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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. ii 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 iii 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. iv 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 v 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. vi 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 vii Page CONCLUSIONS.........................................................................................................…209 GENERAL LIST OF REFERENCES................................................................................215 APPENDIX A: INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE APPROVAL……………………………….......................................................................222 viii 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 ix 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 xi 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 xii 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 xiii 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 xiv 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. 2 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%). 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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 (30N, 85.5W) 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. 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Influence of carbohydrate and fat intake on dietinduced thermogenesis and brown fat activity in rats fed low protein diets. The Journal of nutrition, 117(10), 1721-1726. Schlosser, S. C., Lupatsch, I., Lawrence, J. M., Lawrence, A. L., & Shpigel, M., 2005. Protein and energy digestibility and gonad development of the European sea urchin Paracentrotus lividus (Lamarck) fed algal and prepared diets during spring and fall. Aquaculture Research, 36(10), 972-982. Senaratna, M., Evans, L., Southam, L., & Tsvetnenko, E., 2005. Effect of different feed formulations on feed efficiency, gonad yield and gonad quality in the purple sