter Two will examine requirements for specific proximate nutrients (protein
and carbohydrate) in the sea urchin L. variegatus. Growth and production efficiencies
will be evaluated across a range of protein levels at low and mid-level carbohydrate. Feed
ingredient costs and outcomes for growth and efficiencies will be used to evaluate
economic viability of feeds for sea urchin aquaculture.
Chapter Three will focus on feed management techniques among L. variegatus.
Identifying required ranges for dietary nutrients is critical in developing aquaculture
15
techniques for sea urchins. However, feed management techniques will also be important
factors in maximizing return on investment (ROI). Feed intake and utilization is often
influenced by circadian feeding patterns, frequency of feeding and ration per feeding
event. Therefore, even with proper nutrition, unsuitable feed management techniques
could reduce financial return from an aquaculture enterprise. Urchins will be fed different
rations at different frequencies and times of day. Growth and production outcomes will
be measured across treatments and recommendations for feed management will be
presented based on defined responses.
Chapter Four will focus on identification of intake targets, if they exist, among
adult sea urchins. Knowledge of dietary intake targets may provide valuable in the
development of aquaculture techniques for sea urchins by assisting culturists in
improving (ROI) in feed. Limited ingredient gel based diets containing different nutrient
ratio (protein and carbohydrate) and concentration will be prepared using fish meal and
wheat starch. Using the GF, diets will be offered to adult urchins in pairwise
combinations and daily intake of protein and carbohydrate will be assessed to determine
whether intake targets for protein and/or carbohydrate exist.
Chapter Five will continue investigation of dietary intake targets among wildcaught juvenile sea urchins. Additional complexity (as compared to Chapter Four) will be
added with the addition of other feed ingredients. In formulated diets, specific nutrients
may be more difficult to identify and intake targets may vary depending on dietary
inclusion of other nutrients (vitamins and minerals). The GF will be used as a tool to
identify intake targets for protein and carbohydrate using formulated feeds. Juvenile sea
urchins will be offered gel based formulated feeds. Diets will differ in both nutrient
16
content (protein, carbohydrate and lipid) and nutrient concentration (4, 8 or 12%). Some
individuals will be offered pairwise diet combinations, whereas others will be offered a
single diet. Diet intake will be measured to test the hypothesis that individuals will adjust
feeding patterns to reach optimal intake targets for corresponding dietary nutrients.
17
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Amsterdam. pp. 155-169.
Woods, C.M., James, P.J., Moss, G.A., Wright, J., Siikavuopio S., 2008. A comparison of
the effect of urchin size and diet on gonad yield and quality in the sea urchin
Evechinus chloroticus Valenciennes. Aquaculture International, 16, 49-68.
Zhao, C., Zhang, W., Chang, Y., Zhou, H., Song, J., Luo, S., 2013. Effects of continuous
and diel intermittent feeding regimes on food consumption, growth and gonad
production of the sea urchin Strongylocentrotus intermedius of different size
classes. Aquaculture International, 21, 699-708.
31
CHAPTER 2
EFFECT OF LEVELS OF DIETARY PROTEIN AND CARBOHYDRATE ON THE
CULTURE OF JUVENILE SEA URCHIN Lytechinus variegatus AND ECONOMIC
CONSIDERATIONS FOR DIETARY FORMULATIONS
by:
LAURA E. HEFLIN, ROBERT MAKOWSKY, J. CHRISTOPHER TAYLOR,
MICHAEL B. WILLIAMS, ADDISON L. LAWRENCE AND STEPHEN A. WATTS
In preparation for Aquaculture
Format adapted for dissertation
32
ABSTRACT
Juvenile Lytechinus variegatus (ca. 3.95+/- 0.54 g) were fed 10 formulated diets
with different protein (ranging from 11- 43%) and carbohydrate (12 or 18%) levels.
Urchins (n= 16 per treatment) were fed a daily sub-satiation ration equivalent to 2.0% of
average body weight for 10 weeks. Data were used to create predictive models of growth,
production and efficiency outcomes for juvenile L. variegatus and to generate economic
analysis models (diet costs per unit weight gain) for sea urchins held in culture. At dietary
protein levels below ca. 30%, models for most growth and production outcomes predicted
increased rates of growth and production among urchins fed diets containing 18% dietary
carbohydrate levels as compared to urchins fed diets containing12% dietary
carbohydrate. For most outcomes, growth and production was predicted to increase with
increasing level of dietary protein up to ca. 30%, after which, no further increase in
growth and production were predicted. Likewise, dry matter production efficiency was
predicted to increase with increasing protein level up to ca. 30%, with urchins fed diets
with 18% carbohydrate exhibiting greater efficiency than those fed diets with 12%
carbohydrate. The energetic cost of dry matter production was optimal at protein levels
less than those required for maximal weight gain and gonad production, suggesting an
increased energetic cost (decreased energy efficiency) is perhaps required to increase
gonad production relative to somatic growth, exclusively. Economic analysis models
predict when cost of feed ingredients are low, the lowest cost per gram of wet weight
gain will occur at 18% dietary carbohydrate and ca. 25- 30% dietary protein. In contrast,
33
lowest cost per gram of wet weight gain will occur at 12% dietary carbohydrate and ca.
35- 40% dietary protein when feed ingredient costs are high or average. For both 18 and
12% levels of dietary carbohydrate, cost per gram of wet weight gain is predicted to be
maximized at low dietary protein levels, regardless of feed ingredient costs.
34
INTRODUCTION
The increasing demand for sea urchin roe and concomitant decrease in wild sea
urchin stocks have led to interest in the development of large-scale commercial sea
urchin aquaculture. In a commercial aquaculture facility, feed-related costs commonly
represent one of the principal expenditures (New 1987, Myers, 1994; Patil et al., 2007).
Successful culture of sea urchins will therefore require the development of a least-cost,
nutritionally complete formulated diet. Various studies have investigated nutritional
requirements among sea urchins (reviewed by Watts et al., 2013). However, exact
requirements for both macro and micronutrients essentially remain undetermined and
feed costs associated with production are therefore lacking.
Protein is commonly the most expensive ingredient in formulated aqua feeds and
is a critical macronutrient required by sea urchins to maintain proper physiological
function (Marsh et. al, 2013). Results of previous studies have shown a correlation
between increased protein consumption and increased somatic growth (Fernandez, 1997;
Cook et al. 1998; Fernandez and Bourdouresque, 1998; Fernandez and Pergent ,1998;
Meidel and Scheibling, 1999; Agatsuma 2000; Akiyama et al., 2001; Hammer et al.,
2004; Hammer et al., 2006; Taylor, 2006; Hammer et al. 2012; Heflin et al., 2012a) and
roe production (de Jong-Westman et al., 1995; Fernandez, 1997; Barker et al., 1998;
Cook et al., 1998; Meidel and Scheibling, 1999; Schlosser et al., 2005; Pearce et al.,
2002a; Hammer et al., 2004; Chang et al., 2005; Hammer et al., 2006; Marsh and Watts,
2007; Woods et al., 2008; Hammer et al. 2012; Heflin et al., 2012a). However, elevated
35
levels of dietary protein also lead to reduced food consumption among sea urchins
(Frantzis and Gremare, 1992; Fernandez and Bourdouresque, 1998; McBride et al., 1998;
Meidel and Scheibling, 1999; Agatsuma, 2000; Fernandez and Bourdouresque, 2000;
Hammer et al., 2004; Daggett et al., 2005; Hammer et al., 2006; Hammer et al., 2012),
suggesting that fulfillment of dietary protein requirements (considered separately from
energetic requirements) may promote satiety. It is hypothesized that many organisms
have ‘intake targets’ for one or more nutrients and may become satiated once those
consumptive targets are achieved (Simpson and Raubenheimer, 2012).
Optimal dietary protein levels for species of sea urchins are not yet identified.
Previous data suggest that protein requirements may fall within a 20-30% range of dry
dietary intake. Pseudocentrotus depressus exhibit highest test growth at dietary protein
levels between 20-50%, but feed efficiency decreases at protein levels above 40%
(Akiyama et al., 2001). Both adult (Pearce 2002a) and juvenile (Pearce 2004; Eddy et al.,
2012) Strongylocentrotus droebachiensis appear to have a requirement for ca. 20%
dietary protein. Juvenile (Hammer et al., 2004) and adult (Hammer et al., 2006; Heflin,
2010) Lytechinus variegatus appear to require a minimum of 20- 21% dietary protein for
optimal growth. Dry matter production among adult L. variegatus increases with
increasing level of dietary protein, but feed efficiency is correspondingly reduced
(Hammer et al., 2012; Heflin et al., 2012a).In contrast, wet weight gain among juvenile S.
droebachiensis decreases with increasing level of dietary protein (Eddy et al., 2012),
indicating that protein levels above ca. 20% may inhibit growth in this cold water species.
In addition to a protein source, sea urchins require a source of energy. Dietary
protein consists of amino acids which can be catabolized through transamination to
36
provide cellular energy. However, the use of protein as an energy source is inefficient
(Marsh et al., 2013), will increase feed costs, and adversely affect the environment
through the production of nitrogenous waste products. Carbohydrates exert less
environmental impact and are more economical ingredients to include in diet
formulations as a source of energy. Many carbohydrases have been identified within the
urchin gut (Lawrence et al., 2013), and many animals including sea urchins appear to
utilize dietary carbohydrates as the primary energy source (Marsh et al. 2013). When
considered exclusively, dietary carbohydrate level is a poor predictor of sea urchin
growth (Heflin et al. 2012a). However, inclusion of adequate levels of carbohydrate in a
sea urchin diet has a sparing effect on the use of protein as an energy source (Hammer et
al., 2012; Heflin et al., 2012a), to positively affect growth, production, and nutrient
utilization. Consequently, protein: carbohydrate ratio must also be considered when
preparing formulated diets.
Many nutrients interact extensively with other nutrients, possibly resulting in poor
outcomes (Simpson and Raubenheimer, 2012). Nutrient ratios that occur in formulated
diets affect nutrient utilization (feed efficiency) in sea urchins (Hammer et al., 2012;
Heflin et al., 2012a). Protein efficiency ratio (PER) decreased among adult L. variegatus
fed a diet high in both dietary carbohydrate (30%) and dietary protein (28%) compared to
that of urchins fed diets containing comparatively high levels of dietary protein but low
levels of carbohydrate (Heflin et al., 2012a). Hammer et al. (2012) reported decreased
feed conversion ratio (FCR) as the level of dietary protein increased but increased FCR
when the level of carbohydrate increased.
37
Among all organisms, feed utilization can reasonably be predicted to be optimal
when nutrient and energy requirements are efficiently met. Any deviation from those
requirements should result in a reduction in assimilation (production) efficiency. An
excess of a nutrient generally requires additional energy to process and either store or
excrete, whereas a deficit of a specific nutrient will likely limit metabolic processes. To
date, previous studies examining the relationship between dietary protein and
carbohydrate have not provided a clear understanding of the interaction of these two
macronutrients. Understanding this relationship will be important in the formulation of a
diet that will provide dietary carbohydrate at levels that are high enough to spare protein
as an energy source, yet low enough to maximize production efficiencies.
Although researchers have investigated various aspects of sea urchin nutrition and
husbandry, the economic viability of raising sea urchins in commercial culture is
uncertain. Reliable economic estimates of major variable costs are required to establish
goals, initiate sound business plans, and allow investments to be profitable (De Silva and
Anderson, 1995). In both terrestrial and aquatic farms, profitability is determined by
expenditures and return (Shang, 1986). Therefore, economic analysis of feed costs will be
important in maximizing return on investment (ROI) in establishing commercial sea
urchin farming as a viable industry.
This study seeks to increase existing knowledge of protein and carbohydrate
utilization among sea urchins by examining the effects of dietary protein at two
carbohydrate levels on somatic growth, organ production and production efficiencies in
juveniles of the sea urchin L. variegatus. These data will be used to create predictive
models of growth and production outcomes for juvenile L. variegatus held under similar
38
conditions and fed diets varying in protein and carbohydrate at levels approaching those
found to be optimal (Hammer et al., 2012; Heflin et al., 2012a). A secondary goal of this
study is to generate economic analysis models (diet costs per unit weight gain) for sea
urchins held in culture.
MATERIALS AND METHODS
Collection and initial measurements
Juvenile (ca. 3.95+/- 0.54 g) Lytechinus variegatus were collected from Saint
Joseph Bay, FL (30N, 85.5W) in June, 2013 and transported to the University of
Alabama at Birmingham in aerated coolers. The following week sixteen urchins were
randomly selected for initial evaluation. Individual urchins were weighed to the nearest
mg and dissected by a circular incision around the peristomial membrane. The gonads
were removed and the gut (esophagus, stomach, and intestine combined) was removed
and cleaned in seawater to eliminate food pellets. The test and Aristotle’s lantern were
rinsed in deionized water to remove salt. The organs were blotted on a clean paper towel
and weighed individually to the nearest mg. Organs were dried at 50°C for 72 hours to
constant weight, and dry weights were recorded. Mean dry organ and total dry weights
(the sum of the organ dry weights) were calculated for the initial sub-sample and used as
estimated initial dry organ and total dry weights for the remaining 160 urchins held in the
trial. Remaining individuals were weighed to the nearest mg and assigned randomly to 1
of 10 dietary treatments (n=16 per diet).
39
Culture Conditions
Urchins were placed individually into plastic, cylindrical cages (ca. 8.5 cm
diameter, 25 cm high, with 3 mm open mesh on sides, a 3 mm open mesh bottom secured
by plastic cable-ties, and a 2 mm open mesh circle over-laid on bottom). The mesh cages
were fitted into 8.7 cm ID PVC couplings. Thirty-two cages were randomly placed in
each of five fiberglass raceways (235 cm x 53 cm x 31 cm, L x W x H, Fig. 1, as
described by Taylor (2006). Three empty cages were added to each raceway to ensure
even water flow throughout, resulting in a total of 35 cages in each raceway. A 160 x 23
cm (L x H) center baffle in the center of each raceway allowed for recirculating water
flow by an in-line utility pump (Supreme® Mag Drive Utility Pump, Danner™
Manufacturing, Inc., Islandia, NY, USA, 700 gallons of water/hour, Fig. 1; Taylor 2006).
The utility pump removed saltwater from the raceway on one side of the baffle. Water
then passed through a mechanical and biological filter and returned to the raceway on the
opposite side of the baffle. The flow rate of the resulting current was approximately 9.7 –
12.6 cm s-1. Water passed independently through a 10 watt UV sterilizer (Lifegard®
Aquatics, Cerritos, CA, USA). Water depth was maintained at 15.0 cm. The floor of each
cage was ca. 5.5 cm from the bottom of the raceway. Each cage was fitted on the bottom
with three small Tygon® spacers (ca. 0.5 cm thick) to allow water circulation underneath.
Due to the mesh nature of the cage, water circulation through the cage wall was ca. 93712.6 cm s-1. Cages were coded so that each individual could be tracked over the course of
the study. In these cages, feed pellets were retained, but feces fell through the mesh.
Cages were rotated within and between raceways weekly to eliminate any possible tank
or position effects.
40
Total ammonia nitrogen, nitrite, nitrate, pH and alkalinity levels were checked
weekly using saltwater test kits from Aqua Pharmaceuticals, LLC (Malvern, PA, USA)
for ammonia and nitrogen and La Motte Company (Chestertown, MD, USA) for
alkalinity. Photo-period and water temperature were held constant (12:12 light: dark,
22±2 °C).
41
Figure 1: Schematic of recirculating system. A) Side view of one of the five fiberglass
raceway (235 cm x 53 cm x 31 cm, L x W x H) with a 160 x 23 cm (L x H) center baffle
and 35 individual flow-through cages. B) Top view of one fiberglass raceway (schematic
is not drawn to scale. Arrows indicate water flow).
42
Diets and Diet Preparation
Ten semi-purified diets were formulated and produced using both chemically
defined and practical ingredients (Table 1). Levels of dietary protein and carbohydrate
(Table 2) ranged from 11 to 43 % protein (using a purified plant protein source; Table 1)
and 12 and 18% carbohydrate (using a purified plant starch source; Table 1). Dietary
levels of protein and carbohydrate were chosen to accordingly to the levels reported
previously to promote best growth (Hammer et al., 2012; Heflin et al., 2012a). To
compensate for the different combined levels of protein and carbohydrate within the
experimental diets acid washed diatomaceous earth (DE) was added as appropriate. DE
has been observed to exert no effect on sea urchins at the levels used in this study
(unpublished data). All other nutrients remained constant among treatments. Dry
ingredients were mixed with a PK twin shell® blender (Patterson-Kelley Co., East
Stroudsburg, PA) for 10 minutes and were then transferred to a Hobart stand mixer
(Model A-200, Hobart Corporation, Troy, OH) and blended for 40 minutes. Liquid
ingredients (500 mL kg-1 dry mix) were added, and the mixture was blended for 10
minutes to a mash-like consistency. The diets were extruded using a meat chopper
attachment (Model A-200, Hobart Corporation, Troy, OH) fitted with a 4.8 mm die. Diet
strands were separated and dried on wire trays in a forced air oven (35°C) for 48 hours.
Final moisture content of all diets were 8–10%. Diets were stored individually in air-tight
storage bags at 4°C until used.
43
Table 1. Calculated nutrient levels on an “as fed” basis for the base experimental diet.
*Empirically derived levels by Eurofins Scientific, Inc.
Base
Nutrients
Experimental Diet
Crude Protein*
27.75%
Carbohydrate
31.34%
Crude Fiber*
2.5%
Total Ash*
23.53%
Crude Fat*
3.15-12.5%
Cholesterol
0.32%
Carotenoid
0.97%
Calcium*
4.02%
Phosphorus*
1.99%
Sodium
1.29%
Potassium
1.34%
Magnesium
0.41%
Iron
327 ppm
Zinc
92.7 ppm
Manganese
83.0 ppm
Copper
57.8 ppm
Selenium
0.413 ppm
Arginine
2.08%
Histidine
0.74%
Isoleucine
1.33%
Leucine
2.36%
Lysine
1.91%
Methionine
0.57%
Cystine
0.29%
Phenylalanine
1.47%
Tyrosine
1.20%
Valine
1.38%
Vitamin A
4800 IU
Vitamin D
3000 IU
Vitamin E
241 ppm
Vitamin C
8-921 ppm
Thiamine
36 ppm
Riboflavin
48 ppm
Pyridoxine
96.3 ppm
Niacin
99.3 ppm
Pantothenic Acid
36.5 ppm
Biotin
0.971 ppm
Inositol
128 ppm
Choline
487 ppm
Folic Acid
24.0 ppm
Vitamin B12
0.181 ppm
**All diets contain approximately 4% animal ingredients, 28% marine ingredients, 29.1% plant
ingredients, 0.5% crude fat, 1.7% carotenoids, 0.7% vitamin premix, 21.76% mineral premix, and
4.2% binder + antioxidant.
44
Table 2. Calculated protein and carbohydrate levels (as were fed), total energy, protein:
energy, and protein: carbohydrate caloric ratios in each of the ten diets tested.
Protein
(%)
Carbohydrate
(%)
Total Energy
(cal/g)
Protein: Energy
(mg P/kcal)
43
35
27
19
11
43
35
27
19
11
12
12
12
12
12
18
18
18
18
18
3117
2660
2202
1744
1287
3477
3020
2562
2104
1647
138
131
121
107
82
124
115
104
89
64
Protein:
Carbohydrate
Caloric Ratio
4.76
3.85
2.95
2.06
1.17
2.38
1.92
1.49
1.03
0.59
Feeding Rate
Each sea urchin was fed a limiting daily ration equal to 2.0% of the initial average
wet body weight for a period of ten weeks. Feed was weighed as fed (8- 10% moisture
content). Approximately 2.0% of wet body weight per day is a sub-satiation ration for
juvenile Lytechinus variegatus of this size class fed similar diets with comparable dietary
protein and carbohydrate levels and comparable caloric content, whereas a ration
equivalent to or above 3% of body weight is ad libitum (unpublished data). Feeding at
sub-satiation ensures that urchins consume all of their food in a 24 hour period and
facilitates direct measure of diet intake. A sub-satiation feeding regime also prevents
individuals from compensating for a dietary deficiency by increasing consumption. Each
urchin was individually weighed every two weeks and diet rations were correspondingly
adjusted to be equivalent to 2.0% of the average body weight within the two-week time
45
period. Intake of the presented diet was confirmed by direct observation. Each day, feces
were removed by siphon just prior to feeding.
Daily feeding rate was calculated as:
(1)Average wet weight of individuals (g) x 0.020
Protein: energy ratio of each diet was calculated as:
(2) Protein (mg) / energy content (kcal)
Total energy content of each diet (per g) was calculated based on the methods of Phillips
(1972):
(3) % protein / 100 x 5650 (cal g-1) + % carbohydrate / 100 x 4000 (cal g-1) + % lipid /
100 x 9450 (cal g-1)
Final Dissection
After ten weeks, urchins were photographed and dissected following the
procedure described for the pre-experiment evaluation.
Weight Gain and Production
Every other week, urchins were photographed for image analysis and weighed
individually to the nearest mg to assess growth among treatments. Wet weight gain over
the 10-week period was calculated as:
(4) Final wet weight (g) – initial wet weight (g)
Estimated total dry matter production was calculated as:
(5) Final dry weight (g) - average initial dry weight (g)
46
Estimated protein efficiency ratio (PER) for each individual was calculated as:
(6) Dry matter produced (g) / dry weight protein consumed (g)
Production efficiency (PE), representing the dry matter produced relative to the dry
matter consumed, was calculated for each individual as:
(7) [Final dry weight (g) – initial dry weight (g)/dry diet intake (g)] x 100
Production energy efficiency (PEE), representing the amount of dry tissue produced
relative to the amount of energy consumed, was calculated for each individual as:
(8) Final dry weight (mg) – initial dry weight (mg)/total energy intake (cal)
Estimated organ (gut and gonad) dry matter production for each individual was calculated
as:
(9) Final dry weight of organ (g) – initial average dry weight of organ (g)
Final dry organ gut and gonad index for each individual was calculated as:
(10) Final dry weight of organ (g) / final dry weight of individual (g) x 100
Feed Conversion Ratio (FCR) for each individual was calculated as:
(11) Total diet consumed (g, as fed) / wet weight gain (g)
Economic analysis
Economic analysis was performed using a range of current bulk market prices (as
of December, 2014) for practical feed ingredients from various manufacturers in the US
and abroad. Costs were categorized as high, average and low and like costs among
ingredients were grouped together (e.g. high with high). Ranges were compared with
47
growth outcomes to generate economic analysis plots as predictive models of cost per
gram of wet weight gained for urchins held under the respective treatments at high,
average and low diet ingredient prices.
Statistics
To model the effect of carbohydrate and protein level on various urchin growth
measurements, efficiency and economic outcomes, general linear models were conducted
in R 3.1.2 (www.r-project.org) using the "glm" function (Table 3). Inclusion of predictor
variables was based on 1) a p value less than or equal to 0.05 or 2) being a component of
a higher order interaction. All data were checked for normality and homoscedasticity
using the “glm.diag.plots” function in R 3.1.2. Models with non-normal or
heteroscedastic residuals were log transformed as appropriate. Table 3 shows parameter
estimates, tests of significance and goodness of fit outcomes for various measures of
growth models.
48
Table 3. Parameter estimates, tests of significance and goodness of fit for various
measures of Lytechinus variegatus growth models. For each model, L indicates the
variable was log transformed and - indicates that variable was set to 0 (not estimated).
Associated p-values for parameter estimates being significantly different than 0 are
included as *p < 0.05, **p < 0.01, and ***p < 0.001
Outcome
Wet Weight
Gain
Total Dry
Weight
Dry Weight
Gain
Dry Test
Weight
Dry Test
Gain
Dry Lantern
Weight
Dry Lantern
Gain
Dry GonadL
Dry Gonad
Index
Dry Gut
Weight
Dry Gut
GainL
Dry Gut
Index
Protein
Efficiency
Production
Efficiency
FCR
Protein
21.5***L
Carb.
2.48***
Protein * Carb
-0.72***L
Baseline
2.85***
Adjusted R2
0.77
4.55***L
0.51***
-0.14***L
0.61***
0.78
4.55***L
0.51***
-0.14***L
0.61***
0.78
3.75***L
0.43***
-0.12***L
0.45**
0.74
0.29***
0.169***
-0.004**
-
0.72
0.016***
0.013***
-0.00035***
0.046***
0.50
0.016***
0.013***
-0.00035***
0.046***
0.50
0.27***
1.01***
0.15***
0.31***
-0.0035***
-
-
0.81
0.59
0.009***
0.006***
-0.0002***
0.018**
0.56
0.14***
0.10***
-0.003***
-
0.60
0.023
0.048
-0.002*
0.216*
0.06
0.071***
0.133***
-0.0036***
0.168**
0.58
0.31***L
0.035***
-0.0095***L
0.039**
0.78
-0.17***
-0.12***
0.0034***
-0.126*
0.79
49
RESULTS
Water quality
Water quality parameters were as follows: 32±0.5 ppt salinity, 22±2°C, D.O. 7±2
mg L-1, ammonia 0 mg L-1, nitrite 0 mg L-1, nitrate 0 mg L-1, alkalinity ≥200 mg L-1, and
pH 8.2. All were within the ranges suitable for sea urchins (Basuyaux and Mathieu 1999).
Survival was 100% in all dietary treatments.
Total Wet Weight Gain
Regardless of the different carbohydrate: protein level combinations, urchins in
all dietary treatments increased in weight during the 10-week study, with weight gain
ranging across treatments from 172 to 593% of initial wet weight (Fig. 2). The model for
total wet gain at 12 and 18% dietary carbohydrate predicted an increase in wet weight
gain with increasing protein level (p < 0.001, Fig. 3). At dietary protein levels below ca.
23%, the model predicted wet weight gain among urchins fed diets containing 18%
dietary carbohydrate to be significantly higher than that of individuals fed diets
containing 12% dietary carbohydrate (p < 0.05, Fig.3). Although not significant, a
reduction in weight gain was suggested when both dietary protein and carbohydrate
levels were high (Fig. 3).
Gonad
The models for gonad dry matter production at 12 and 18% dietary carbohydrate
predicted an increase in gonad dry matter production with increasing level of dietary
protein up to ca. 35 and 30%, respectively (p < 0.001, Fig. 4). At dietary protein levels
below ca. 34%, gonad dry matter production was predicted to be significantly greater
among urchins fed diets containing 18% dietary carbohydrate (p < 0.05, Fig. 4). At
50
dietary protein levels above 35%, a non-significant reduction in gonad dry matter
production was suggested, particularly among urchins in the 18% dietary carbohydrate
treatments (Fig.4).
At dietary protein levels below ca. 38%, the model predicted significantly higher
gonad index among urchins fed diets containing 18% dietary carbohydrate as compared
to urchins fed diets containing 12% dietary carbohydrate (p < 0.001, Fig. 5). Among
urchins in both the 12 and 18% carbohydrate treatments, the model for gonad index
predicted an increase in gonad index with increasing protein level up to ca. 32 and 34%,
respectively (p <0.001, Fig. 5). Additional increases in dietary protein did not yield
further increases in gonad index, regardless of dietary carbohydrate level (Fig. 5).
51
900
800
% Wet Weight Gain
700
600
500
400
300
200
Carbohydrate
12
%
100
0
10
20
30
40
50
Protein (%)
Figure 2: Percent wet weight gain. Dashed line shows percent wet weight gain among
urchins fed diets with 18% dietary carbohydrate. Solid line shows percent wet weight
gain among urchins fed diets with 12% dietary carbohydrate.
52
Figure 3: Total wet weight gain. Dashed line is predictive of wet weight gain among
urchins fed diets with 18% dietary carbohydrate. Solid line predicts wet weight gain
among urchins fed diets with 12% dietary carbohydrate. Hash-marked areas indicate 95%
confidence intervals.
53
Figure 4: Gonad dry matter production. Dashed line is predictive of gonad dry matter
production among urchins fed diets with 18% dietary carbohydrate. Solid line predicts
gonad dry matter production among urchins fed diets with 12% dietary carbohydrate.
Hash-marked areas indicate 95% confidence intervals.
54
Figure 5: Dry gonad index. Dashed line is predictive of gonad index among urchins fed
diets with 18% dietary carbohydrate. Solid line predicts gonad index among urchins fed
diets with 12% dietary carbohydrate. Hash-marked areas indicate 95% confidence
intervals.
Gut
The model for gut dry matter production at 18% dietary carbohydrate predicted an
increase in gut dry matter production with increasing protein level up to ca. 32% (p <
0.001, Fig. 6). However, at dietary protein levels above ca. 32%, gut dry matter
55
production did not increase (Fig 6). Likewise, the model for gut dry matter production at
12% dietary carbohydrate predicted an increase in gut dry matter production with
increasing protein level up to ca. 36% (p < 0.001), after which gut dry matter production
did not increase (Fig. 6). At dietary protein levels below ca. 26%, the model predicted
significantly higher gut dry matter production among urchins fed diets containing 18%
dietary carbohydrate as compared to that of urchins fed diets with 12% dietary
carbohydrate (p < 0.05, Fig. 6).
The model predicted gut index would be maximized at ca. 17% dietary protein
among urchins fed diets containing 18% carbohydrate, but among individuals fed diets
containing 12% carbohydrate, gut index was predicted to increase as protein level
increased to ca. 27% (Fig.7).
Aristotle’s Lantern and Test
The model for lantern dry matter production for diets containing 12 and 18%
dietary carbohydrate predicted an increase in lantern dry matter production with
increasing level of dietary protein up to ca. 36 and 32%, respectively (p < 0.001, Fig. 8).
Further increases in dietary protein level did not result in additional increases in lantern
dry matter production (Fig. 8).
The model for test dry matter production at levels of 12 and 18% dietary
carbohydrate predicted an increase in test dry matter production as level of dietary
protein increased to ca.40 and 37%, respectively (p < 0.001, Fig. 9). Further increases in
dietary protein did not yield further increases in dry test weight gain (Fig. 9). At dietary
protein levels below ca. 25%, test dry matter production was predicted to be significantly
56
higher among urchins fed diets containing 18% dietary carbohydrate as compared to that
of individuals fed diets containing 12% dietary carbohydrate (p < 0.05, Fig. 9).
Figure 6: Gut dry matter production. Dashed line is predictive of gut dry matter
production among urchins fed diets with 18% dietary carbohydrate. Solid line predicts
gut dry matter production among urchins fed diets with 12% dietary carbohydrate. Hashmarked areas indicate 95% confidence intervals.
57
Figure 7: Dry gut index. Dashed line is predictive of dry gut index among urchins fed
diets with 18% dietary carbohydrate. Solid line predicts dry gut index among urchins fed
diets with 12% dietary carbohydrate. Hash-marked areas indicate 95% confidence
intervals.
58
Figure 8: Lantern dry matter production. Dashed line is predictive of lantern dry matter
production among urchins fed diets with 18% dietary carbohydrate. Solid line predicts
lantern dry matter production among urchins fed diets with 12% dietary carbohydrate.
Hash-marked areas indicate 95% confidence intervals.
59
Figure 9: Test dry matter production. Dashed line is predictive of test dry matter
production among urchins fed diets with 18% dietary carbohydrate. Solid line predicts
test dry matter production among urchins fed diets with 12% dietary carbohydrate. Hashmarked areas indicate 95% confidence intervals.
60
Food Conversion Ratio and Efficiencies
At 18% dietary carbohydrate, the model for FCR predicted a decrease in FCR as
protein level increased to ca. 32% dietary protein, after which, FCR was predicted to rise
(p < 0.001, Fig. 10). At 12% dietary carbohydrate, FCR decreased with increasing protein
level up to ca. 38% (Fig. 10). Below ca. 27% dietary protein, FCR was predicted to be
significantly lower among urchins fed diets containing 18% dietary carbohydrate as
compared to that of individuals fed diets containing 12% dietary carbohydrate (p < 0.05,
Fig. 10). Although not significant, at dietary protein levels above ca. 33%, FCR among
urchins fed diets containing 18% dietary carbohydrate trended higher than that of urchins
fed diets containing 12% dietary carbohydrate (Fig. 10).
The model for PE at 18% dietary carbohydrate predicted an increase in efficiency
with increasing protein level up to 34% dietary protein, after which, PE no longer
increased (p < 0.001, Fig 11). For urchins fed diets containing 12% carbohydrate, the
model for PE projected an increase in efficiency with increasing protein level up to ca.
40% (Fig. 11). At dietary protein levels above 40%, PE no longer increased (Fig. 11). At
dietary protein levels below ca. 34%, PE among urchins fed diets containing 18% dietary
carbohydrate was projected to be significantly higher than that of individuals fed diets
containing 12% dietary carbohydrate (p < 0.05, Fig. 11).
Below ca. 32% protein, the model for PER predicted significantly higher
efficiency among urchins fed diets with 18% dietary carbohydrate as compared to those
fed diets with 12% dietary carbohydrate (p < 0.05, Fig. 12). However at dietary protein
levels higher than ca. 32%, protein efficiency did not vary with dietary carbohydrate level
(Fig. 12). At the18% level of dietary carbohydrate, protein efficiency decreased with
61
increasing dietary protein level (Fig. 12). A rapid rate of decrease in PER was observed
when urchins were fed diets with high protein and high carbohydrate (Fig. 12).
Regardless of dietary carbohydrate level, the model for PEE predicted increased
efficiency with increasing dietary protein level up to ca. 18- 20% dietary protein (Fig 13),
with PEE decreasing as dietary protein increased from that level. At dietary protein levels
above 30%, PEE was significantly lower among urchins fed diets containing 18% dietary
carbohydrate as compared to those fed diets containing12% dietary carbohydrate (p <
0.05, Fig. 13).
62
Figure 10: Food conversion ratio (FCR); Dashed line is predictive of FCR among urchins
fed diets with 18% dietary carbohydrate. Solid line predicts FCR among urchins fed diets
with 12% dietary carbohydrate. Hash-marked areas indicate 95% confidence intervals.
63
Figure 11: Production efficiency (PE); Dashed line is predictive of PE among urchins fed
diets with 18% dietary carbohydrate. Solid line predicts PE among urchins fed diets with
12% dietary carbohydrate. Hash-marked areas indicate 95% confidence intervals.
64
Figure 12: Protein efficiency ratio (PER); Dashed line is predictive of PER among
urchins fed diets with 18% dietary carbohydrate. Solid line predicts PER among urchins
fed diets with 12% dietary carbohydrate. Hash-marked areas indicate 95% confidence
intervals.
65
Figure 13: Production energy efficiency (PEE); Dashed line is predictive of PEE among
urchins fed diets with 18% dietary carbohydrate. Solid line predicts PEE among urchins
fed diets with 12% dietary carbohydrate. Hash-marked areas indicate 95% confidence
intervals.
66
Economic Analysis
The models for economic analysis predicted that lowest cost per gram of wet
weight gain will occur at 12% dietary carbohydrate and ca. 35- 40% dietary protein when
feed ingredient costs are high or average (Fig. 13). When feed ingredient costs are low,
the lowest cost per gram of wet weight gain was predicted to occur at 18% dietary
carbohydrate and ca. 25- 30% dietary protein (Fig. 13). For both 18 and 12% dietary
carbohydrate, cost per gram of wet weight gain was predicted to be highest at low dietary
protein levels, regardless of feed ingredient costs (Fig. 13). At dietary protein levels
below ca. 25%, the economic analysis models predicted significantly higher cost per
gram of wet weight gain at 12% dietary carbohydrate as compared to 18% dietary
carbohydrate for all feed ingredient ranges (p < 0.05, Fig. 13).
67
Figure 14: Economic analysis of sea urchin diets. Dashed line is predictive of cost per
gram wet weight gain among urchins fed diets with 18% dietary carbohydrate. Solid line
predicts cost per gram wet weight gain among urchins fed diets with 12% dietary
carbohydrate. Hash-marked areas indicate 95% confidence intervals.
DISCUSSION
Urchins in all dietary treatments grew throughout the 10-week study and survival
was 100% in all dietary treatments, indicating that all diets were adequate for
maintenance and growth. Feeding at sub-satiation ensured that all urchins consumed
equal amounts of the respective diets and were not able to increase consumption to
compensate for potential nutritional deficiencies in the diets.
Among the experimental diets in this study, total dietary protein and carbohydrate
levels were adjusted by varying the level of DE; levels of DE used in this study exert no
effect on growth in sea urchins (unpublished data). Therefore, differences in growth and
68
efficiencies among dietary treatments can be attributed to variations in the level (amount)
of protein, carbohydrate or energy consumed and/or interactions between dietary protein
and carbohydrate utilization. Using empirical data from the current study, we can provide
a predictive tool (model) for growth, production and feed costs/ efficiencies among
juvenile L. variegatus under the conditions of this study. These results can be used to
evaluate economic return on investment (ROI) in feed for young L. variegatus held in
culture.
Somatic Growth
Optimization of sea urchin growth through the regulation of dietary nutrient levels
is challenging. Dietary protein level (inclusive of essential amino acids) is a particularly
critical consideration in formulating an aquafeed for growing urchins. Protein must be
provided in adequate quantities to support maintenance (turnover) requirements and
optimize new tissue growth. However, excess dietary protein should be avoided because
protein is one of the most expensive components of formulated aquatic diets. In addition,
nitrogenous waste produced from the catabolism of protein to produce energy can reduce
the quality of the water for culture. Furthermore, excessive protein (Pearce et al., 2002a;
Woods et al., 2008) or perhaps specific amino acids (Hoshikawa et al., 1998; Murata et
al., 2001, 2002; Pearce et al. 2002a, 2002b; Robinson et al., 2002; Senaratna et al., 2005;
Woods et al. 2008) may contribute a bitter flavor to roe, which is highly undesirable in
the food industry. Ideally, dietary protein should be included at levels which optimally
balance growth and efficiency, while sparing as much as possible the use of protein as an
energy source.
69
Sea urchins survived and increased in weight at dietary protein levels of 11 and
19%, but these levels were insufficient to facilitate maximum weight gain, regardless of
carbohydrate content. However, PER in the 11% protein diet was maximized at 18%
carbohydrate, but was significantly reduced when fed diets containing a low level (12%)
of carbohydrate. These data indicate that sufficient energy (from carbohydrate) should be
present to utilize protein effectively and prevent catabolism of protein for use as an
energy source (protein sparing). Poor FCR and PE observed in urchins fed 11% dietary
protein and 12% carbohydrate further corroborate that, although protein was used
efficiently, both protein and energy levels were too low to support high rates of growth.
In addition, the production: energy efficiency ratio (PEE, dry matter produced per unit of
energy consumed) suggests energy was not utilized efficiently when protein and
carbohydrate were minimal because these represent levels below which urchins would
most likely not survive or be productive long term. However, an increase in protein and
carbohydrate energy (19% protein; 18% carbohydrate) significantly increased all growth
parameters and efficiencies. Likewise, Hammer et al. (2004) reports a reduction in
growth and survival and an increase in FCR among juvenile L. variegatus fed low levels
(9 or 15%) of dietary protein.
Growth among juvenile sea urchins may be maximized at dietary protein levels
around 20% or greater (Akiyama et al., 2001; Hammer et al., 2004; Kennedy et al.,
2005). Juvenile Pseudocentrotus depressus fed purified diets exhibited highest growth at
dietary protein levels between 20-50% and highest feed efficiency at protein levels
between 20-40% (Akiyama et al., 2001), indicating that 20% protein may be adequate for
juveniles of this species. Similarly, both adult and juvenile Strongylocentrotus
70
droebachiensis are reported to require around 20% dietary protein (Pearce et al., 2002a).
Among juvenile L. variegatus, a minimum of 21% dietary protein is recommended for
growth and survival (Hammer et al., 2004). Hammer et al. (2012) observed low feed
conversion ratio (FCR), increased dry matter production, and increased production
efficiency at 31% dietary protein in small adult L. variegatus.
Models from our study predict that, for juvenile L. variegatus raised in culture,
diets containing protein levels between 25 and 30% will most likely be appropriate to
provide satisfactory tissue growth and promote good feed efficiency outcomes. Under the
conditions of this study, and for urchins of this size class, this represents a daily protein
intake of ca. 2.4 to 2.8 g protein per kilogram of urchins. When dietary protein and
carbohydrate were both low, the low values in PER, PE and PEE as well as the high
values of FCR suggested that 12% dietary carbohydrate was insufficient to supply energy
needs and dietary protein was catabolized to meet energy demands. When carbohydrate
was increased to 18%, elevated weight gain suggests protein was spared as an energy
source and was most likely used in tissue production, suggesting an important relation
between intake of dietary protein relative to carbohydrate. In fact, the value of PEE
increased substantially when carbohydrate increased from 12 to 18% at low protein,
suggesting tissue production required substantially less energy (increased efficiency)
when additional carbohydrate was available. In contrast, when protein levels were higher
(> 25%), the higher carbohydrate diets did not promote the efficient use of protein.
Above these levels we hypothesize energy is in excess and reduced efficiency could
reflect the increased cost of energy storage or, as protein levels increase further, the
increased cost of protein catabolism. Previous data show a reduced effect of increasing
71
protein: carbohydrate ratio on protein utilization among adult L. variegatus (Heflin et al.,
2012a). Consequently, both too little or too much dietary protein lead to increased
maintenance cost and reduced efficiency (Erlenbach et al., 2014).
Gonad analysis
Numerous studies show a direct correlation between dietary protein level and
gonad production in sea urchins (Barker et al., 1998; Chang et al., 2005; Cook et al.,
1998; de Jong-Westman et al., 1995; Fernandez, 1997; Hammer et al., 2004; Hammer et
al., 2006; Marsh et al., 2013; Meidel and Scheibling, 1999; Olave et al., 2001; Pearce et
al., 2002a; Schlosser et al., 2005; Woods et al., 2008; Hammer et al., 2012; Heflin et al.,
2012a). Among urchins in this study, both gonad production and gonad index were
directly correlated with dietary protein level. However, data from the current and other
(de Jong-Westman et al., 1995; Fernandez, 1997; McBride et al., 1998; Pearce et al.,
2002a) studies indicate that there is a level of dietary protein, most likely respective for
each species, above which no further increases in gonad production and/or index will
occur.
Although protein was directly correlated with increased gonad production (at low
to moderate levels of dietary inclusion), carbohydrate remained an important contributor
to increased gonad mass. Among urchins in this study, increased gonad production of
urchins fed diets containing 18% dietary carbohydrate can be attributed to an increase in
body size of urchins. Additionally, the gonad index was also significantly higher among
individuals fed diets containing the higher carbohydrate level, suggesting that additional
nutrients, perhaps carbohydrates, may have been stored in the gonads relative to other
body components (unpublished data). We hypothesize that these higher levels of
72
carbohydrate promoted a more efficient use of ingested protein to support this increased
growth.
These data support the premise that optimization of dietary carbohydrate levels
will be important for urchins held in culture. Levels must be sufficiently high to fulfill
energy requirements and a slight excess may contribute to increased roe production and
quality. However, production data from this and other studies suggest that excess
carbohydrate (ca. 18% or higher) should be avoided, particularly at high levels of dietary
protein. Predictive models generated from these data indicate that dietary carbohydrate
levels of around 18% are appropriate for juvenile L. variegatus held in culture, provided
that dietary protein levels do not exceed ca. 30%.
Among sea urchins, dietary carbohydrate is primarily stored in gonad tissue
(Marsh et al., 2013). Taste studies reveal that an increase in the sweet taste of the roe of
Evechinus chloroticus is correlated with an increase in carbohydrate storage (Chen 2005;
Woods et al., 2008), suggesting that slight excesses of dietary carbohydrate may also
improve roe quality. Additionally, carbohydrate storage may affect the quantity of roe
produced. Schlosser et al. (2005) reported reduced gonad dry matter production and
gonad index in P. lividus are attributed to limitations in digestible dietary energy
(presumably carbohydrate).
Gut Analysis
Among sea urchins, gut size is variable and correlated with food availability
(Bishop and Watts, 1992; Hammer et al., 2006). When food is plentiful, gut tissue
increases in size, perhaps to increase digestive capacity (Bishop and Watts, 1992). The
stomach is also used for nutrient storage in sea urchins and the number and size of
73
storage cells within the gut tissue increases as food availability increases, possibly
contributing to an increase in gut mass (Bishop and Watts 1992). In this study, feed
rations were equivalent among treatments but varied in nutrient content. Gut dry matter
production varied with protein and carbohydrate level, but differences among treatments
correspond to other somatic tissue growth patterns and thus, cannot be specifically
attributed to variations in nutrient storage or in digestive capacity. Heflin et al. (2012a)
reported differences in gut index due to an interaction effect between protein and
carbohydrate; however, the possible physiological significance of these manifestations is
unknown.
Test and Aristotle’s lantern
Among sea urchins, increased resource allocation to the test in response to an
increase in dietary protein level has been reported (Heflin et al., 2012b). The Aristotle’s
lantern is generally less variable and often grows to the same size regardless of diet
intake (Heflin et al. 2012b). At protein levels below 30%, test and lantern growth
increased with intake of dietary protein, leading to the trends observed for the total body
mass change. At these low to moderate protein levels (ca. < 25%), increased carbohydrate
intake further increased weight gain. However, increases in test growth at protein levels
above 30% were not affected by carbohydrate intake. In fact, the efficiency of protein use
(PER) as well as PEE falls dramatically with high dietary protein intake, further
suggesting that excess protein, while promoting a slight increase in growth, is used
inefficiently and is energetically expensive to incorporate into tissues.
74
Economic Analysis
To our knowledge, we have presented the first economic analysis of feed
utilization for application in commercial sea urchin production. Because of the
proportionally high cost of feed, return on investment relative to this significant variable
cost will have a significant impact on overall production costs and the difference between
expenses and revenue. Among diets offered in this study, cost of production increased
with increasing dietary protein level. Therefore, diets with 11- 19% protein were most
economical to manufacture. However, reduced growth among urchins receiving these
diets indicates that individuals would have to consume more to reach the same body
weight as that of individuals in treatments with higher dietary protein. Economic analysis
reveals that utilization of low protein diets in that manner would be inefficient and would,
over time, not provide the best ROI. Depending on ingredient prices, cost to produce 1
gram of urchin body weight will be minimized (and therefore provide maximum
profitability) within a range of 20 – 35% dietary protein. Not surprisingly, this is also the
range wherein urchins in this study exhibited the highest PE and PEE, and lowest FCR.
We suggest that these predictive economic analysis models can be used to estimate cost
of rearing cultured urchins to market size (i.e. depending on ingredient costs within the
parameters listed, the expense of providing feed to raise one urchin to 120 g would be
estimated between $1.80 and $13.20 USD.) and assess the economic viability of raising
sea urchins in commercial culture.
As nutritional requirements of adult L. variegatus may vary from those of
juveniles, dietary requirements and efficiencies among marketable adults must be
evaluated at their respective size and/or age classes. Data reported by Eddy et al. (2012)
75
indicate that economic returns among other species of interest may vary from that of L.
variegatus. Therefore, predictive modeling and economic analysis will be necessary for
urchin species with life history and/or feeding strategies that differ from those of L.
variegatus.
76
ACKNOWLEDGEMENTS
The authors thank the staff at the Texas AgriLIFE Research Mariculture
Laboratory for support. We would like to thank Jeff Barry, Mickie Powell, Lacey Dennis,
Adele Fowler, Karen Jensen, Kate Kohlenberg and Susan Sewell for providing technical
assistance. Approval for this study was granted by the Institutional Animal Care and Use
Committee of the University of Alabama at Birmingham. Animal resources were
supported by the NORC Aquatic Animal Research Core within the Animal Models Core
(NIH P30DK056336). R. Makowsky was supported by NIH training grant T32HL072757
UAB Statistical Genetics Post-doctoral Training Program. The statements, findings,
conclusions, and recommendations expressed herein are those of the authors' and do not
necessarily reflect the views of NORC or NIH.
77
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Taylor, A., 2006. Effects of dietary carbohydrates on weight gain and gonad production
in small sea urchins, Lytechinus variegatus. Masters Thesis. University of
Alabama at Birmingham, Birmingham, Alabama, USA.
Trier, T. M., & Mattson, W. J., 2003. Diet-induced thermogenesis in insects: a
developing concept in nutritional ecology. Environmental Entomology, 32(1), 18.
Watts, S., Lawrence, A., & Lawrence, J., 2013. Nutrition. In J. Lawrence (Ed.), Sea
Urchins: Biology and Ecology (3rd ed., pp. 155- 166). Amsterdam, The
Netherlands: Elsevier BV.
Woods, C. M., James, P. J., Moss, G. A., Wright, J., & Siikavuopio, S., 2008. A
comparison of the effect of urchin size and diet on gonad yield and quality in the
sea urchin Evechinus chloroticus Valenciennes. Aquaculture International, 16(1),
49-68.
85
CHAPTER 3
FEEDING TIME, FREQUENCY AND RATION AFFECTS GROWTH AND ENERGY
ALLOCATION IN YOUNG ADULTS OF THE SEA URCHIN Lytechinus variegatus
by
LAURA E. HEFLIN, STEPHEN A. WATTS
Aquaculture Nutrition: DOI: 10.1111/anu.12323
Copyright
2015
by
Wiley Blackwell Publishing
Used by permission
Format adapted for dissertation
86
ABSTRACT
Frequency of feeding, diurnal feeding patterns, and feed ration are important
considerations in the optimization of feeding strategies to maximize growth for sea
urchins held in culture. In 30 day and 62 day growth trials, wild caught Lytechinus
variegatus (ca. 18g) were fed as follows: 1) ad libitum ration (3% of average body
weight) once per day in the morning (0930 hr, AM treatment); 2) ad libitum ration once
per day in the evening (1730 hr, PM treatment); 3) one-half of ad libitum (1.5% of
average body weight) twice per day (morning and evening, AM/PM treatment), 4) ration
equivalent to 3% of average body weight proffered as a single ration every other evening
(EODSR/PM); or 5) ration equivalent to 6% (double ration) of average body weight
proffered every other evening (EODDR/PM). At 30 and 62 days wet weight gain among
urchins fed daily was significantly higher than that of urchins fed every other day,
regardless of ration size. Dry matter production among urchins in the EODDR/PM
treatment was significantly less than that of individuals in the PM and AM/PM treatments
but not significantly different from the AM or EODSR/PM treatments. At 62 days,
reduced gonad dry matter production and gonad index were observed among individuals
fed every other day, regardless of ration size. At 30 days, production efficiency was
significantly higher among individuals fed daily compared to those fed every other day.
87
However, at 62 days, PE among individuals in the EODDR/PM did not vary significantly
from that of individuals in the AM or PM treatments. At 30 and 62 days, feed conversion
ratio was optimized among individuals fed daily and individuals in the EODSR/PM
treatment. These data suggest that, for sea urchin culture, daily feeding will be necessary
for the optimization of growth, gonad production, and feed efficiency regardless of ration
size or time of feeding.
88
INTRODUCTION
The success and economic practicality of commercial sea urchin culture will be
dependent on multiple factors. Among these, some of the most important considerations
will include feed and feed management strategies. Feed often represents one of the
primary expenses in a commercial aquaculture enterprise, with costs accounting for as
much as 40-60% of total variable expenditures (Meyers, 1994). Feed is generally
provided in a manner (frequency, time of day and ration) that is convenient or practical
for culturists with little consideration of the natural feeding habits of the organism being
cultured (Tacon, 1995). Feed provided indiscriminately may remain uneaten for a period
of time and may be subject to nutrient loss due to loss of integrity combined with
leaching, often resulting in water fouling and poor efficiency. To maximize return on
investment in feed, it will be important to understand intrinsic feeding patterns that occur
in sea urchin populations so that feed intake under culture conditions can be optimized.
In the wild, sea urchin feeding strategies vary among species (Zhao et al., 2013)
and even among conspecifics (McCarron et al., 2009). Variations in feeding strategies are
attributed to differences in predator-prey interactions (Dance, 1987; Barnes and Crook,
2001; Boudouresque and Verlaque, 2001) or availability of food (Dix, 1970; McCarron et
al., 2009). Some species such as Paracentrotus lividus and Evechinus chloroticus often
inhabit areas where food is frequently unavailable causing them to feed intermittently
(Dix, 1970). Such species may continue to feed intermittently even when food is not
89
limited (McCarron et al., 2009). However, in a laboratory study, McCarron et al. (2009)
reported reduced gonad production in P. lividus fed intermittently as compared to those
fed daily, indicating that daily feeding may be necessary to optimize production. Reduced
gonad index is also reported in Strongylocentrotus droebachiensis (Minor and
Scheibling, 1997; James and Siikavuopio 2012), Strongylocentrotus intermedius (Zhao et
al., 2013) and Lytechinus variegatus (Lawrence et al., 2003) fed intermittently as
compared to more frequent feeding. Among reptiles, intermittent feeding is reported to
affect gut size and morphology with fasting conditions linked to a reduced gut size (Secor
and Diamond, 1995; Starck and Beese, 2001; Lignot et al., 2005). Upon resumption of
feeding, gut size may increase to expand digestive capacity (Bishop and Watts, 1992;
Secor and Diamond, 1995; Starck and Beese, 2001; Lignot et al., 2005).
Circadian feeding patterns (time of feeding relative to photoperiod) should also be
considered. Circadian feeding patterns are associated with hormonal fluctuations and
affect feed utilization and efficiency among mammals (Nelson et al., 1975; Philippens et
al., 1977; Arble et al., 2009) and fish (Spieler, 1977; Delahunty et al., 1978; Sundararaj et
al., 1982; Noeske and Spieler, 1984; Perez et al., 1988; Azzaydi et al., 2000; Bolliet et al.,
2001). Investigations have found circadian feeding patterns (usually nocturnal) among
sea urchins (Ebling et al., 1966; Lewis, 1964; Lawrence and Hughes-Games, 1972;
Ogden et al. 1973; Dance 1987; Mills et al., 2000; Barnes and Crook, 2001;
Boudouresque and Verlaque 2001; Vaïtilingon et al., 2003). However, these patterns are
generally attributed to predator-prey interactions (Nelson and Vance, 1979; Dance, 1987;
Barnes and Crook, 2001; Boudouresque and Verlaque, 2001) rather than hormonal
fluctuations and any effects of diel feeding rhythms on feed utilization and efficiency are
90
generally not reported. Zhao, et al. (2013) examined the effect of diel feeding rhythms on
production of S. intermedius and found no correlations but such effects may exist under
different circumstances or among other species.
Aquatic organisms held under commercial culture conditions should be fed an
appropriate ration and feeding events should be scheduled so that feed is rapidly and
completely consumed (Tacon, 1995) and both feed assimilation and production efficiency
are optimized (Boujard and Leatherland, 1992). The objective of this study is to evaluate
the effects of different feeding rates and feeding at different time periods on growth and
production efficiency among young adults of the sea urchin L. variegatus fed a
formulated diet.
MATERIALS AND METHODS
Collection and Initial Measurements
Juvenile (ca. 18.9+/- 1.5 g) Lytechinus variegatus were collected from Saint
Joseph Bay, FL (30N, 85.5W) in June and transported in aerated coolers to the
University of Alabama at Birmingham. The following day fourteen individuals were
randomly selected for initial evaluation. Individuals were weighed to the nearest mg and
dissected by a circular incision around the peristomial membrane. The gut (esophagus,
stomach, and intestine combined), and gonads were removed and cleaned in seawater to
remove food pellets. The organs were blotted on a clean paper towel and weighed to the
nearest mg. Organs were dried at 50°C for 72 hours to constant weight, and dry weights
were recorded. Mean dry organ and total dry weights (the sum of the organ dry weights)
91
were calculated for the initial sub-sample and used as an estimate of initial dry organ and
total dry weights of the experimental urchins. Seventy urchins selected for the trial were
weighed to the nearest mg and assigned randomly to 1 of 5 treatments (n=14 per
treatment). Treatments were as follows: 1) ration equivalent to 3% of average body
weight proffered once per day (24 hr. intervals) in the morning (AM treatment); 2) ration
equivalent to 3% of average body weight proffered once per day (24 hr. intervals) in the
evening (PM treatment); 3) ration equivalent to 1.5% (half ration) of average body
weight proffered twice per day (morning and evening, every 12 hrs., AM/PM treatment),
4) ration equivalent to 3% of average body weight proffered as a single ration on
alternate days (every other day) in the evening (EODSR/PM); or 5) ration equivalent to
6% (double ration) of average body weight proffered on alternate days (every other day)
in the evening (EODDR/PM). These treatments are summarized in Table 1.
Urchins were placed individually into plastic, cylindrical cages (ca. 8.5 cm
diameter, 25 cm high, with 3 mm open mesh on sides, a 3 mm open mesh bottom secured
by plastic cable-ties, and a 2 mm open mesh circle over-laid on bottom). The mesh cages
were fitted into 8.7 cm ID PVC couplings. Thirty-five cages were randomly placed in
each of two fiberglass raceways (235 cm x 53 cm x 31 cm, L x W x H, Fig. 1, as
described by Taylor (2006). A 160 x 23 cm (L x H) center baffle in the center of the
raceway allowed for recirculating water flow by an in-line utility pump (Supreme® Mag
Drive Utility Pump, 700 gallons of water/hour, Fig. 1; Taylor, 2006). The utility pump
removed saltwater from the raceway on one side of the baffle. Water then passed through
a mechanical and biological filter and returned to the raceway on the opposite side of the
baffle. The flow rate of the resulting current was approximately 9.7 – 12.6 cm s-1. Water
92
passed independently through a 10 watt UV sterilizer (Lifegard® Aquatics, Cerritos, CA).
Water depth was maintained at 15.0 cm. The floor of each cage was ca. 5.5 cm from the
bottom of the raceway. Each cage was fitted on the bottom with three small Tygon®
spacers (ca. 0.5 cm thick) to allow water circulation underneath. Cages were coded so
that each individual could be tracked over the course of the study. In these cages, feed
pellets were retained, but feces fell through the mesh. Cages were rotated within and
between raceways weekly to prevent tank or position effects.
All individuals were fed a formulated diet shown previously to promote good
growth and survival (Heflin et al., 2013, Table 2). Feed rations were adjusted every other
week to maintain rations at their respective percentage of body weight (1.5, 3.0 or 6.0%,
Table 1).
Total ammonia nitrogen, nitrite, nitrate, pH and alkalinity levels were checked
weekly using saltwater test kits from Aqua Pharmaceuticals, LLC (Malvern, PA, USA)
for ammonia and nitrogen and La Motte Company (Chestertown, MD, USA) for
alkalinity. Photo-period and water temperature were held constant (12:12 light: dark,
23±1 °C).
Table 1. Feeding treatments. AM indicates individuals fed once per day in the morning (0930 hr).
PM indicates individuals fed once per day in the evening (1730 hr). AM/PM indicates individuals
fed in the morning and evening. EODSR/PM indicates individuals fed every other day in the
evening at a single ration. EODDR/PM indicates individuals fed every other day in the evening at
a double ration.
Dietary
Treatment
Feed Ration per Feeding
Event (as a % of average body
weight, adjusted biweekly)
Time(s)
of
Feeding
Frequency of Feeding
AM
PM
AM/PM
EODSR/PM
EODDR/PM
3.0%
3.0%
1.5%
3.0%
6.0%
AM
PM
AM/PM
PM
PM
once per day
once per day
twice per day
once every other day
once every other day
93
Figure 1: Schematic of recirculating system. A) Side view of one of the two fiberglass
raceway (235 cm x 53 cm x 31 cm, L x W x H) with a 160 x 23 cm (L x H) center baffle
and 35 individual flow-through cages. B) Top view of one fiberglass raceway (schematic
is not drawn to scale. Arrows indicate water flow).
94
Table 2. Calculated nutrient levels on an “as fed” basis for the base experimental diet.
*Empirically derived levels by Eurofins Scientific, Inc.
Base
Nutrients
Experimental Diet
Crude Protein*
27.75%
Carbohydrate
31.34%
Crude Fiber*
2.5%
Total Ash*
23.53%
Crude Fat*
3.15-12.5%
Cholesterol
0.32%
Carotenoid
0.97%
Calcium*
4.02%
Phosphorus*
1.99%
Sodium
1.29%
Potassium
1.34%
Magnesium
0.41%
Iron
327 ppm
Zinc
92.7 ppm
Manganese
83.0 ppm
Copper
57.8 ppm
Selenium
0.413 ppm
Arginine
2.08%
Histidine
0.74%
Isoleucine
1.33%
Leucine
2.36%
Lysine
1.91%
Methionine
0.57%
Cystine
0.29%
Phenylalanine
1.47%
Tyrosine
1.20%
Valine
1.38%
Vitamin A
4800 IU
Vitamin D
3000 IU
Vitamin E
241 ppm
Vitamin C
8-921 ppm
Thiamine
36 ppm
Riboflavin
48 ppm
Pyridoxine
96.3 ppm
Niacin
99.3 ppm
Pantothenic Acid
36.5 ppm
Biotin
0.971 ppm
Inositol
128 ppm
Choline
487 ppm
Folic Acid
24.0 ppm
Vitamin B12
0.181 ppm
**All diets contain approximately 4% animal ingredients, 28% marine ingredients, 29.1% plant
ingredients, 0.5% crude fat, 1.7% carotenoids, 0.7% vitamin premix, 21.76% mineral premix, and
4.2% binder + antioxidant.
95
Growth and Production
Every other week, urchins were photographed for image analysis and weighed to
the nearest mg to assess growth among treatments.
Wet weight gain over the 12 week study period was calculated for each individual as:
(1) Final wet weight (g) – initial wet weight (g)
After 30 days, ½ of urchins in each treatment were haphazardly selected and
dissected following the procedure described for the pre-experiment evaluation. The
remaining urchins were maintained in their respective feeding regimes for an additional
32 days, after which time they were dissected as described previously. Estimated total dry
matter production for each individual was calculated as:
(2) Final dry weight (g) - average initial dry weight (g)
Estimated dry organ (test, lantern, gonad, and gut) production for each individual was
calculated as:
(3) Final dry weight of organ (g) – average initial dry weight of organ (g)
Final dry gonad and gut index for each individual was calculated as:
(4) Final dry weight of organ (g) / final dry weight of individual (g) x 100
Dry matter production efficiency (PE) for each individual was calculated as:
(5) [Final dry weight (g) – initial dry weight (g)]/dry feed proffered (g, as fed) x 100
Feed Conversion Ratio (FCR) for each individual was calculated as:
(6) Dry feed proffered (g, as fed)/ wet weight gain (g)
96
Statistical analyses of growth parameters were performed in IBM SPSS Statistics
22. P values ≤ 0.05 were considered significant. Data were tested for normality and
homoscedasticity using Shapiro-Wilk and Levene’s tests, respectively, and found to be
normal and parametric. Data were then analyzed with ANCOVA using the GLM
procedure with frequency of feeding as the fixed factor and time of feeding as the
covariate. If a significant difference was detected, a Tukey’s HSD test was used to
compare means. Outcomes examined were total dry matter production, organ dry matter
production, lantern index, gonad index, PE and FCR.
RESULTS
Water Quality
Water conditions were maintained as follows: 32±0.5 ppt salinity, 23±1°C, D.O.
7±2 ppm, ammonia 0 ppm, nitrite 0 ppm, nitrate 0 ppm, alkalinity ≥200 ppm, and pH 8.2.
Water quality parameters maintained in this study were within the ranges suitable for sea
urchins (Basuyaux and Mathieu, 1999).
Total Wet Weight Gain and Dry Matter Production
There were no significant differences in mean initial wet weight (19 ± 2.0 g, n =
14 treatment-1) among treatment groups at the start of the feeding trial. Mean wet weight
gain of all urchins increased for the 62 day trial (Fig. 2), regardless of treatment. Total
wet weight gain at 30 days was highest among urchins in the AM/PM or the PM
treatment (Fig. 2, p≤0.039). At 30 days, but not at 62, mean wet weight gain of urchins in
97
the AM treatment was significantly lower than that of those in the AM/PM (Fig. 2, p=
0.039). At 30 and 62 days, wet weight gain of urchins fed every other day did not vary
between rations but was less than half that of those fed daily (Fig. 2, p≤ 0.001, p≤ 0.007).
Mean total dry matter production (dry weight gain) increased in all feeding
regimes during the 62 day trial. Mean total dry matter production did not differ at 30 or
62 days among individuals in the AM, PM and AM/PM treatments (Fig. 3, p≤ 0.027). At
30 and 62 days, total dry matter production for those urchins in the EODDR/PM
treatment did not differ significantly from that of those in the AM treatment (Fig. 3). The
mean total dry matter production of urchins in both the EOSDR/PM and EODDR/PM
treatments did not differ significantly at 30 or 62 days (Figure 3).
Gonad Analysis
At 30 days, both mean gonad dry matter production and mean gonad indices were
highest among urchins fed daily (Fig. 4, Fig 5, p≤ 0.05, p≤ 0.001). However, gonad dry
matter production and gonad indices among urchins in the AM treatment did not differ
from that of those in the EODDR/PM and EDOSR/PM treatments (Fig. 4, Fig. 5). At 62
days, both gonad dry matter production and gonad indices of all individuals fed daily
were significantly higher than that of urchins fed every other day, regardless of ration
size (Fig.4, Fig. 5, p≤ 0.026, p≤ 0.034). For urchins held 30 or 62 days, there were no
significant differences between gonad dry matter production or gonad indices of urchins
fed every other day, regardless of ration (Fig. 4, Fig. 5).
98
Total Wet Weight Gain (g)
30
a
25
30 day
a
a
62 day
20
15
b
10
B
AB
5
b
A
C
C
0
AM
PM
AM/PM
EODSR/PM
Feeding Schedule
EODDR/PM
Figure 2: Total wet weight gain (g, ±SEM) by treatment. AM indicates individuals fed ad
libitum once daily in the morning. PM indicates individuals fed ad libitum once daily in
the evening. AM/PM indicates individuals fed twice daily at ½ ad libitum. EODSR/PM
indicates individuals fed every other day in the evening at the daily ad libitum ration.
EODDR/PM indicates individuals fed every other day in the evening at twice the daily ad
libitum ration. Pale gray bars indicate total wet weight gain at 30 days. Dark gray bars
indicate total wet weight gain at 62 days. Differential capital letters designate significant
differences at 30 days. Differential lower case letters designate significant differences at
60 days.
99
Total Dry Matter Production (g)
6
a
5
a
ab
30 Day
62 Day
4
bc
3
A
2
AB
c
A
1
C
BC
0
AM
PM
AM/PM
EODSR/PM
Feeding Schedule
EODDR/PM
Figure 3: Total dry matter production (g, ±SEM) by treatment. AM indicates individuals
fed ad libitum once daily in the morning. PM indicates individuals fed ad libitum once
daily in the evening. AM/PM indicates individuals fed twice daily at ½ ad libitum.
EODSR/PM indicates individuals fed every other day in the evening at the daily ad
libitum ration. EODDR/PM indicates individuals fed every other day in the morning at
twice the daily ad libitum ration. Pale gray bars indicate total dry matter production at 30
days. Dark gray bars indicate total dry matter production at 62 days. Differential capital
letters designate significant differences at 30 days. Differential lower case letters
designate significant differences at 60 days.
100
Gonad Dry Matter Production (g)
2.0
a
a
a
1.5
30 Day
62 Day
1.0
A
0.5
b
A
b
AB
B
0.0
AM
PM
AM/PM
EODSR/PM
Feeding Schedule
B
EODDR/PM
Figure 4: Gonad dry matter production (g, ±SEM) by treatment. AM indicates individuals
fed ad libitum once daily in the morning. PM indicates individuals fed ad libitum once
daily in the evening. AM/PM indicates individuals fed twice daily at ½ ad libitum.
EODSR/PM indicates individuals fed every other day in the evening at the daily ad
libitum ration. EODDR/PM indicates individuals fed every other day in the evening at
twice the daily ad libitum ration. Pale gray bars indicate gonad dry matter production at
30 days. Dark gray bars indicate gonad dry matter production at 62 days. Differential
capital letters designate significant differences at 30 days. Differential lower case letters
designate significant differences at 60 days.
101
20
a
a
18
a
30 Day
Gonad Index (%)
16
14
62 Day
A
12
b
A
10
8
b
AB
6
4
BC
C
2
0
AM
PM
AM/PM
EODSR/PM
Feeding Schedule
EODDR/PM
Figure 5: Gonad index (%, ±SEM) by treatment. AM indicates individuals fed ad libitum
once daily in the morning. PM indicates individuals fed ad libitum once daily in the
evening. AM/PM indicates individuals fed twice daily at ½ ad libitum. EODSR/PM
indicates individuals fed every other day in the evening at the daily ad libitum ration.
EODDR/PM indicates individuals fed every other day in the evening at twice the daily ad
libitum ration. Pale gray bars indicate gonad index at 30 days. Dark gray bars indicate
gonad index at 62 days. Differential capital letters designate significant differences at 30
days. Differential lower case letters designate significant differences at 60 days.
Gut Analysis
After 62 days, mean gut dry matter production of urchins in the EODSR/PM
treatment was significantly lower than that of urchins in every other treatment except
those in the AM treatment (Fig. 6, p≤ 0.039). However, after 62 days the mean gut index
of individuals in the EODDR/PM treatment was significantly higher than that of urchins
in each of the other treatments (Fig. 7, p≤ 0.025).
102
62 Day Gut Dry Matter Production (g)
0.25
0.20
a
ab
a
a
0.15
b
0.10
0.05
0.00
AM
PM
AM/PM
EODSR/PM
EODDR/PM
Feeding Schedule
Figure 6: 62 day gut dry matter production (g, ±SEM) by treatment. AM indicates
individuals fed ad libitum once daily in the morning. PM indicates individuals fed ad
libitum once daily in the evening. AM/PM indicates individuals fed twice daily at ½ ad
libitum. EODSR/PM indicates individuals fed every other day in the evening at the daily
ad libitum ration. EODDR/PM indicates individuals fed every other day in the evening at
twice the daily ad libitum ration. Differential lower case letters designate significant
differences.
103
3.5
b
62 Day Gut Index (%)
3.0
2.5
a
a
a
a
2.0
1.5
1.0
0.5
0.0
AM
PM
AM/PM
EODSR/PM
Feeding Schedule
EODDR/PM
Figure 7: 62 day gut index (%, ±SEM) by treatment. AM indicates individuals fed ad
libitum once daily in the morning. PM indicates individuals fed ad libitum once daily in
the evening. AM/PM indicates individuals fed twice daily at ½ ad libitum. EODSR/PM
indicates individuals fed every other day in the evening at the daily ad libitum ration.
EODDR/PM indicates individuals fed every other day in the evening at twice the daily ad
libitum ration. Differential lower case letters designate significant differences.
Test Analysis
At 30 days, mean test dry matter among urchins in the EODSR/PM treatment had
not increased from estimates at the initiation of the study (Fig. 8). However, by day 62,
mean test dry matter production of individuals in the EODSR/PM treatment was not
significantly different than that of individuals in the EODDR/PM treatment (Fig. 8). At
both 30 and 62 days, mean test dry matter production was not different among those
treatments in which urchins were fed every day, regardless of frequency or time of
feeding (Fig. 8).
104
Aristotle’s Lantern Analysis
Mean dry matter production of the Aristotle’s lantern did not vary among feeding
regimes at either day 30 or day 62 (Fig. 9). Likewise, lantern: test indices did not vary
among treatments at 30 days, regardless of feeding frequency or time of feeding (Fig.
10). At day 62, however, lantern: test index of individuals in the AM/PM treatment was
significantly lower than that of urchins in the EODSR/PM treatment (Fig. 10, p= 0.013).
4.0
a
Test Dry Matter Production (g)
3.5
3.0
30 Day
ab
ab
62 Day
2.5
bc
2.0
1.5
1.0
A
A
A
c
B
0.5
B
0.0
-0.5
-1.0
AM
PM
AM/PM
EODSR/PM
EODDR/PM
Feeding Schedule
Figure 8: Test dry matter production (g, ±SEM) by treatment. AM indicates individuals
fed ad libitum once daily in the morning. PM indicates individuals fed ad libitum once
daily in the evening. AM/PM indicates individuals fed twice daily at ½ ad libitum.
EODSR/PM indicates individuals fed every other day at the daily ad libitum ration.
EODDR/PM indicates individuals fed every other day at twice the daily ad libitum ration.
Pale gray bars indicate test dry matter production at 30 days. Dark gray bars indicate test
dry matter production 62 days. Differential capital letters designate significant
differences at 30 days. Differential lower case letters designate significant differences at
60 days.
105
Lantern Dry Matter Production (g)
0.30
a
a
0.25
30 Day
a
62 Day
0.20
0.10
a
a
0.15
A
A
A
A
A
0.05
0.00
AM
PM
AM/PM
EODSR/PM
Feeding Schedule
EODDR/PM
Figure 9: Lantern dry matter production (g, ±SEM) by treatment. AM indicates
individuals fed ad libitum once daily in the morning. PM indicates individuals fed ad
libitum once daily in the evening. AM/PM indicates individuals fed twice daily at ½ ad
libitum. EODSR/PM indicates individuals fed every other day in the evening at the daily
ad libitum ration. EODDR/PM indicates individuals fed every other day in the evening at
twice the daily ad libitum ration. Pale gray bars indicate lantern dry matter production at
30 days. Dark gray bars indicate lantern dry matter production at 62 days. Differential
capital letters designate significant differences at 30 days. Differential lower case letters
designate significant differences at 60 days.
106
14
30 Day
Lantern: Test Index (%)
12
10
A
A
a
62 Day
a
A
a
A
A
a
b
8
6
4
2
0
AM
PM
AM/PM
EODSR/PM
Feeding Schedule
EODDR/PM
Figure 10: Lantern: test index (%, ±SEM) by treatment. AM indicates individuals fed ad
libitum once daily in the morning. PM indicates individuals fed ad libitum once daily in
the evening. AM/PM indicates individuals fed twice daily at ½ ad libitum. EODSR/PM
indicates individuals fed every other day in the evening at the daily ad libitum ration.
EODDR/PM indicates individuals fed every other day in the evening at twice the daily ad
libitum ration. Pale gray bars indicate lantern: test index at 30 days. Dark gray bars
indicate lantern: test index at 62 days. Differential capital letters designate significant
differences at 30 days. Differential lower case letters designate significant differences at
60 days.
107
Dry Matter Production Efficiency and Feed Conversion Ratio
Mean dry matter production efficiency (PE) of urchins in the EODSR/PM and
EODDR/PM treatments was significantly lower at 30 days than that of those fed daily,
regardless of frequency or time of feeding (Fig 11, p≤ 0.05). Mean dry matter production
efficiency among individuals fed every other day did not vary significantly regardless of
ration (Fig 11). At 62 days, mean PE among urchins in the AM/PM treatment was
significantly higher than that of urchins in the EODDR/PM treatment, despite having
received equal amounts of food throughout the duration of the study (Fig 11, p= 0.05).
Mean feed conversion ratio (FCR) of urchins in the EODDR/PM treatment were
significantly higher at 30 and 62 days than those fed daily, regardless of frequency or
time of feeding (Fig 12, p≤0.01 , p≤ 0.006). At 30 and 62 days, mean FCR did not vary
significantly among individuals fed every other day, regardless of ration (Fig 12). Feed
conversion ratio did not vary among urchins fed daily regardless of frequency or time of
feeding (Fig. 12). At 62 days, mean FCR of urchins in the EODSR/PM treatment was not
significantly different than that of urchins fed daily (Fig 12).
108
Dry Matter Production Efficiency (%)
16
14
A
12
10
ab
A
30 Day
a
ab
62 Day
A
b
b
8
6
4
B
B
2
0
-2
-4
AM
PM
AM/PM
EODSR/PM
EODDR/PM
Feeding Schedule
Figure 11: Dry matter production efficiency (%, ±SEM) by treatment. AM indicates
individuals fed ad libitum once daily in the morning. PM indicates individuals fed ad
libitum once daily in the evening. AM/PM indicates individuals fed twice daily at ½ ad
libitum. EODSR/PM indicates individuals fed every other day in the evening at the daily
ad libitum ration. EODDR/PM indicates individuals fed every other day in the evening at
twice the daily ad libitum ration. Pale gray bars indicate dry matter production efficiency
at 30 days. Dark gray bars indicate dry matter production efficiency at 62 days.
Differential capital letters designate significant differences at 30 days. Differential lower
case letters designate significant differences at 60 days.
109
Feed Conversion Ratio
10
9
30 Day
8
62 Day
A
AB
7
6
a
5
4
3
ab
BC
b
C
b
C
b
2
1
0
AM
PM
AM/PM
EODSR/PM
Feeding Schedule
EODDR/PM
Figure 12: Feed conversion ratio by treatment (±SEM). AM indicates individuals fed ad
libitum once daily in the morning. PM indicates individuals fed ad libitum once daily in
the evening. AM/PM indicates individuals fed twice daily at ½ ad libitum. EODSR/PM
indicates individuals fed every other day in the evening at the daily ad libitum ration.
EODDR/PM indicates individuals fed every other day in the evening at twice the daily ad
libitum ration. Pale gray bars indicate feed conversion ratio at 30 days. Dark gray bars
indicate feed conversion ratio at 62 days. Differential capital letters designate significant
differences at 30 days. Differential lower case letters designate significant differences at
60 days.
DISCUSSION
Growth and Production
Throughout the study, urchins in all treatments grew, indicating the adequacy of
all treatments for maintenance and growth. Reduced growth and production observed for
urchins in the EODDR/PM treatment indicate that intermittent feeding may adversely
affect nutrient uptake and assimilation in L. variegatus held in culture, even when urchins
are fed (and consume) amounts of feed equivalent to that of urchins fed daily.
110
Zhao et al. (2013) found no association between circadian feeding patterns and
growth and production in S. intermedius, although circadian feeding patterns have been
shown to be positively correlated with growth and production in fish (Spieler, 1977;
Delahunty et al., 1978; Sundararaj et al., 1982; Noeske and Spieler, 1984; Perez et al.,
1988; Azzaydi et al., 2000; Bolliet et al., 2001) and mammals (Nelson et al., 1975;
Philippens et al., 1977; Arble et al., 2009). Data from this study indicate that, among
urchins fed daily, wet weight gain was initially controlled by circadian feeding patterns,
as those fed an entire or partial daily ration in the PM showed higher production.
However, by 62 days, urchins in the AM treatment appeared to have adjusted to the
feeding regime and had compensated for the initial reduction in weight gain. We suggest
that circadian feeding patterns do exist in wild L. variegatus but that individuals held in
culture have the ability to adjust to changes in daily feeding regime and will exhibit
compensatory weight gain.
Gut and Gonad Production
Among many organisms, including mammals (McDonagh et al., 1984; Taylor et
al., 1996; Salvador and Savageau, 2003), fish (Fudge et al., 2001) and perhaps, most
notably, snakes (Secor and Diamond, 1995; Starck and Beese, 2001; Lignot et al., 2005;
Cox and Secor, 2008) organ morphology and capacity will vary in response to the
conditions imposed on the particular tissue. Previous studies report that among sea
urchins, gut size varies in response to conditions (in the form of food availability, Bishop
and Watts, 1992; Hammer et al., 2006). When fed ad libitum, increases in thickness of
gut mucosal folds, mucosal depressions, and visceral peritoneum occur in the stomach,
all of which are thought to contribute to an increase in digestive capacity (Bishop and
111
Watts, 1992). Additionally, the number and size of cells increase as nutrients are
absorbed and stored (Bishop and Watts, 1992). In the present study, that difference in gut
size among urchins was, in part, correlated with overall body size. However, for those in
the EODDR/PM treatment, gut index was also directly influenced by feeding regime.
These data suggest that feeding regime, as it relates to frequency and amount, affects
digestive processes as influenced by the size of the gut relative to the size of the urchin
(gut index). We hypothesize that those fed a large ration every other day increased either
the storage capacity of the gut or its digestive capacity. Proximal control of these
alterations is not known.
Among sea urchin species, gonad production varies directly with frequency of
feeding (Lawrence et al., 2003). Under conditions of episodic feeding, urchins are
reported to allocate proportionally more resources to maintenance activities than to
growth and gonad production, regardless of the quantity of feed proffered (McBride et
al., 1999; Zhao et al., 2013). Urchins in this study exhibited similar patterns, as those fed
daily increased gonad production and indices relative to those fed every other day. This
response occurred regardless of ration size, as observed in the EODDR/PM treatment.
However, we suggest that the duration of this study may have been insufficient to
observe the growth response of the gonad tissue to variations in feed ration among
urchins fed every other day. These data suggest that feeding every other day will be
limiting for urchins in the grow-out phase of culture.
Test and Aristotle’s Lantern
Urchins subjected to severe food limitations are reported to remodel the test to
retrieve stored nutrients for use in metabolic processes, thereby resulting in a smaller test
112
size (Ebert 1968, Ebert 1980, Levitan, 1991, Guillou et al., 2000). While urchins in the
EODSR/PM treatment were not held under conditions of severe limitation of food, lack
of increase in test weight among urchins in this treatment suggests that, at least initially,
the feeding regime was only sufficient for maintenance requirements. By day 62,
however, test growth of urchins in this feeding regime did occur, suggesting that some
mechanism is used to adjust their metabolic processes to adapt to the feed limitations.
Among sea urchins, it has been suggested that the Aristotle’s lantern will grow to
a pre-determined size, regardless of food and nutrient availability and growth of other
body components (Heflin et al., 2012). Growth of the Aristotle’s lantern did not vary
among urchins representing each treatment. However, the size of the lantern relative to
the test (lantern: test index) did vary between urchins fed twice daily and those in the
EODSR/PM treatment. Among sea urchins, lantern: test index will often vary in response
to feed (Ebert 1968; Ebert 1980; Black et al., 1982; Fansler, 1983; Black et al., 1984;
Levitan, 1991; Constable, 1993; Fernandez and Boudouresque, 1997; McShane and
Anderson, 1997; Guillou et al. 2000; Hagen, 2008; Lau et al., 2009) or nutrient (Jones et
al., 2010; Heflin et al., 2012) limitations. We suggest that feed and/or nutrient limitations
in the EODSR/PM treatment were adequate to initiate increased resource allocation of
nutrients to the Aristole’s lantern of individuals in this treatment.
Dry Matter Production Efficiency and Feed Conversion Ratio
Data from this study and from that of Lawrence et al. (2003) indicate that
production efficiency will decrease if L. variegatus are fed every other day rather than
daily. Despite their larger gut index that, presumably, increases capacity to process and/or
store nutrients), urchins in the EODDR/PM treatment were unable to assimilate dietary
113
nutrients as efficiently as those fed daily, resulting in reduced PE and increased FCR.
Among aquatic organisms, poor feed utilization can be the result of poor nutrient
content/availability of feed or poor management of feed resulting from inadequate
knowledge of energetic needs, and inadequate distribution and application of feeds
(Alanara et al., 2001). These data indicate that sea urchins that are fed large quantities of
feed every other day may be unable to process and assimilate consumed nutrients
efficiently, despite an increase in short-term feed intake. Although no studies have
compared FCR among urchins fed daily and those fed every other day, Heflin et al.
(2012) report that FCR among L. variegatus may decrease when dietary energy becomes
limiting and James and Siikavuopio (2012) report increased FCR among S.
droebachiensis fed continuously as compared to those fed and then starved. Studies with
other sea urchin species, including Strongylocentrotus franciscanus (McBride et al.,
1999) and Paracentrotus lividus (Frantzis and Grémare, 1992) have shown a correlation
between increased consumption rate and decreased absorption efficiency. However,
neither McBride et al. (1999) nor Frantzis and Grémare (1992) report effects on urchins
subjected to episodic feeding regimes.
The efficiency at which sea urchins utilize proffered feeds will be a key factor
determining the sustainability and economic attractiveness of farming sea urchins. Based
on data collected from this study, we recommend that L. variegatus held in culture be fed
a daily ad libitum ration to maximize both growth and efficiency. As feed is commonly a
significant cost in aquaculture operations, these feed management strategies can help
optimize growth as well as the respective return on investment.
114
ACKNOWLEDGEMENTS
The authors thank Courtney Duncan, Priya Patel, Amanda Monroe, and the rest of
the Watts’ lab at the University of Alabama at Birmingham for providing technical
support for this study. We thank Louis D’Abramo, Daniel Smith, Daniel Warner and Jim
McClintock for editorial comments. We also thank John Lawrence and Addison
Lawrence for related discussions. This report was prepared by S.A.W. under award
NA07OAR4170449 from the University of Alabama at Birmingham, U.S. Department of
Commerce. The statements, findings, conclusions, and recommendations are those of the
authors' and do not necessarily reflect the views of NOAA or the U.S. Department of
Commerce.
115
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CHAPTER 4
BALANCING MACRONUTRIENT INTAKE IN CULTURED Lytechinus variegatus
by:
LAURA E. HEFLIN, DAVID RAUBENHEIMER,
STEPHEN J. SIMPSON, STEPHEN A.WATTS
Submitted to Aquaculture
Format adapted for dissertation
125
ABSTRACT
Among aquatic organisms, nutritional requirements are historically assessed by
evaluating formulated diets with changes in a single nutrient profile. These studies
provide organisms with the option of regulating the total feed intake but do not enable
individuals to regulate intake of specific nutrient ratios. An alternate experimental
approach, known as the geometric framework (GF) for nutrition, tests whether organisms
held under specific conditions have optimal target intake levels for particular nutrients
and whether, when provided with diet choices, they will self-regulate nutrient intake to
reach these specific targets. In this study, we used the GF to assess intake levels of
dietary protein and carbohydrate in cultured adults of the sea urchin Lytechinus
variegatus provided choices between diets varying in both dry matter and specific
macronutrient concentrations. Adult urchins (ca. 120 g) held previously on formulated
diets were offered moist gel-based diets in pairwise combinations. Diets varied in levels
of fish meal isolate (as a protein source) and wheat starch (as a carbohydrate source) and
in nutrient concentration (5 or 10% dry matter). Regardless of diet combination,
individuals maintained an average dietary protein intake of ca. 0.047- 0.061 g day-1.
Dietary carbohydrate intake was not regulated to a specific level and ranged from 0.0420.136 g day-1. These data suggest that large adult L. variegatus held in culture have a
tightly regulated intake target for dietary protein but not carbohydrate. Regardless of
nutrient ratios or macronutrient concentration, individuals adjusted intake patterns to
126
defend this target. We suggest that the GF will be an important tool in evaluating
macronutrient requirements in cultured sea urchins and other aquatic species.
127
INTRODUCTION
The nutritional requirements of animals, whether terrestrial or aquatic, are
complex and include a variety of both macro and micronutrients. However, due to lack of
convenient access, nutritional research among aquatic animals presents additional
challenges in natural observations that are not characteristically encountered in the
terrestrial environment. For example, in the wild, identification of natural prey items and
observation and quantification of food intake of aquatic animals are unavoidably difficult.
When food consumption can be assessed, an estimation of the quality of food items is
difficult to achieve. Likewise, it is challenging for researchers to estimate the nutritional
requirements of aquatic animals held in culture with the goal of formulating a practical
diet that will satisfy requirements. Therefore, knowledge of the nutrient requirements of
many aquatic species remains lacking.
For many animals held in culture, a single diet will often be formulated to
theoretically provide the daily nutritional requirements of that organism. That single diet
will then contain a fixed ratio of macronutrients (protein, fat and carbohydrate). An
individual fed that single diet may choose to eat more or less but cannot control any
change in the ratio of dietary macronutrient intake. In addition, an individual restricted to
one diet may be compelled to sacrifice an important nutrient target to avoid over or under
intake of another nutrient (Hewson-Hughes et al., 2013; Raubenheimer and Simpson,
1993; Simpson and Raubenheimer, 1993, 2012). Conversely, when an individual has
128
access to two or more food choices with different nutrient profiles, it may modify feed
intake among the different individual foods to reach the intake target for one or more
nutrients through consumptive combinations of the available diets (Raubenheimer and
Simpson, 1993; Simpson and Raubenheimer, 1993, 1995, 2012).
Nutritional requirements are dynamic, and change due to environmental
conditions, reproductive state, and interactions with other nutrients (Simpson and
Raubenheimer, 2012). At any one time, organisms in the wild may have access to a
variety of foods that vary in both nutrient composition and nutrient ratio and can thus
compensate for possible changes in nutritional requirements. In an aquaculture facility,
failure to provide a feed that allows organisms to reach individual intake targets without
exceeding or falling short of others can potentially have an adverse effect on organismal
health, carcass yield and reproduction, resulting in loss of profit for culturists (Ruohonen
et al., 2007; Simpson and Raubenheimer, 2001). To be effective, a fixed diet formulation
must have the capacity to match nutritional requirements integrated over time and across
environmental conditions.
The eventual establishment of commercial aquaculture facilities for sea urchins
has great potential. Of paramount importance is the development of a cost-effective,
practical diet. Such a diet will be designed to optimize health, growth and reproduction
while minimizing cost and environmental impact. Protein is one of the most costly and
potentially environmentally harmful ingredients, when fed in excess, in aquafeeds but
adequate dietary protein is necessary for growth, tissue maintenance and repair, digestion
and metabolism. Provision of adequate levels of dietary protein is correlated with a
decrease in feed intake among sea urchins (Agatsuma, 2000; Daggett et al., 2005;
129
Fernandez and Bourdouresque, 1998, 2000; Frantzis and Gremare, 1992; B.W. Hammer
et al., 2004; H.S. Hammer et al., 2006, 2012; Meidel and Scheibling, 1999; McBride et
al., 1999), suggesting that an intake target (optimal nutrient requirement, Simpson and
Raubenheimer, 2012) for protein may exist among urchins. Other reports suggest that sea
urchins also eat to fulfill an energy (principally carbohydrate) requirement (Hammer,
2006; Lawrence et al., 2009; Otero-Villanueva et al., 2004; Taylor, 2006). Historically,
nutritional studies have sought to establish nutrient requirements among sea urchins by
offering individuals one of several feeds that vary in the concentration of one or two
nutrients and measuring outcomes such as growth and production among treatments.
While these studies assist researchers in establishing required ranges for particular
nutrients, the Geometric Framework (GF) approach is suggested as an approach to
complement current research efforts and expand upon existing knowledge of nutritional
requirements among aquatic organisms (Ruohonen et al., 2007; Simpson and
Raubenheimer, 2001). Identification of intake targets for specific nutrients will allow for
the formulation of a practical sea urchin diet that satisfies nutritional requirements for
urchins held in culture.
Studies utilizing the GF model suggest that animals have the ability to adjust
foraging and feed intake patterns to fulfill nutritional requirements (Chambers et al.,
1995; Dussutour et al, 2010; Felton et al., 2009; Hewson-Hughes et al., 2012, 2013; Lee
et al, 2002; Mayntz et al., 2009; Raubenheimer and Simpson, 1993; Simpson and
Raubenheimer, 2012). When presented with food choices, many organisms will selfregulate feed intake whereby intake targets for one or more nutrients are achieved
(Raubenheimer and Simpson, 1993; Simpson and Raubenheimer, 2012). The nutrient
130
composition of available foods will determine whether an organism can reach a particular
intake target through adjustment of the consumption of those foods (Chambers et al.,
1995; Simpson and Raubenheimer, 1995, 2012). A single food will contain a certain set
ratio of nutrients (eg. protein and carbohydrate). The nutrient content of that food is
represented graphically as a vector projecting from the origin through nutrient space and
is referred to as a “nutrient rail” (Raubenheimer and Simpson, 1993; Simpson and
Raubenheimer, 1993, 1995, 2012). The nutritional intake of an animal feeding on a single
food must lie along the trajectory of the nutrient rail created by that food (Raubenheimer
and Simpson, 1993; Simpson and Raubenheimer 1993, 1995, 2012). When an individual
is restricted to only one food, it may choose to eat more or less but cannot vary the ratio
of dietary nutrients consumed. Conversely, when an individual has access to two or more
complementary foods (and thus, two or more nutrient rails), it may vary eating patterns
whereby the intake target for one or more nutrients can be realized as long as the nutrient
targets lie between the rails created by the available foods (Raubenheimer and Simpson,
1993; Simpson and Raubenheimer, 1993, 1995, 2012).
In this study, we used the GF to assess feed intake of cultured adult sea urchins
Lytechinus variegatus to determine whether intake targets for dietary protein and
carbohydrate exist when provided choices between diets that vary in both dry matter and
specific macronutrient concentrations.
131
MATERIALS AND METHODS
Collection and Culture Conditions
Adult (ca. 120 g) Lytechinus variegatus were selected from stock collected
previously from Saint Joseph Bay, FL (30N, 85.5W) and transported to the University
of Alabama at Birmingham. Urchins had been held on a maintenance diet previously
shown to maintain survival and health (Hammer et al., 2012; Heflin et al., 2012). Urchins
were assigned randomly to 1 of 4 treatments (n =5 urchins per treatment) and were then
placed individually into rectangular plastic mesh cages (14.4 x24.8 x 15.2 cm, L x W x
H). Cages were coded so that each individual could be tracked over the course of the 8day study. The floors of the mesh enclosures were elevated ca. 3.81 cm to allow water
circulation underneath. In these cages, feed cubes were retained, but feces fell through
the mesh. The cages were randomly placed in a fiberglass raceway (235 cm x 53 cm x 31
cm, L x W x H, as described by Taylor (2006). A 160 x 23 cm (L x H) center baffle in the
center of the raceway allowed for recirculating water flow by an in-line utility pump
(Supreme® Mag Drive Utility Pump, 700 gallons of water/hour). The utility pump
removed synthetic seawater (Instant Ocean) from the raceway on one side of the baffle.
Water then passed through a mechanical and biological filter and returned to the raceway
on the opposite side of the baffle. The flow rate of the resulting current was
approximately 9.7 – 12.6 cm s-1. Water passed independently through a 10 watt UV
sterilizer (Lifegard® Aquatics, Cerritos, CA). Water depth was maintained at 15.0 cm.
Total ammonia nitrogen, nitrite, nitrate, pH and alkalinity levels were checked every
other day using saltwater test kits from Aqua Pharmaceuticals, LLC (Malvern, PA, USA)
132
for ammonia and nitrogen and La Motte Company (Chestertown, MD, USA) for
alkalinity. Photo-period and temperature were held constant (12:12 light: dark, 24±1 °C).
Salinity was maintained at 32 ± 0.5 ppt) using synthetic seawater (Instant Ocean).
Feed Preparation and Feeding
Four moist diets (Table1) were prepared using three ingredients: non-nutritive
agar (Sigma A7002), wheat starch (ca. 98% carbohydrate, MP Biomedical, cat no.
902952) and fish meal hydrolysate (ca. 80% protein, The Scoular Company, SopropecheC.P.S.P.90). The fish meal hydrosylate contained ca. 10% lipid (by analysis, Scoular) and
7% ash (as fed). Consequently, the level of residual oil or ash co-varied with the protein
content. Diets were prepared by boiling agar (final concentration of 1.5%) in synthetic
seawater (34 ppt.). After boiling, the agar solution was allowed to cool to 60° C and then
combined with dry ingredients (fish meal and wheat starch) in ratios indicated in Table 1,
resulting in four diets which varied in dry matter content and in protein and carbohydrate
concentration. This feed emulsion was poured into a baking dish (23 x 23 cm, L x W) and
placed on the surface of an ice bath (ca. 0°C) to solidify. Each prepared gel was placed in
a refrigerator overnight and then cut into food cubes (ca. 3.2 x 3.2 x 1.5 cm, L x W x H)
and stored in air tight containers at 4º C to prevent water evaporation. Diets were
prepared fresh every four days.
133
Table 1. Calculated fish meal and wheat starch levels (as fed), agar, synthetic seawater,
salt, and percent moisture of diets. As a code, P and C represent the predominant nutrient
(protein or carbohydrate, respectively), in the diet; and p and c represent the lesser
nutrient.
Diet
10% Pc
5% Pc
10% pC
5% pC
Fish
Meal
(%)
9
4.5
1
0.5
Wheat
Starch
(%)
1
0.5
9
4.5
Agar
(%)
1.5
1.5
1.5
1.5
Synthetic
Seawater
(%)
88.5
93.5
88.5
93.5
Salt
(%)
Moisture
(%)
0.03
0.03
0.03
0.03
88.47
93.47
88.47
93.47
Individual urchins were offered pairwise diet combinations (Table 2). Both diets
in each pairwise combination were provided in excess allowing unbiased choice of intake
of either diet. Food cubes representing each diet were weighed individually prior to
feeding and offered simultaneously to an individual urchin. For each individual, food
cubes were placed side by side in cages in direct contact and equal proximity to the
urchin, enabling urchins the opportunity to consume both of the diets. To avoid positional
bias, the location of each diet was alternated daily. After 24 hours, the portions of the
food cubes representing each diet that remained, were recovered, rinsed in deionized
water, blotted and re-weighed to calculate feed intake (by subtraction). Leaching of
ingredients (estimated by weight loss or gain of the food cube in the absence of an
urchin) in the 24 hour period was determined to be negligible. Total feed and specific
macronutrient intake of each diet and the combined total nutrient intake from each
pairwise combination were recorded daily during an 8-day period.
Daily intake of each diet was calculated as:
(1) Initial weight of food cube (g) – final weight of food cube (g)
134
Cumulative intake of each diet was calculated as:
8
(2) Σ f(i) Where f(i)= total weight of food cube (g) consumed per day
i=1
Protein intake per day was calculated as:
(3) [Total weight (g) of Pc food cube consumed x food concentration (5 or 10%) x fish
meal concentration (90%) x protein content of fish meal (80%)] + [Total weight (g)
of pC food consumed x food concentration (5 or 10%) x fish meal concentration
(10%) x protein content of fish meal (80%)]
Cumulative protein intake was calculated as:
8
(4) Σ f(i) Where f(i)= total protein consumed per day (g)
i=1
Carbohydrate intake per day was calculated as:
(5) [Total weight (g) of Pc food consumed x food concentration (5 or 10%) x wheat
starch concentration (10%) x carbohydrate content of wheat starch (98%)] + [Total
weight (g) of pC food consumed x food concentration (5 or 10%) x wheat starch
concentration (90%) x carbohydrate content of wheat starch (98%)]
Cumulative carbohydrate intake was calculated as:
8
(6) Σ f(i) Where f(i)= total carbohydrate consumed per day (g)
i=1
Percent protein intake per day was calculated as:
135
(7) [Total weight (g) of Pc food cube consumed x food concentration (5 or 10%) x fish
meal concentration (90%) x protein content of fish meal (80%)] + [Total weight (g)
of pC food consumed x food concentration (5 or 10%) x fish meal concentration
(10%) x protein content of fish meal (80%)]/ [Total weight (g) of Pc food cube
consumed + Total weight (g) of pC food consumed](all percentages are used in
calculations as a fraction)
Percent carbohydrate intake per day was calculated as:
(8) [Total weight (g) of Pc food consumed x food concentration (5 or 10%) x wheat
starch concentration (10%) x carbohydrate content of wheat starch (98%)] + [Total
weight (g) of pC food consumed x food concentration (5 or 10%) x wheat starch
concentration (90%) x carbohydrate content of wheat starch (98%)] / [Total weight
(g) of Pc food cube consumed + Total weight (g) of pC food consumed] (all
percentages are used in calculations as a fraction)
136
Table 2. Pairwise diet combinations (4 treatments) offered to urchins (n = 5 per
treatment).
Diet combinations
9:1 fish meal: wheat
starch ratio (Pc)
High Protein/ High
Carbohydrate
(10%Pc/10%pC)
10% Pc
9:1 wheat starch: fish
meal ratio
(pC)
10% pC
High Protein/ Low
Carbohydrate
(10%Pc/5%pC)
10% Pc
5% pC
Low Protein/ High
Carbohydrate
(5%Pc/10%pC)
5% Pc
10% pC
Low Protein/ Low
Carbohydrate
(5%Pc/5%pC)
5% Pc
5% pC
Statistics
Statistical analyses of macronutrient intake were performed in IBM SPSS
Statistics 22. P values ≤ 0.05 were considered significant. Data were tested for normality
and homoscedasticity using Shapiro-Wilk and Levene’s tests, respectively. Non –
homoscedastic data were log transformed and were analyzed with ANOVA using the
GLM procedure. Data that were found to be normal and equal in variance were analyzed
with ANOVA using the GLM procedure. If a significant difference was detected, a
Tukey’s HSD test was used to compare means. Outcomes reported included diet intake
137
(as fed) and total feed intake (for each combination) as well as total protein intake and
total carbohydrate intake.
RESULTS
Water Quality
Water conditions were maintained as follows: 32±0.5 ppt salinity, 22±2°C, D.O.
7±2 ppm, ammonia 0 ppm, nitrite 0 ppm, nitrate 0 ppm, alkalinity ≥200 ppm, and pH 8.2.
Water quality parameters maintained in this study were within the ranges suitable for sea
urchins (Basuyaux and Mathieu, 1998).
Food and nutrient intake
Urchins in all diet combinations readily consumed all experimental diets. Feed
intake among sea urchins offered diets with a high protein level (10%Pc combined with
5% pC 10%pC) was generally less than that of individuals offered diets with low protein
levels (Fig 1). Feed intake among sea urchins offered the high carbohydrate level diet
(10%pC) was significantly higher than that of those offered the low carbohydrate level
diet (5%pC, p= 0.03, Fig. 1); however, protein level in pC diets co-varied with
carbohydrate. Regardless of diet combination, individuals in all treatments consumed the
respective diet at the same rate (Fig. 1).
138
2.0
Average Daily Intake (g)
1.8
a
ab
1.6
B
1.4
AB
1.2
1.0
b
A
b
A
0.8
0.6
10%Pc
5%Pc
10%pC
5%pC
0.4
0.2
0.0
10%Pc/10%pC
10%Pc/5%pC
5%Pc/10%pC
5%Pc/5%pC
Diet Combinations
Figure 1. Average daily diet intake (g, ± SEM) in the pairwise diet combinations. White
bars indicate the 10%Pc diet. Pale gray bars indicate the 5% Pc diet. Dark gray bars
indicate the 10%pC diet and black bars indicate the 5%pC diet. As a code, P and C
represent the predominant nutrient (protein or carbohydrate, respectively), in the diet; and
p and c represent the lesser nutrient. Percentages (5% or 10%) represent the feed
concentration in the agar-bound diet. Diet combinations are represented as diet 1/diet 2
(e.g. 10%Pc/10%pC represents the diet at 10% feed concentration with fish meal as the
predominate ingredient in combination with the diet at 10% feed concentration with
wheat starch as the predominant nutrient). Significant differences are designated among
high protein (capital letters) or high carbohydrate (lower-case letters) diets.
Individuals offered the 10%pC diet consumed a higher dietary carbohydrate
percentage (and thus, a lower protein: carbohydrate ratio) than individuals offered the
5%pC diet (p< 0.001, Fig. 2). Levels of diluents (agar or synthetic seawater) in the diets
were not significantly correlated with nutrient intake patterns and, thus, are not shown.
Dietary lipid and ash intake for those consuming protein co-varied with dietary protein
intake and are not shown.
139
Individuals in all diet combinations consumed proportions of both diets and
maintained an average dietary protein intake of ca. 0.047- 0.061 g day-1 regardless of the
diet combination (Fig. 3). Dietary carbohydrate intake was not regulated, and ranged
from 0.042- 0.136 g day-1 (p= 0.01, Fig. 3). Variance in average daily carbohydrate intake
was higher among individuals receiving the 10%pC diet than those receiving the 5%pC
diet (p< .001, Fig. 2, Fig. 3).
140
8
10%Pc
7
6
Protein (%)
5
4
10%Pc/10%p
C
10%Pc/5%pC
5%Pc
3
5%Pc/10%pC
2
1
10%pC
5%pC
0
0
2
4
6
8
10
Carbohydrate (%)
Figure 2: Percent daily protein and carbohydrate intake by diet combination. As a code, P
and C represent the predominant nutrient (protein or carbohydrate, respectively), in the
diet; and p and c represent the lesser nutrient. Percentages (5% or 10%) represent the feed
concentration (% dry matter) in the diet. Diets are represented by solid circles. Thin solid
lines indicate diet combinations offered and thus, represent possible protein carbohydrate
intake ratios available to individuals in the treatment. Bold gray lines indicate protein:
carbohydrate intake between treatments in which 10% pC was an option. Bold black
lined indicate protein: carbohydrate intake between treatments in which 5%pC was an
option. Dashed lines indicate protein: carbohydrate rails selected by urchins. Diet
combinations are represented as diet 1/diet 2. Squares indicate average daily protein and
carbohydrate intake for urchins in each diet combination. Horizontal error bars represent
dietary carbohydrate (SEM). Vertical error bars represent dietary protein (SEM).
141
Average daily protein intake (mg)
0.08
0.07
5%Pc/10%pC
0.06
10%Pc/10%pC
10%Pc/5%pC
0.05
5%Pc/5%pC
0.04
0.03
0.02
0.01
0
0
0.05
0.1
0.15
Average daily carbohydrate intake (mg)
Figure 3: Average daily macronutrient intake (mg, ±SEM) by treatment. As a code, P and
C represent the predominant nutrient (protein or carbohydrate, respectively), in the diet;
and p and c represent the lesser nutrient. Percentages (5% or 10%) represent the feed
concentration in the diet. Diet combinations are represented as diet 1/diet 2. Dots indicate
average daily protein and carbohydrate intake for urchins in each diet combination.
Horizontal error bars represent dietary carbohydrate (SEM). Vertical error bars represent
dietary protein (SEM).Solid line indicates trend. Dashed lines indicate nutrient rails
created by the feed combinations.
DISCUSSION
The geometric framework is an applied nutritional science that seeks to reveal
how animals use nutritional choices to relate to and thrive within their environment
(Raubenheimer and Simpson, 1993; Simpson and Raubenheimer, 2012). The GF model
has provided an effective tool for the study of nutritional responses of different
organisms, including humans (Blumfield et al. 2013; Gosby et al. 2014), non-human
142
mammals (Felton et al., 2009; Hewson-Hughes et al., 2012, 2013; Mayntz et al., 2009;
Solon-Biet et al., 2014; Sorensen et al., 2008), fish (Ruohonen et al., 2007), insects
(Chambers et al., 1995; Lee et al., 2002; Raubenheimer and Simpson, 1993;
Raubenheimer and Simpson, 2003) and even a cellular slime mold (Dussutour et al.
2009). The results of this study establish that the GF can be used successfully to evaluate
nutrient intake targets in sea urchins.
Dietary intake targets for protein have been identified across many animal taxa
(Chambers et al., 1995; Felton et al., 2009; Hewson-Hughes et al., 2012, 2013; Lee et al.,
2002; Mayntz et al., 2009; Raubenheimer and Simpson, 2003; Solon-Biet et al., 2014;
Sorensen et al., 2008). Previous studies indicate that sea urchins will adjust feed intake,
presumably to satisfy nutritional requirements (Fernandez and Boudouresque, 2000;
McBride et al., 1998; Taylor, 2006; Wallace, 2001). However, exact dietary requirements
(intake targets) for protein or other nutrients have not been identified for sea urchins.
Data from this study suggest that large, adult L. variegatus have a tightly regulated intake
target for dietary protein. Across treatments, individuals adjusted intake patterns to
defend this target regardless of nutrient ratios or feed concentrations. In fact, for diets
containing high carbohydrate and low protein levels, urchins over consumed
carbohydrates to achieve the protein intake target. Urchins fed a high-protein diet are
reported to become satiated more quickly than those fed a low-protein diet (Agatsuma,
2000; Daggett et al., 2005; Fernandez and Bourdouresque, 1998, 2000; Frantzis and
Gremare, 1992; B.W. Hammer et al., 2004; H.S. Hammer et al., 2006, 2012; McBride et
al., 1998; Meidel and Scheibling, 1999), further supporting the suggestion of a protein
intake target. In sea urchins, as with other eukaryotes, protein plays an essential role in
143
many biological processes, including reproduction, early development, growth, and repair
and maintenance of body tissues. Given the importance of dietary protein, it seems
reasonable to conclude that L. variegatus have an intake target for dietary protein and that
this target is closely regulated to maintain proper physiological function.
Although present in the fish meal isolate, the small quantities of dietary lipid and
ash intake among sea urchins in this study co-varied directly with dietary protein levels.
However, we suggest that individuals consumed the fish meal hydrolysate as a protein
source to fulfill a specific requirement for dietary protein, as opposed to lipid or ash.
Protein is the primary component of the fish meal hydrolysate. In addition, dietary ash
has been observed to exert no effect on sea urchins at the dietary levels found in the
experimental diets used in this experiment (Watts, unpublished data). Previous studies
indicate that lipid requirements among sea urchins are low (Gibbs et al., 2009) and excess
intake of dietary lipid restricts growth in L. variegatus (Gibbs et al., 2009). In contrast,
increases in levels of dietary protein are associated with increased growth and production
(Hammer et al., 2012; Heflin et al., 2012).
Carbohydrates provide the principal source of chemical energy for many animals,
including sea urchins (Marsh et al., 2013). The sea urchin gut contains numerous
carbohydrases (although no cellulases have been identified, Lawrence et al., 2013),
indicating that sea urchins can most likely utilize carbohydrates contained within a wide
array of sources. However, sea urchins have low respiration rates (Lawrence and Lane,
1982), perhaps a consequence of their lack of thermoregulation and low activity level.
Thus, dietary energy, while easily obtained, may not be needed in large quantities.
Despite the lack of a comparatively high energy requirement, previous studies suggest
144
that sea urchins will still consume food to fulfill an energy requirement (Hammer, 2006;
Lawrence et al., 2009; Otero-Villanueva et al., 2004; Taylor, 2006). Lytechinus
variegatus in this study did not actively regulate carbohydrate intake. Instead,
carbohydrate intake was variable and appeared to be dependent, in part, on protein
regulation and the level of dietary carbohydrate in the diet (10% pC vs. 5% pC).
Sea urchins are generalist feeders and are opportunistic omnivores: Feed intake
habits in wild urchin populations depend largely on the availability of a variety of foods,
which varies with season and habitat (De Ridder and Lawrence, 1982; Lawrence et al.,
2013). Many of these diets are proportionately high in plant matter and, as a
consequence, low in protein, suggesting that urchins consume excess carbohydrate
(starches) to meet intake targets for protein. Among other organisms, a mechanism for
prioritizing the intake target for one nutrient while over or under consuming another
(perhaps less important) nutrient (referred to as prioritization; Felton et al., 2009; Mayntz
et al., 2009; Simpson and Raubenheimer, 2012) appears to exist. We suggest that
individuals consuming high levels of dietary carbohydrate in this study did so, in part, to
achieve needed intake levels for dietary protein (protein leveraging). The consequence of
the de facto consumption of excess carbohydrate by sea urchins is unknown; however,
protein prioritization alone cannot completely explain the lack of carbohydrate regulation
observed. Prioritization of a particular intake target will generally only occur among
organisms when available food choices provide no prospect of reaching intake targets.
Both humans (Gosby et al. 2014; Simpson and Raubenheimer, 2005) and nonhuman primates (Felton et al., 2009) are reported to utilize a protein leverage intake
strategy. Under this strategy, consumption/intake of dietary protein, which cannot be
145
stored by some organisms, must be tightly regulated to meet needs (Felton et al., 2009;
Gosby et al., 2014; Simpson and Raubenheimer, 2005). In contrast, non-protein energy is
easily stored and, thus, intake fluctuates relative to both protein availability and the
availability of high energy food (Felton et al., 2009; Gosby et al., 2014; Simpson and
Raubenheimer, 2005). Although sea urchins can store proteins in the form of major yolk
protein in the gonad (Unuma et al., 1998), we suggest that protein leverage also occurs in
sea urchins. Individual urchins in this study were fed diets which contained different
amounts of protein and carbohydrate and in theory, could have speculated about an intake
target for carbohydrate while still reaching intake targets for dietary protein. Nonetheless,
urchins in this study consumed a higher percentage of dietary carbohydrate (relative to
dietary protein) when offered the 10%pC diet as opposed to the 5%pC diet. Sea urchins
store excess energy in their gonad tissue in the form of glycogen (Marsh et al., 2013). We
suggest that individuals in this study prioritized protein intake but opportunistically
consumed excess carbohydrate when it was in abundance. As generalists feeders, sea
urchins are predicted to have a high degree of dietary flexibility (Erlenbach et al., 2014;
Lee et al., 2003; Raubenheimer and Jones, 2006) and are better able to cope with
nutritional imbalance than specialists (Raubenheimer and Jones, 2006; Raubenheimer and
Simpson 2003). In the wild, urchins are often food limited and composition of available
foods is unpredictable. We suggest that urchins in this study may have consumed and
stored excess carbohydrate (when it was abundant in the diet) to compensate for the
possibility of a later occurrence of a deficit.
Future studies will be required to establish intake targets for protein,
carbohydrate, lipid and/or other nutrients in sea urchins. We hypothesize nutrient targets
146
(requirements) will vary with age, health status, reproductive status, and stress. In
addition, we hypothesize that nutrient intake may be positively influenced by familiarity
with the diet. The sea urchins used in this study were previously habituated to both fish
meal and starch in the maintenance diet. It is possible that naïve urchins may respond
differently, than what has been observed in other species (Hewson-Hughes et al., 2009).
We suggest a series of studies in which nutritionally complete diets that vary in
macronutrient levels are provided in both choice (pairwise combinations) and no choice
(single feed offered) treatments to better define and understand nutrient intake targets for
urchins held in culture. These data would provide necessary information for daily intake
metrics, which is important in feed formulation and production. Under the parameters of
the GF experimental design, researchers can examine the effect of nutrient levels, nutrient
ratios, and abiotic interactions simultaneously (Ruohonen et al., 2007; Simpson and
Raubenheimer, 2001), thus greatly decreasing time and cost in the formulation of optimal
aquafeeds for organisms held in culture.
147
ACKOWLEDGEMENTS
The authors would like to thank Lacey Dennis and the rest of the Watts’ lab at the
University of Alabama at Birmingham for providing technical support for this study.
Approval for this study was granted by the Institutional Animal Care and Use Committee
of the University of Alabama at Birmingham. Animal resources were supported by the
NORC Aquatic Animal Research Core within the Animal Models Core (NIH
P30DK056336). The statements, findings, conclusions, and recommendations expressed
herein are those of the authors' and do not necessarily reflect the views of NORC.
148
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CHAPTER 5
GENERALIST FEEDING STRATEGIES UTILIZED BY JUVENILES OF THE SEA
URCHIN Lytechinus variegatus
by
LAURA E. HEFLIN, MARLEE D. HAYES, KAREN E. JENSEN
DAVID RAUBENHEIMER, STEPHEN J. SIMPSON, STEPHEN A. WATTS
In Preparation for Proceedings of the Royal Society of the UK
Format adapted for dissertation
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ABSTRACT
Sea urchins are important macro-invertebrates within marine habitats, and
often influence community structure via their population structure and feeding activities.
As such, their population structure and success is often dependent on the availability of
nutrients. To better understand nutritional acquisition strategies, we employed the
geometric framework (GF) to evaluate protein and carbohydrate intake in wild-caught
juveniles (ca. 11±0.9g) of the sea urchin Lytechinus variegatus. Two concurrent
experiments (21 total diet treatments, n=8 per treatment) were performed. The first was a
no-choice experiment in which urchins were offered ad libitum one of 15 single diets
varying in protein and carbohydrate concentration and in feed dilution (4, 8 or 12%, as
fed). Variation in nutrient concentration (protein: carbohydrate ratio) allowed for the
evaluation of intake targets for dietary protein or carbohydrate. The second was a choice
experiment in which individual urchins were offered pairwise combinations of the diets
described previously (8% food concentrations). Within each diet combination, both diets
were offered ad libitum. Total feed and specific nutrient intake from each treatment and
the combined total nutrient intake from each pairwise combination were recorded daily
over a 43-day period. Urchins fed the most dilute (4% food concentration) in the nochoice experiment generally altered intake strategies to that of an extreme generalist and
ate to volume (but not nutrient) satiation. Urchins in the no-choice experiment fed an
increased nutrient density (8 or 12% food concentration) diet demonstrated nutrient
recognition and utilized the fixed proportion regulatory pattern to maintain a constant
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ratio of error in protein to error in carbohydrate, regardless of whether protein or
carbohydrate was in excess. Error in dietary protein intake was maintained at a lower
level than error in dietary carbohydrate intake, indicting a tendency for protein leverage.
Urchins offered diet combinations made choices between the respective diets and
generally preferred the diet with the most balanced (equi-proportioned) protein:
carbohydrate ratio. Intake arrays show regulation around a diffuse protein target range
(ca. 26- 34% of dietary intake, 0.024-0.033 g day-1). Urchins in treatments with noncomplementary diet combinations were prevented from reaching this target range but still
preferentially consumed more of the less extreme food in the respective diet combination,
indicating nutrient recognition and an attempt to regulate protein intake. This study is
among the first to utilize the GF to examine feed intake strategies in benthic marine
generalists and we believe that data from these studies contribute to existing
understanding of feed intake strategies of an extreme generalist. The sea urchin is of
particular interest due to its role and apparent success in marine communities for millions
of years, and has evolved a feeding and nutrient storage strategy to tolerate frequent bouts
of food limitation.
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INTRODUCTION
Darwinian fitness is proposed to be increased among individuals that have the
ability to regulate food intake (Westoby, 1978). However, key factors that motivate
organisms to regulate dietary intake are not entirely understood. Several theories which
attempt to explain intake strategies based on various intake parameters and ecosystem
interactions are in place. Optimal foraging theory (OFT) suggests that animals will alter
intake patterns to optimize energy acquisition, thus increasing fitness (Emlen, 1966;
Pianka, 1966). Optimal foraging theory considers trade-offs involving the energy
acquired from a particular food item and the energy required to attain that food item, with
consideration also given to predation. Although fulfillment of energy requirements is
important for all organisms, OFT is likely too simplistic to fully explain the driving
forces that govern nutrient intake among organisms: Most animals require very specific
macro and micronutrients for proper physiological function (Simpson and Raubenheimer,
2012) and few of these requirements are satisfied through energy intake alone. Therefore,
other motivating factors must also exist.
An alternate theory, ecological stoichiometry (ES), considers the balance of both
energy and nutrient intake and examines its effect on an organism and on the organism’s
interaction within the ecosystem (Sterner and Elser, 2002). Ecological stoichiometry
utilizes an elemental level (as opposed to macro and micronutrient level) approach to
explain intake patterns and is based on the hypothesis that an organism will thrive on a
diet with elemental composition similar to that of its own body tissues (Raubenheimer et
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al., 2009). Ecological stoichiometry attempts to explain why elemental imbalances exist
in feed intake patterns and what consequences they may impose upon a biological system
(Sterner and Elser, 2002). While ES often provides a more parsimonious explanation of
feed intake strategies than OFT, it neglects to consider that structurally similar molecules
may be functionally distinct and, consequently, be utilized differentially (Raubenheimer
et al., 2009).
A third nutritional framework, the geometric framework (GF), is useful in
understanding feed intake strategies and the consequences of differential nutrient intake
on fitness within an ecosystem. Geometric framework demonstrates the relationship
between one or more food components (macro and micronutrients) within a geometric
space and considers the impact of an organism’s intake strategy on performance and
fitness outcomes (Raubenheimer et al., 2009). Under the GF, self-regulation of feed
intake patterns allows individuals to reach an ‘intake target’ for one or more nutrients
and/or make nutritional ‘trade-offs’ (in the form of over or under-ingestion of nutrients)
to optimize nutrition and minimize long-term consequences. Such regulation presumably
leads to nutrient optimization and increased Darwinian fitness under a particular set of
environmental conditions (Simpson and Raubenheimer, 2012).
Strategies for regulation of feed intake and acquisition of intake targets differ
between generalist and specialist feeders (Lee et al., 2003). Generalist feeders have a
higher degree of dietary heterogeneity than specialists (Lee et al., 2003) and will eat a
variety of foods. As compared to specialists, generalists also have increased flexibility to
deviate from one or more nutrient intake targets and may utilize a wider variety of forage
(Simpson et al., 2002). Among organisms that periodically struggle to find food, adoption
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of a generalist feeding strategy may have evolutionary origin: Dependence on the
availability of one particular food is reduced and the likelihood of encountering other
edible items is increased (Raubenheimer and Jones, 2006), leading to an increase in
fitness.
Most sea urchins are generalist feeders (Meidel and Scheibling, 1999). However,
despite the heterogeneous nature of their diet, some species are often food limited in the
wild (Dix, 1970; Lawrence, 2013). Failure to frequently encounter food items has
potentially dire consequence in many organisms but may be of reduced concern for sea
urchins, especially adults. Urchins store nutrients in the form of protein, glycogen and
lipid in their tissues (primarily in the gonad but also in the gut and test) and retrieve and
utilize accumulated nutrients to survive extended (usually seasonal) periods of food
limitation (Lawrence and Lane, 1982). When food availability is high, urchins may
choose food items that optimize their current nutritional status and also provide additional
nutrients/energy for storage and subsequent utilization during periods of nutrient
restriction (Chapter 4). We hypothesize such an organism would become more selective
when levels of stored nutrients were high and would, thus, become more focused on
intake targets for select nutrients. Data from a recent study indicate that well-fed adult L.
variegatus (high levels of stored nutrients) regulate the intake of individual nutrients,
specifically protein (Chapter 4).
Food-limited juvenile urchins in an exponential growth phase may be obligated to
employ different feed intake strategies than their well-fed adult counterparts. Juvenile sea
urchins are faced with the challenge of obtaining and consuming adequate nutrients for
maintenance, growth and gonad production whereas nutrient requirements among adult
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sea urchins will be primarily limited to maintenance (both somatic and reproductive).
Young sea urchins may face additional obstacles in that they have very little to no gonad
tissue for nutrient storage and thus, cannot rely heavily on nutrient reserves during times
of food limitation. Consequently, we hypothesize they may be less discerning in food
choice than well-fed adults. For a generalist with high capacity for nutrient storage, a
more effective strategy may be that of an extreme (Raubenheimer and Jones, 2006)
generalist. An organism utilizing an extreme generalist strategy will eat to satisfy intake
targets for one nutrient and in doing so, may consume more than the intake target for
another nutrient (Raubenheimer and Jones, 2006). Hypothetically, such an individual
would store a supply of the excess nutrient for later utilization when food resources were
limited. Among juvenile sea urchins, Darwinian fitness could be expected to increase
among individuals that utilize an extreme generalist feed intake strategy. Variation in
feeding strategy at different lifestages is documented in other species (Simpson et al.,
2002) and seems reasonable as physiological needs (and nutrient stores) are expected to
change throughout the lifetime of an individual.
In this study we used the GF to assess feed and macronutrient intake in wildcaught pre-gonadal juveniles of the sea urchin L. variegatus. Sea urchins are widely
considered to be important macro-invertebrates inhabiting marine habitats, and often
influence community structure via their population structure and feeding activities. As
such, their population structure is often dependent on the availability of nutrients.
Conversely, their population structure can also influence the availability of nutrients and
even habitat for themselves and for other organisms in that community. To better
understand nutritional acquisition strategies, we employed a controlled approach which
162
utilizes reliable and precise feeding protocols (Watts et al. 2010) not generally available
in a number of aquatic species. This approach was used to determine nutrient intake
strategies in juvenile L. variegatus. These studies will provide a framework of knowledge
for further understanding of sea urchin nutritional ecology and evolution of feeding
strategies. Additionally, we seek to add to existing knowledge of variations in feeding
strategies that may be employed by generalist feeders, specifically those with a high
capacity for nutrient storage.
MATERIALS AND METHODS
Collection and Initial Dissection
Juvenile (ca. 11±0.9g) Lytechinus variegatus were collected from Saint Joseph
Bay, FL (30N, 85.5W) in March, 2014 and transported to the University of Alabama at
Birmingham in aerated coolers. The following week, eight urchins were randomly
selected for initial evaluation. Urchins were weighed to the nearest mg and dissected by a
circular incision around the peristomial membrane. The gonads were removed (they were
minimal or absent at this size class) and the gut (esophagus, stomach, and intestine
combined) was removed and cleaned in seawater to eliminate food pellets. The cleaned
gut, test and Aristotle’s lantern were rinsed in deionized water to remove salt. Organs
were blotted on a clean paper towel and weighed to the nearest mg. Organs were dried at
50°C for 72 hours to constant weight, and dry weights were recorded. Mean dry organ
and total dry weights (the sum of the organ dry weights) were calculated for the initial
sub-sample and used as estimated initial dry organ and total dry weights for the
remaining 168 urchins held in the trial. Remaining urchins were weighed to the nearest
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mg and assigned randomly to 1 of 2 experiments, comprising a total of 21 dietary
treatments (n=8 per treatment).
Culture Conditions
Urchins were placed individually into plastic, cylindrical cages (ca. 8.5 cm
diameter, 25 cm high, with 3 mm open mesh on sides, a 3 mm open mesh bottom secured
by plastic cable-ties, and a 2 mm open mesh circle over-laid on bottom). The mesh cages
were fitted into 8.7 cm ID PVC couplings. Thirty-three or thirty-four cages were
randomly placed in each of five fiberglass raceways (235 cm x 53 cm x 31 cm, L x W x
H, Fig. 1, as described by Taylor (2006). Empty cages were placed in each raceway to
bring the total number of cages to thirty- five per raceway. Addition of the empty cages
ensured even water flow throughout. A 160 x 23 cm (L x H) baffle in the center of each
raceway allowed for recirculating water flow by an in-line utility pump (Supreme® Mag
Drive Utility Pump, Danner™ Manufacturing, Inc., Islandia, NY, USA, 700 gallons of
water/hour, Fig. 1; Taylor 2006). The utility pump removed saltwater from the raceway
on one side of the baffle. Water then passed through a mechanical and biological filter
and returned to the raceway on the opposite side of the baffle. The flow rate of the
resulting current was approximately 9.7 – 12.6 cm s-1. Water depth was maintained at
15.0 cm. The floor of each cage was ca. 5.5 cm from the bottom of the raceway. Each
cage was fitted on the bottom with three small Tygon® spacers (ca. 0.5 cm thick) to allow
water circulation underneath. Cages were coded so that each individual could be tracked
over the course of the study. In these cages, feed was retained, but feces fell through the
mesh. Cages were rotated within and between raceways weekly to prevent tank or
position effects.
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Figure 1. Schematic of recirculating system. A) Side view of one of the five fiberglass
raceway (235 cm x 53 cm x 31 cm, L x W x H) with a 160 x 23 cm (L x H) center baffle
and 35 individual flow-through cages. B) Top view of one fiberglass raceway (schematic
is not drawn to scale. Arrows indicate water flow).
165
Total ammonia nitrogen, nitrite, nitrate, pH and alkalinity levels were checked
weekly using saltwater test kits from Aqua Pharmaceuticals, LLC (Malvern, PA, USA)
for ammonia and nitrogen and La Motte Company (Chestertown, MD, USA) for
alkalinity. Photo-period and water temperature were held constant (12:12 light: dark,
23±1 °C).
Feed and Feed Preparation
Five semi-purified diets were formulated and produced using both purified and practical
ingredients (Table 1). Levels of dietary protein and carbohydrate (Table 2) ranged from
10 to 42 % protein (using a purified protein source) and 13 to 42% carbohydrate (using a
purified starch source). Protein and carbohydrate were exchanged reciprocally (by
weight) and all other ingredients remained constant among treatments. Due to the
presence of lipid in some protein sources utilized, lipid levels varied slightly among diets.
Dry ingredients were mixed with a PK twin shell® blender (Patterson-Kelley Co., East
Stroudsburg, PA) for 10 minutes. Dry ingredients were then transferred to a Hobart stand
mixer (Model A-200, Hobart Corporation, Troy, OH) and blended for 40 minutes. Liquid
ingredients were added, and the mixture was blended for an additional 10 minutes to a
mash-like consistency. The diets were extruded using a meat chopper attachment (Model
A-200, Hobart Corporation, Troy, OH) fitted with a 4.8 mm die. Feed strands were
separated and dried on wire trays in a forced air at room temperature (22°C) for 48 hours.
Final moisture content of all feed treatments was 8–10%. Feed was stored in air-tight
storage bags at 4°C until used. Feeds were dried once more for 12 hours at 30°C and
were ground in a coffee grinder to a fine grind (≤300 µm) prior to inclusion in the agarbased gel diets.
166
Table 1. Calculated nutrient levels on an “as fed” basis for the base experimental diet.
*Empirically derived levels by Eurofins Scientific, Inc.
Nutrients
Crude Protein*
Carbohydrate
Crude Fiber*
Total Ash*
Crude Fat*
Cholesterol
Carotenoid
Calcium*
Phosphorus*
Sodium
Potassium
Magnesium
Iron
Zinc
Manganese
Copper
Selenium
Arginine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Cystine
Phenylalanine
Tyrosine
Valine
Vitamin A
Vitamin D
Vitamin E
Vitamin C
Thiamine
Riboflavin
Pyridoxine
Niacin
Pantothenic Acid
Biotin
Inositol
Choline
Folic Acid
Vitamin B12
Base
Experimental Feed
10-42%
42-13%
2.5%
23.53%
6.55-7.78%
0.32%
0.97%
4.02%
1.99%
1.29%
1.34%
0.41%
327 ppm
92.7 ppm
83.0 ppm
57.8 ppm
0.413 ppm
2.08%
0.74%
1.33%
2.36%
1.91%
0.57%
0.29%
1.47%
1.20%
1.38%
4800 IU
3000 IU
241 ppm
8-921 ppm
36 ppm
48 ppm
96.3 ppm
99.3 ppm
36.5 ppm
0.971 ppm
128 ppm
487 ppm
24.0 ppm
0.181 ppm
**All feeds contain approximately 4 parts animal ingredients, 28 parts marine
ingredients, 29.1 parts plant ingredients, 0.5 parts crude fat, 1.7% carotenoids, 0.7%
vitamin premix, 21.76% mineral premix, and 4.2% binder + antioxidant.
167
Fifteen moist diets (Table 2) were prepared using non-nutritive agar (Sigma
A7002) and the ground feed. Diets were prepared by boiling agar (final concentration of
1.5%) in synthetic seawater (34 ppt). After boiling, the agar solution was allowed to cool
to 40° C and then combined with dry feed in concentrations indicated in Table 2,
resulting in fifteen diets with one of five protein (10, 18, 26, 34, or 42% dry matter) and
carbohydrate concentrations (13, 22, 31, 40 or 49% dry matter) at one of three (4, 8 or
12%) food concentrations (representing three diet dilutions) within each of the nutrient
combinations. This diet emulsion was poured into a baking pan (27.5 x 17.5 cm, L x W)
and placed on the surface of an ice bath (ca. 0°C) to solidify. The gel was refrigerated
overnight and then the solidified preparation was cut into cubes (ca. 3.2 x 3.2 x 1.5 cm, L
x W x H) and stored in air tight containers at 4º C to prevent water evaporation and
minimize bacterial growth. Diets were made fresh every four days.
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Table 2. Calculated protein (% dry matter, lipid from animal sources removed),
carbohydrate (% dry matter) and lipid (% dry matter, including lipid from animal
sources) levels in each of the five feeds tested and non-nutritive ingredients and food
concentrations of moist diets prepared.
Nutritive Ingredients
Diet
4% 10:49
8% 10:49
12% 10:49
4% 18:40
8% 18:40
12% 18:40
4% 26:31
8% 26:31
12% 26:31
4% 34:22
8% 34:22
12% 34:22
4% 42:13
8% 42:13
12% 42:13
Protein Carbohydrate
(%)
(%)
10
49
10
49
10
49
18
40
18
40
18
40
26
31
26
31
26
31
34
22
34
22
34
22
42
13
42
13
42
13
Lipid
(%)
6.55
6.55
6.55
6.85
6.85
6.85
7.16
7.16
7.16
7.47
7.47
7.47
7.78
7.78
7.78
Non-Nutritive Ingredients
Concentration
Agar
Synthetic
of Feed in Diet
(%)
Seawater (%)
(%)
1.5
94.5
4
1.5
90.5
8
1.5
86.5
12
1.5
94.5
4
1.5
90.5
8
1.5
86.5
12
1.5
94.5
4
1.5
90.5
8
1.5
86.5
12
1.5
94.5
4
1.5
90.5
8
1.5
86.5
12
1.5
94.5
4
1.5
90.5
8
1.5
86.5
12
Two experiments were performed concurrently. The first (exp. 1) was a no-choice
experiment in which urchins were offered one of 15 single diets that differed in protein
and carbohydrate concentration and in feed dilution (Fig. 2). Differences in nutrient
concentration (protein: carbohydrate ratio) permitted evaluation of intake targets for
dietary protein or carbohydrate. As nutrient intake balance could not be adjusted within
the respective diets, urchins were expected to adjust feed intake amounts to prioritize
either a protein or a carbohydrate intake target. Food concentration (4, 8 or 12%) within
the respective diets differed to determine whether urchins would detect differences in
nutrient density within the respective diets and correspondingly, adjust intake to reach
prospective intake targets. The second (exp. 2) was a choice experiment whereby
169
individual urchins were offered pairwise diet combinations, all of which contained 8%
food concentration in the gel cube (Figure 2). For both experiments, cubes representing
each diet were weighed individually prior to feeding. For individuals receiving pairwise
combinations (exp. 2) diet cubes were offered simultaneously to an individual urchin and
were placed side by side in cages in direct contact and equal proximity to the urchin,
enabling urchins to readily sample and consume either or both of their diets. The edge of
one diet cube in each combination was clipped (on alternate days) for identification. To
avoid positional bias, the location of each diet was alternated daily. In both exp. 1 and
exp. 2, the remaining portions of the cubes representing each diet were recovered after 24
hours, rinsed in deionized water, blotted and re-weighed to calculate diet and feed intake
(by subtraction). Leaching of ingredients (estimated by weight loss or gain of the food
cube in the absence of an urchin) in the 24 hour period was determined to be negligible.
Total feed and specific nutrient intake from each diet (exp. 1 and 2) and the
combined total nutrient intake from each pairwise combination (exp. 2) were recorded
daily over a 43-day period.
Daily intake of each diet was calculated as:
(1) Initial weight of food cube (g, as fed) – final weight of food cube (g, as fed)
Cumulative intake of each diet was calculated as:
43
(2) Σ f(i) Where f(i)= total weight of food cube (g) consumed per day
i=1
Protein intake per diet per day was calculated as:
170
(3) Total weight (g) of food cube consumed x food concentration (4, 8 or 12%) x
[protein concentration of diet - lipid content of animal protein sources]
For individuals offered pairwise diet combinations (exp. 2), the sum of protein intake
from both diets was calculated singularly and in combination.
Cumulative protein intake was calculated as:
43
(4) Σ f(i) Where f(i)= total protein consumed per day (g)
i=1
Carbohydrate intake per day was calculated as:
(5) Total weight (g) of food cube consumed x food concentration (4, 8 or 12%) x
carbohydrate concentration of diet
For individuals offered pairwise diet combinations (exp. 2), the sum of carbohydrate
intake from both diets was used.
Cumulative carbohydrate intake was calculated as:
43
(6) Σ f(i) Where f(i)= total carbohydrate consumed per day (g)
i=1
Lipid intake per day was calculated as:
(7) Total weight (g) of food cube consumed x food concentration (4, 8 or 12%) x
[lipid concentration of diet + lipid contributed from animal protein sources]
For individuals offered pairwise diet combinations (exp. 2), the sum of lipid intake from
both diets was used.
Cumulative lipid intake was calculated as:
171
43
(8) Σ f(i) Where f(i)= total lipid consumed per day (g)
i=1
Total Energy Intake was calculated as:
(9) Total protein consumed (g) x 5650 + total carbohydrate consumed (g) x 4000
+ total lipid consumed (g) x 9450
Caloric values were obtained from Phillips (1972).
A.
6
10:49
5
Carbohydrate (g)
18:40
4
26:31
3
34:22
2
1
42:13
0
0
1
2
3
Protein (g)
4
5
6
172
B.
6
10:49
5
Carbohydrate (g)
18:40
4
26:31
3
34:22
2
1
42:13
0
0
1
2
3
4
5
6
Protein (g)
Figure 2: No choice diets and pairwise diet combinations. A. No choice diets offered to
urchins in experiment 1. Each trajectory represents a single nutrient rail (single feed).
Gray boxes indicate macronutrient ratios of the respective diets (i.e. 42:13 represents the
feed with 42% protein and 13% carbohydrate). For each feed, diets were offered at 4, 8
and 12% feed concentration for a total of 15 diet treatments. B. Pairwise diet
combinations offered to urchins in experiment 2. Diagonal colored lines connect diet
combinations offered and indicate nutrient space for those diet combinations. Each
trajectory represents a single nutrient rail (single feed). Gray boxes indicate
macronutrient ratios of the respective diets (i.e. 42:13 represents the feed with 42%
protein and 13% carbohydrate)
Statistics
For exp. 1 and 2, statistical analyses of macronutrient intake were performed in
IBM SPSS Statistics 22. P values ≤ 0.05 were considered significant. Data were tested for
173
normality and homoscedasticity using Shapiro-Wilk and Levene’s tests, respectively.
Data were found to be normal and equal in variance and were analyzed with ANCOVA
and MANCOVA using Pillai’s trace statistic and with ANOVA. If a significant
difference was detected, a Tukey’s HSD test was used to compare means. Outcomes
reported included total mass of gel-based diet consumed, feed intake (dry matter), total
protein intake, total carbohydrate intake, total lipid intake (all macronutrient intake
reported on dry matter basis), and total energy intake.
Results
Experiment 1, No-Choice Experiment
Diet and Feed Intake
Among urchins in exp. 1, total diet intake (dry feed plus water and agar binding
matrix) varied significantly with food concentration for most treatments (Fig. 3). With the
exception of urchins in the 4% 10:49 and 4% 34:22 treatments, total diet intake increased
among urchins fed diets with 4% food concentration, as compared to those offered the
same feed at 8% concentration in the diet (p≤ 0.01, Fig. 3).
Figure 4 shows nutrient rails created by the diets and nutrient intake points for sea
urchins receiving each diet at the respective food concentration. As urchins in exp. 1 were
limited to a single diet, all nutrient intake points were restricted to the nutrient rail created
by that diet regardless of food concentration (Fig. 4). Diet intake (as fed) at 4% food
concentration was generally higher than at 8 and 12%; however, total nutrient (protein
and carbohydrate) intake was lower at 4% compared to 8 and 12% food concentration
(Fig. 4). We hypothesize that total mean diet intake (Fig. 3) and total mean macronutrient
intake (Fig. 4) indicate that urchins in the 4% food concentrations (with the exception of
174
the 4% 10:49 diet) most likely reached volume satiation and, thus, were unable or not
stimulated to consume sufficient dietary nutrients due to nutritional dilution.
Consequently, data for urchins receiving diets with 4% food concentration were removed
from further analysis in exp. 1.
Some variation between nutrient intake values at 8 and 12% was observed;
however, ANOVA indicated no interaction between food concentration and diet, thus,
values could be averaged for further analysis (Fig. 5). Ratio of average protein:
carbohydrate intake of urchin fed diets with 8 and 12% food concentrations was
equivalent among all treatments within each nutrient rail. The slope of the line (slope = 0.84; R2= 0.998) representing the continuum of protein: carbohydrate intake ratio for
each diet (8 and 12% diets averaged for each nutrient combination) is less than one (Fig.
5), indicating that the error for the intake of carbohydrate was greater than the error for
protein intake. Dry matter intake was similar at 8 and 12% food concentration (Fig. 6).
As expected, intake targets for dietary protein or carbohydrate are not discernable in the
intake array observed in Figure 4.
175
120
G
100
FG
FG
Total Mean Diet Intake (g)
DEF
80
CDE BCD
DE
CDE
CD
ABCD
60
AB
ABC
A
A
A
40
20
0
Diet and Concentration
Figure 3: Total mean diet intake (g, as fed, includes feed plus water and agar binding
matrix, ±SEM) of protein: carbohydrate diets fed at 4, 8 and 12% food concentration
among urchins in exp. 1. Differential capital letters designate significant differences
among diet treatments.
176
4% feed concentration
4.0
Total Mean Carbohydrate Intake (g)
8% feed concentration
10: 49
18: 40
12% feed concentration
26: 31
3.0
34: 22
2.0
42: 13
1.0
0.0
0.0
1.0
2.0
3.0
Total Mean Protein Intake (g)
4.0
Figure 4: Total mean macronutrient intake points (g, ±SEM) among urchins in exp. 1.
Solid lines represent nutrient rails created by each of the diets. Intake points associated
with respective diets lie along the rails.
177
4.0
Average Total Mean Carbohydrate Intake (g)
10:49
3.5
10:49
18:40
26:31
18:40
3.0
34:22
26:31
42:13
2.5
B
2.0
1.5
34:22
A
42:13
1.0
0.5
0.0
0.0
1.0
2.0
3.0
4.0
Average Total Mean Protein Intake (g)
Figure 5: Average total mean macronutrient intake points (g, ±SEM) among urchins fed
diets with 8 and 12% food concentration in exp. 1. Solid black lines represent nutrient
rails created by each of the diets. Solid blue line represents slope of intake points among
respective diets. Dashed orange lines represent average error in protein (A) and
carbohydrate (B).
178
Total Mean Dry Feed Intake (g)
8
AB
7
6
B
AB
AB
AB
AB
A
AB
AB
AB
5
4
3
2
1
0
Diet Treatment
Figure 6: Total mean dry feed intake (g, dry matter, no water or agar binding matrix,
±SEM) among exp. 1, 8 and 12% food concentrations. Differential capital letters
designate significant differences among diet treatments.
Macronutrient and Energy Intake
Total mean dietary protein (p≤ 0.05) and total mean dietary carbohydrate (p≤
0.05) intake varied with level of inclusion in the feed (Fig. 7). However, protein and
carbohydrate intake did not vary with food concentration (8 vs 12%) within any diet.
Figure 8 shows the ratio of total mean protein energy intake to total mean nonprotein energy intake (PE: NPE). Some difference between nutrient intake values at 8 and
12% in the 18:40 diet was observed; however, ANOVA indicated no interaction between
food concentration and diet, thus, values could be combined for further analysis. Ratio of
average PE: NPE carbohydrate intake of urchin fed diets with 8 and 12% food
concentrations was equivalent among all treatments within each nutrient rail. The slope
of the line (slope = -0.78; R2= 0.994) representing the continuum of PE: NPE intake ratio
179
for each diet (PE and NPE for 8 and 12% diets averaged for each nutrient combination) is
less than one (Fig. 9), indicating that the error for the intake of PE was greater (higher
variability) than the error for NPE intake (lower variability).
Total Mean Macronutrient
Consumption (g)
4.0
E
3.5
3.0
E
D
2.5
CD
d
1.0
bc
a
e
C
C
2.0
1.5
f
f
e
d
B
c
B
A
ab
A
0.5
0.0
Diet Treatment
Total mean carbohydrate intake (g)
Total mean protein intake (g)
Figure 7: Total mean protein (dark bars) and carbohydrate (pale bars) intake (g, ±SEM)
among urchins in exp. 1, 8 and 12% food concentrations. Differential lower case letters
designate significant differences in protein intake among diet treatments. Differential
capital letters designate significant differences in carbohydrate intake among diet
treatments.
180
Total Mean Non-Protein Energy Intake (cal)
18000
8% Feed Concentration
12% Feed Concentration
16000
14000
18: 40
10: 49
26: 31
12000
34: 22
10000
42: 13
8000
6000
4000
2000
0
0
2000 4000 6000 8000 10000 12000 14000 16000 18000
Total Mean Protein Energy Intake (cal)
Figure 8: Total mean protein to non-protein energy intake (cal, ±SEM) among urchins in
exp. 1. Solid lines represent nutrient rails created by each of the diets. Intake points lie
along the rail created by the respective diets. Solid circles designate mean intake for
urchins fed diets with 12% food concentration. Dotted triangles designate mean intake for
urchins fed diets with 8% food concentration.
181
18000
Total Mean Non-Protein Energy Intake (cal)
16000
42:13
14000
34:22
12000
R² = 0.994
10000
26:31
B
18:40
8000
A
6000
10:49
4000
2000
0
0
5000
10000
15000
20000
Total Mean Protein Energy Intake (cal)
Figure 9: Average total mean protein to non-protein energy intake (cal, ±SEM) among
urchins fed diets with 8 and 12% food concentration in exp. 1. Solid black lines represent
nutrient rails created by each of the diets. Solid blue line represents slope of intake points
among respective diets. Dashed orange lines represent average error in protein energy (A)
and non-protein energy (B).
Experiment 2, Choice Experiment
Diet and Feed Intake
Figure 10 shows nutrient rails and intake points for urchins in exp. 2. Urchins in
each diet combination consumed both diets (Fig. 11). Urchins in the 42:13/26:31,
42:13/18:40 and 34:22/18:40 diets demonstrated a loose intake target range for dietary
182
protein (Fig. 10). Within most diet combinations, urchins preferentially consumed more
of the diet with the least extreme nutrient ratios (e.g. urchins in the 42:13/26:31 favored
the 26:31 diet, p≤ .001). However, collective feed intake of both wet (as fed, Fig. 12) and
dry matter (Fig. 13) for each diet combination was equivalent across all diet
combinations.
Macronutrient and Energy Intake
Both total mean protein and total mean carbohydrate intake (Fig. 14a and b)
varied with level of dietary inclusion. Trends indicate that urchins with high protein or
high carbohydrate diet combinations consumed less of the macronutrient in excess than
would be expected if equivalent amounts of each diet had been consumed at random
(equivalent consumption is shown as hypothetical intake estimates, Fig. 14). Likewise,
urchins with low protein or low carbohydrate diet combinations consumed more of the
deficient macronutrient than would be expected (Fig. 14). Combined total mean protein
plus carbohydrate intake did not vary among diet combinations (Fig. 15). Total mean
lipid intake and total energy intake did not vary across diet combinations (Fig. 16 and 17,
respectively).
Comparison of PE: NPE intake indicated that energy source varied among the
respective diet combinations (Fig. 18). Among all diet combinations, PE: NPE intake
varied with levels of protein and carbohydrate inclusion in the diets (Fig. 18). Among
urchins in diet combinations that allowed acquisition of intake targets for protein and
carbohydrate, PE: NPE intake was more closely balanced than that of urchins in other
(more extreme) diet combinations.
183
42:13/34:22
Average Total Mean Carbohydrate Intake (g)
4.0
42:13/ 26:31
18:40
3.5
42:13/18:40
10:49
26:31
34:22/18:40
3.0
26:31/10:49
18:40/10:49
2.5
34:22
2.0
1.5
42:13
1.0
0.5
0.0
0.0
1.0
2.0
3.0
4.0
Average Total Mean Protein Intake (g)
Figure 10: Total mean macronutrient intake points for urchins in exp. 2. Solid black lines
represent nutrient rails created by each of the diets (labeled, respectively). Dotted colored
lines indicate hypothetical nutrient rails along which urchins in each treatment would
have eaten if diet intake had been indiscriminate (no choice made). Solid circles in
corresponding colors indicate actual intake points for urchins in each treatment. Error
bars represent SEM. Blue oval encircles range of intake targets for protein and
carbohydrate.
184
70
Total Diet Intake (g)
B
B
50
40
B
B
60
A
A
A
A
A
A
A
A
30
20
10
0
42: 13/34: 22
42: 13/ 26: 31
42: 13/18: 40
34: 22/18: 40
26: 31/10: 49
18: 40/10: 49
Diet Combination
Figure 11: Total mean intake (g, as fed, includes feed plus water and agar binding matrix,
±SEM) of protein: carbohydrate diets within the respective diet combinations among
urchins in exp. 2. Pale bars represent the lowest protein diet within the combination. Dark
bars represent the highest protein diet within the combination. Differential capital letters
designate significant differences within but not among diet combinations.
185
120
Total Mean Diet Intake (g)
100
A
A
A
A
A
26:31/10:49
18:40/10:49
A
80
60
40
20
0
42:13/34:22
42:13/26:31
42:13/18:40
34:22/18:40
Diet Combination
Figure 12: Total mean diet intake (g, as fed, includes feed plus water and agar binding
matrix, ±SEM) among urchins in exp. 2. Capital letters designate significant differences
among diet combinations.
186
Total Mean Dry Feed Intake (g)
9
8
7
A
A
42:13/34:22
42:13/26:31
A
A
A
26:31/10:49
18:40/10:49
A
6
5
4
3
2
1
0
42:13/18:40
34:22/18:40
Diet Combination
Figure 13: Total mean dry feed intake (g, dry matter, no water or agar binding matrix,
±SEM) among urchins in exp. 2. Capital letters designate significant differences among
diet combinations.
A
Actual vs. Hypothetical Protein Intake
(g)
187
3.0
D
C
2.5
C
2.0
B
B
1.5
A
1.0
0.5
0.0
42:13/34:22 42:13/26:31 42:13/18:40 34:22/18:40 26:31/10:49 18:40/10:49
Diet Combination
B
Actual vs Hypothetical Carbohydrate
Intake (g)
Actual
Hypothetical
4.0
3.5
D
D
3.0
2.5
C
2.0
1.5
C
B
A
1.0
0.5
0.0
42:13/34:22 42:13/26:31 42:13/18:40 34:22/18:40 26:31/10:49 18:40/10:49
Diet Combination
Actual
Hypothetical
Figure 14: Actual total mean protein (A) and carbohydrate (B) intake among urchins in
exp. 2 vs. hypothetical intake expected if urchins had eaten equivalent amounts of each
diet within a diet combination. Pale bars represent actual intake (±SEM). Dark bars
represent hypothetical intake. Capital letters designate significant differences in protein
(A) or carbohydrate (B) intake among diet combinations.
188
Combined Protein + Carbohydrate
Intake (g)
5.0
4.5
A
A
4.0
A
A
A
A
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
42:13/34:22
42:13/26:31
42:13/18:40
34:22/18:40
26:31/10:49
18:40/10:49
Diet Combinations
Figure 15: Combined total mean protein + total carbohydrate intake (±SEM) among
urchins in exp. 2. Capital letters designate significant differences among diet
combinations.
0.6
A
A
A
A
26:31/10:49
18:40/10:49
A
0.5
Total Lipid Intake (g)
A
0.4
0.3
0.2
0.1
0
42:13/34:22
42:13/26:31
42:13/18:40
34:22/18:40
Diet Combination
Figure 16: Total mean lipid intake (g, ±SEM) among urchins in exp. 2. Capital letters
designate significant differences among diet combinations.
189
30000
A
Total Energy Intake (cal)
25000
A
A
A
A
A
20000
15000
10000
5000
0
42: 13/34: 22 42: 13/ 26: 31 42: 13/18: 40 34: 22/18: 40 26: 31/10: 49 18: 40/10: 49
Diet Combination
Figure 17: Total mean energy intake (cal, ±SEM) among urchins in exp. 2. Capital letters
designate significant differences among diet combinations.
190
42:13/34:22
25000
Total Mean Non-Protein Energy Intake (cal)
42:13/ 26:31
42:13/18:40
26:31
20000
34:22/18:40
18:40
10:49
26:31/10:49
18:40/10:49
15000
34:22
10000
42:13
5000
0
0
5000
10000
15000
20000
25000
Total Mean Protein Energy Intake (cal)
Figure 18: Total mean protein to non-protein energy intake (cal, ±SEM) among urchins in
exp. 2. Solid black lines represent nutrient rails created by each of the diets. Intake points,
represented by colored circles, lie between nutrient rails created by the respective diet
combinations. Colored diagonal arrows connect diet combinations for the respective
treatments. Blue oval encircles treatments in which urchins demonstrated intake targets
for protein and carbohydrate.
Experiment 1 and 2, Combined Data
Figure 19 shows total mean protein and total mean carbohydrate intake for
urchins fed diets with 8% food concentration in exp. 1 and for urchins in all diet
combinations in exp. 2 (reminder, all diets in combination were at 8% food
concentration). Visualization of combined data allows us to establish that both the 8%
26:31 and the 8% 34:22 diets confined urchins to nutrient rails that were within the target
191
area for protein and carbohydrate (Fig. 19). Notably, urchins in exp. 2 consumed more
total dry matter (feed) and, consequently, 20% more energy (cal) from protein and
carbohydrate than urchins in exp. 1 fed diets with 8% food concentrations (Fig. 20).
42:13/34:22
42:13/ 26:31
42:13/18:40
34:22/18:40
26:31/10:49
18:40/10:49
8% 42: 13
8% 34: 22
8% 26: 31
8% 18: 40
8% 10: 49
4.0
18:40
Average Total Carbohydrate Intake (g)
3.5
10:49
26:31
3.0
2.5
2.0
34:22
1.5
42:13
1.0
0.5
0.0
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
Average Total Protein Intake (g)
Figure 19: Total mean macronutrient intake points for urchins in exp. 1 and exp. 2. Solid
black lines represent nutrient rails created by each of the diets (labeled, respectively).
Dashed colored lines indicate hypothetical nutrient rails along which urchins in each
treatment in exp. 2 would have eaten if diet intake had been indiscriminate (no choice
made). Solid circles in corresponding colors indicate actual intake points for urchins in
each treatment in exp. 2. Solid squares indicate intake points for urchins in exp. 1 fed
diets with 8% food concentration. Error bars represent SEM. Solid blue diagonal line
represents average energy intake from protein and carbohydrate for urchins in exp. 1.
Blue oval encircles range of intake targets for protein and carbohydrate.
192
8% 10:49
25000
Total Mean Non-Protein Energy Intake (cal)
8% 18: 40
8% 26: 31
26:31
8% 34: 22
20000
18:40
10:49
8% 42: 13
42:13/34:22
42:13/ 26:31
15000
42:13/18:40
34:22
34:22/18:40
26:31/10:49
10000
18:40/10:49
42:13
5000
0
0
5000
10000
15000
20000
25000
Total Mean Protein Energy Intake (cal)
Figure 20: Total mean protein to non-protein energy intake (cal, ±SEM) among urchins in
exp. 1, 8% food concentrations and exp. 2. Solid black lines represent nutrient rails
created by each of the diets. Intake points for exp. 2, represented by colored circles, lie
between nutrient rails created by the respective diet combinations. Intake points for exp.
1, represented by colored squares, lie along the nutrient rail created by the respective diet.
Colored diagonal arrows connect diet combinations for the respective treatments in exp.
2. Blue oval encircles treatments in which urchins demonstrated intake targets for protein
and carbohydrate.
DISCUSSION
Experiment 1, No-Choice Experiment
Urchins in exp. 1 had free access to a single diet with different food concentration
and were restricted to intake along a single nutrient rail. Consequently, individuals could
193
regulate intake of any single nutrient but could not regulate nutrient balance. Feed intake
was reduced in the lowest protein diet (4% 10:49) as compared to the other diets with 4%
food concentration. The quality of the 4% 10:49 diet was poor (lacking in adequate
protein) and nutrient density was, most likely, outside the range of evolutionary
experience (lower than would typically be encountered) among urchins. Consequently,
this particular diet may not have served as an adequate food source and individuals in this
treatment may have searched to encounter an alternate, more suitable food. In addition,
we hypothesize that, at very low levels of protein and/or energy intake, the energetic cost
of processing the diet outweighed the benefit gained from eating. Among brown bears,
both excess dietary protein and protein deficiency resulted in increased maintenance
costs, which, in turn, led to a decrease in efficiency of energy utilization (Erlenbach et al.,
2014), suggesting that energy requirements may increase when intake targets for dietary
protein cannot be achieved. Dietary protein is important for growth and health among sea
urchins (Hammer et al., 2012; Heflin et al., 2012) and previous data indicate that adult
urchins will prioritize protein intake targets under some conditions (Chapter 4).
As total dry feed intake was regulated among diets with 8 and 12% food
concentration, but not among urchins fed diets with 4% concentration, it is presumed that,
for all other diets with 4% food concentration, urchins were unable to achieve intake
targets for dry feed and, as a consequence, specific nutrient targets. Nonetheless, urchins
continued to consume large volumes of food. This intake behavior suggests that under
conditions of limited food, urchins will shift intake strategies to that of an extreme
generalist. Among extreme generalists, individuals will eat inferior food to volume
satiation when a more suitable food is not available (Simpson et al., 2002). As some diets
194
with 4% food concentration appear to have been too dilute for urchins to reach intake
targets (4% 10:49), urchins fed these diets are omitted from further discussion.
Organisms restricted to a single nutrient rail cannot diversify their diet to reach
exact intake targets for multiple nutrients simultaneously. For organisms restricted to a
single unbalanced diet, acquisition of an intake target for a particular nutrient will only
occur if the organism adjusts volume of feed intake to reach that target (prioritizes that
nutrient). When nutrient balancing is impossible, organisms are forced to alter feeding
strategies: Specialists alter intake in such a way as to find the most tolerable balance
between nutritional ‘errors’ (distance of actual intake from the intake target,
Raubenheimer and Simpson, 1997). Generalists feeders have a higher degree of dietary
flexibility (Lee et al., 2003; Raubenheimer and Jones, 2006; Erlenbach et al., 2014) and
are better able to cope with nutritional error than their specialists counterparts
(Raubenheimer and Simpson 2003; Raubenheimer and Jones, 2006). Therefore, they are
more likely to utilize alternate strategies.
It was hypothesized that urchins in exp. 1 would prioritize the intake of one
nutrient (most likely protein, Chapter 4) and thus, would exceed or fall short of intake
targets for other nutrients depending on nutrient balance in the respective feeds. Slope of
the intake array (as demonstrated in Fig. 5) was not equivalent to -1, indicating that
urchins in the 8 and 12% food concentrations did not feed indiscriminately but exhibited
weak homeostatic macronutrient regulation typical of a generalist feeder (Raubenheimer
and Simpson, 2003). Negative linearity of the slope through the target nutrient space
shows interchangeability among the errors in protein and carbohydrate intake, but not in
protein or carbohydrate alone (Raubenheimer and Simpson, 2003). L. variegatus
195
demonstrated nutrient recognition and utilized the ‘fixed proportion’ regulatory pattern
(Raubenheimer and Simpson, 2003) to maintain a constant ratio of error in protein to
error in carbohydrate, regardless of whether protein or carbohydrate was in excess. Error
in dietary protein intake was maintained at a lower level than error in dietary
carbohydrate intake, indicting a tendency (qualitative observation) for protein leverage.
While lipid intake was similar among treatments, we do not think this was meaningful as
all diets contained similar lipid concentrations with only a small amount of variation
added from animal protein sources. As almost all urchins ate an equivalent amount of
feed, lipid intake would not be expected to vary significantly among treatments.
Total dry feed intake and PE: NPE ratio among urchins in the 8 and 12% food
concentrations first seems indicative of the ‘equal distance rule’ (Simpson and
Raubenheimer, 2002) rather than recognition and regulation of macronutrients. However,
urchins in the 8 and 12% dietary treatments could have consumed a greater volume of
food, and therefore, did not maximize total volume of dry feed or energy intake alone.
Data from a previous study utilizing the geometric framework do not show evidence of
volume or energy regulation among adult urchins (Chapter 4).
Urchins in exp. 1 consumed less dry feed than urchins in exp. 2, indicating
differential intake strategies. In most cases, urchins in exp. 1 either exceeded or fell short
of macronutrient targets. Among some generalists, excess nutrients will be stored to
compensate for a later deficit (Simpson et al., 2002). Such a strategy may offset the
consequences of a complementary imbalanced diet in the future and may allow the
organism to “jump” nutrient rails through differential nutrient utilization and reallocation
(Simpson and Raubenheimer, 1997). However, the organism will suffer costs associated
196
with over-consumption of the excess nutrients if it does not encounter a complementary
food in the future (Simpson et al., 2002).
Lytechinus variegatus is an opportunistic generalist feeder and may sometimes be
food limited in the wild (Watts et al. 2013). Despite food limitations, juveniles must find
adequate food for growth and reproductive effort. When forced to eat a sub-optimal diet,
juvenile urchins may often fail to encounter complementary foods and out of necessity,
may have evolved methods of mitigating the consequences of nutrient imbalance at
critical lifestages. Boersma et al. (2006) hypothesized that benthic organisms have
evolved superior mechanisms for obtaining and storing potentially limiting nutrients. It
seems reasonable to assume that organisms with superior storage capacity may also
experience reduced consequences of excess stores of a particular nutrient. As a benthic
organism, urchins have the somewhat unique ability to store nutrients without
considerable modification of proximate composition (e.g. protein will be stored as protein
in the form of major yolk protein, Unuma et al., 2010). Although biochemical analysis
will be required to identify differences in nutrient storage among urchins in exp. 1, we
suggest that, in the presence of a single food type, juvenile L. variegatus will eat most
food (provided it is not too energetically costly) and regulate error in dietary protein to
error in dietary carbohydrate (sometimes exceeding or falling short of macronutrient
targets) to optimize the ratio of macronutrient intake error as permitted by the respective
diets.
197
Experiment 2, Choice Experiment
L. variegatus in exp. 2 were offered pairwise diet combinations and were thereby
presented with the opportunity to balance macronutrient or energy intake within the
confines of the nutrient space provided by their respective diets. Energy intake and PE:
NPE ratios among urchins in exp. 2 appear indicative of an intake target for dietary
energy (utilizing the equal distance rule), as suggested by the optimal foraging theory.
However, comparison of data from exp. 1 and exp. 2 does not support this hypothesis.
Urchins in exp. 2 consumed more food and, on average, 20% more total calories than
urchins in exp. 1. Urchins in both experiments were from the same cohort and were
handled equally and stocked simultaneously in the same raceways. Caloric differences in
intake between exp. 1 and exp. 2 appear to be a result of total diet consumption rather
than a contribution of additional carbohydrate or protein intake by urchins in exp. 2 and
were dependent on the nutrient composition of the diets in each combination. In addition,
the reduced error associated with non-protein energy sources suggest these sources were
more tightly regulated than protein energy sources, suggesting a metabolic preference for
energy derived from carbohydrates and/or lipids. Combined, these data would suggest
that L. variegatus does not follow optimal foraging theory, choosing instead to consume
to macronutrient intake targets to maintain both nutrient and energy homeostatis.
Variation in intake patterns within diet combinations indicate macronutrient
recognition and regulation in L. variegatus. Individuals made choices between the
respective diets and generally preferred the diet with the most balanced protein:
carbohydrate ratio. Individuals in non-complementary (i.e. 18:40/10:49) diet
combinations were forced to make compromises in which some nutrient targets would be
198
exceeded and others would not be met. As with exp. 1, intake targets for protein and
carbohydrate were not immediately apparent for urchins in exp. 2. However, the intake
array shown in Fig. 10 does show regulation around a diffuse protein target range (ca. 2634% of dietary intake, 0.024-0.033 g day-1). Urchins in treatments with noncomplementary diet combinations were prevented from reaching this target range but still
preferentially consumed more of the less extreme food in the respective diet combination,
indicating nutrient recognition and an attempt to regulate protein intake. Only two diet
treatments, 34:22 and 26:31, in exp. 1 allowed urchins to reach intake targets for dietary
protein.
Previous data indicate that adult urchins that consumed formulated diets, when
provided with choice, will over or under-consume dietary carbohydrate to reach protein
intake targets (Chapter 4). Likewise, urchins in exp. 2 demonstrated greater variation in
dietary carbohydrate intake as compared to dietary protein. When urchins in exp. 2 were
provided choices that allowed them to reach target intake ranges for dietary protein, they
utilized the ‘equal distance rule’ (Simpson and Raubenheimer, 2001) to regulate intake of
protein and carbohydrate relative to one another within the diffuse target range as
permitted by the diets. This does not indicate that protein and carbohydrate are
interchangeable but rather it is a generalist strategy that facilitates maximization of total
nutrient intake up to a combined limit (Simpson and Raubenheimer, 2001). Other
generalists use the equal distance rule as a strategy when available food is sub optimal
but tightly regulate intake of both protein and carbohydrate when food choices permit
(Simpson and Raubenheimer, 2001). We suggest that at least one diet combination in
exp. 2 may have allowed urchins to tightly regulate intake targets for both protein and
199
carbohydrate but exact intake targets are not discernable due to equal distance regulation
in other treatments within the target range. Adult sea urchins demonstrate strict regulation
of protein intake with variable carbohydrate intake (Chapter 4). However, other
conspecifics demonstrate variation in intake strategy with different lifestage (Simpson et
al., 2002). Among L. variegatus, the requirements of juveniles are most likely different
from those of adults. In addition, the intake of other dietary nutrients (fats, minerals,
vitamins) may require further consideration. Therefore, when provided choice of diet that
allows intake within a certain range, juvenile urchins may choose a more diffuse target
for dietary protein to satisfy requirements for other nutrients.
Lack of variation in lipid intake among urchins in exp. 2 most likely occurred
passively as a result of diet intake regulation. Regardless, intake data from exp. 1 and 2
provide evidence that the importance of lipid in the diet cannot be minimized. Lipids are
required by sea urchins (Hammer et al., 2010) but total lipid requirements are low for L.
variegatus (Gibbs et al., 2009; 2015). Additional studies will be necessary to evaluate
intake targets for dietary lipid.
Understanding regulation of protein and carbohydrate intake is best facilitated by
experiments which measure intake across a wide range of nutrient rails and provide both
choice and no choice treatments (Simpson and Raubenheimer, 1997). The GF has proved
useful in analyzing feed intake strategies among both specialists and generalists. This
study is among the first to utilize the GF to examine feed intake strategies in benthic
marine generalists. We believe that data derived from these studies contribute to current
understanding of feed intake strategies of an extreme generalist. The sea urchin is of
particular interest due to its success in marine communities for millions of years, most
200
likely experiencing frequent bouts of food limitation and unique nutrient storage
capabilities. This species may have evolved the capacity to tolerate diffuse intake targets
as a mechanism to compensate for episodic food availability. Future analysis of
proximate composition of body tissues will provide information about differential
nutrient storage, which we presume occurred among urchins in both exp. 1 and exp. 2.
Additionally, analysis of growth and efficiencies among urchins held under choice and
no-choice feeding regimes will further our understanding of nutrient utilization under
restrictive conditions as well as conditions which allow individuals to achieve intake
targets for particular nutrients.
201
Acknowledgements
The authors thank R. Jeffery Barry, J. Christopher Taylor, Michael B. Williams,
Lacy Dennis and the rest of the Watts’ lab at the University of Alabama at Birmingham
for providing technical support for this study. This report was prepared by S.A.W. under
award NA07OAR4170449 from the University of Alabama at Birmingham, U.S.
Department of Commerce. Animal care was supported by the NIH P30DK056336 NORC
Aquatic Animal Research Core of the Animal Models Core. The statements, findings,
conclusions, and recommendations are those of the authors' and do not necessarily reflect
the views of NOAA or the U.S. Department of Commerce.
202
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CHAPTER 6
CONCLUSIONS
Sea urchins demonstrate potential for commercial aquaculture enterprise but
prospective investors must consider operational costs and the level of potential
profitability, if any. Primary determinants of achieving a profitable enterprise will include
factors related to feed costs combined with feed utilization and feed management. This
series of investigations examined macronutrient requirements of sea urchins, economic
viability based on feed costs for sea urchin raised in culture, and feeding techniques to
maximize return on investment (ROI). Additionally, the geometric framework (GF)
concept was examined to evaluate intake targets for protein and carbohydrate at different
lifestages.
Understanding exact nutrient requirements for urchins at all lifestages will be an
important factor in establishing the economic viability of sea urchin aquaculture. Costs of
practical commercial feeds similar to those used in this series of investigations are quite
variable and estimated to range from $1,200.00 to $13,000.00 metric ton-1. This
variability in cost is based on fluctuating prices for feed ingredients, both purified and
practical. Protein, required for normal physiological function and growth, is an expensive
feed ingredient. Therefore, dietary protein must be provided at a level that will be
sufficient to satisfy needs and promote optimum efficiency. However, if dietary protein is
210
provided at a level higher than necessary, cost of feed will correspondingly increase with
a concomitant decrease in production-related efficiencies.
Economic analysis models suggest that the cost of producing 1 gram of urchin
body weight will be minimized within a range of 25 – 30% dietary protein. Previous
studies indicate that apparent optimal protein ranges for L. variegatus (those levels which
promote good growth and optimize production efficiency) at most lifestages will fall
within this range. Similarly, this study has shown that carbohydrate will affect protein
utilization and efficiency. These data indicate that the costs associated with dietary
protein level are influenced by the inclusion of less expensive carbohydrate sources.
However, accurate cost assessment of commercial feeds will require consideration of the
interactive effects among dietary protein, carbohydrate and lipid. In addition, the
development of practical diets and evaluation of least-cost formulation can further reduce
costs associated with feed development in Lytechinus variegatus. Therefore, further
economic analysis is recommended utilizing the GF to test ROI for ranges of protein,
carbohydrate and lipid simultaneously using a variety of purified and practical
ingredients.
To maximize economic return it will also be important to optimize feeding
management strategies in cultured urchins. Few studies have examined the effect of feed
management strategies on sea urchins held in culture; however, indiscriminately applied
feed is subject to nutrient loss and leaching, often leading to poor feed conversion
efficiency and water fouling (both resulting in profit loss). To optimize feeding culturists
should feed whereby as much feed as possible is consumed before excess nutrient
leaching compromises growth. Data derived from these investigations indicate that
211
frequency of feeding and feed ration are probably the most important feed management
considerations for urchins raised in commercial culture. Time of day may also be an
important defining factor. Weight gain and production among L. variegatus was
primarily affected by frequency of feeding, with urchins fed every other day exhibiting
poor growth and production outcomes as compared to individuals fed daily or twice
daily. Ration size was also a determinate of growth and production response. Sea urchins
that were fed large quantities of feed every other day were unable to assimilate nutrients
efficiently, despite an increase in short-term feed intake. For efficient feed management,
sea urchins will require daily feeding and feed rations must be large enough to maintain
satisfactory growth but sufficiently small enough to minimize waste.
Circadian rhythms are known to affect growth and production of other aquatic and
terrestrial species. Among L. variegatus growth and production were initially affected by
circadian feeding patterns, with individuals fed some portion of feed during the evening
demonstrating a higher production response. However, urchins ultimately adjusted to
morning feeding within two months and compensated for an initial reduction in weight
gain. Other sea urchin data show similar results, indicating that circadian feeding patterns
will, most likely, not be a defining factor in feed management among sea urchins held in
culture.
Among cultured large adult urchins provided choice between diets that differ in
levels of fish meal and wheat starch, dietary protein intake appeared to be strongly
regulated. Models for dietary protein intake predicted an increase in somatic growth and
gonad production with increasing dietary protein level. However, protein levels above ca.
32- 35% combined with 18% dietary carbohydrate resulted in a decrease in the rate of
212
growth and level of roe production. These data suggest that adult L. variegatus (ca. 120
g) have an intake target for dietary protein which probably lies around ca. 45- 60 mg per
day per adult urchin (ca. 0.38- 0.54 g kg-1 body weight) but also indicate that protein:
carbohydrate ratio can influence the relative amount of protein utilization. Protein intake
was less tightly regulated among young wild-caught urchins provided gel-based
formulated diets. However, among individuals (ca. 16 g) offered either a choice of diets
(representing several nutrient ranges for protein or carbohydrate), or provided only one
diet with fixed nutrients, these individuals consumed protein within a wide range of 3560 mg per day per urchin (ca. 2.19- 3.75 g kg-1 body weight) with the exception of
urchins fed diets with extreme nutrient dilution or very low protein levels. Individuals fed
extremely low levels of protein may have been unable or not stimulated to consume an
adequate volume of feed to reach intake targets for dietary protein, carbohydrate or lipid.
Although juvenile urchins did not strongly prioritize the intake of dietary protein,
individuals still appeared to eat sufficient quantities of protein relative to carbohydrate
when possible. These data indicate that, on a per gram basis, juvenile urchins have a
higher requirement for dietary protein than that of their adult counterparts. In addition,
adult urchins that have a high nutrient storage capacity appeared to have a more tightly
regulated intake target for dietary protein. Thus, adult sea urchins with high nutrient
stores may have more specialized intake targets than those of juveniles.
Dietary protein intake is regulated for most animal taxa and many species will
preferentially regulate protein intake when limited to a single diet with fixed
macronutrient levels. In the wild, sea urchins consume large quantities of plant and algal
material that are presumed to contain limited protein. Therefore, it is reasonable to
213
assume that urchins in some lifestages may increase fitness by prioritizing protein intake
over that of carbohydrate, especially when nutrient stores of other nutrients are high.
Dietary carbohydrate does not appear to be tightly regulated in sea urchins.
Among urchins provided choice between gel-based diets, carbohydrate intake varied
widely (ranging from 11 to 73 mg day-1) and was not regulated to a specific level.
Efficiencies of urchins fed single low protein diets at a sub-satiation ration were poor at
low (12%) carbohydrate compared to individuals fed similar diets with 18%
carbohydrate. These and previous data suggest that, when dietary carbohydrate intake
among sea urchins is inadequate to supply energy demands, protein may be catabolized to
meet energetic needs. Growth and production models indicate that dietary carbohydrate
levels around 18% provide adequate energy to spare protein requirements of sea urchins.
Few studies have examined the effect of dietary carbohydrate at levels below 18%.
However, these data agree with those derived from a previous investigation which found
that carbohydrate levels of 19% were adequate to support growth and production and to
provide a protein sparing effect.
Data from this dissertation strongly support previous suggestions that the balance
between protein, carbohydrate, and lipid will be important in the development of a
practical sea urchin feed that maximizes growth, production and, consequently, economic
return. Across this series of investigations, dietary protein intake and/or utilization of
protein was affected by dietary carbohydrate intake. When juvenile urchins were
provided choices that allowed them to reach target intake ranges for dietary protein, they
regulated the relative amounts of protein and carbohydrate intake within the diffuse target
range as permitted by the diets. As a result, total nutrient intake was maximized up to a
214
combined limit. Adult L. variegatus demonstrated strict regulation of protein intake with
variable carbohydrate intake. However, reduction in the efficient utilization of both
dietary protein and dietary energy at high protein (32- 35%) levels and 18% dietary
carbohydrate indicate that there are consequences associated with dietary protein:
carbohydrate imbalance. These data indicate that the macronutrient requirements (i.e.
carbohydrate and protein balance) of juveniles are most likely different from those of
cultured adults and dietary macronutrient homeostatic balance will be an important
consideration for L. variegatus reared in culture, and may also be important in wild
populations of urchins.
Knowledge of nutrient balance and feed management techniques will contribute
to the goal of maximizing economic return in commercial sea urchin culture. This series
of investigations increases current knowledge of culture techniques, nutrient interactions
and nutrient targets for L. variegatus. Although further studies are required to optimize
culture conditions for a variety of commercially-important sea urchins, L. variegatus has
demonstrable value as a viable species for sustainable aquaculture and provide new
economic opportunities for industry to meet the growing market demand for sea urchin
roe.
215
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APPENDIX A
INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE APPROVAL
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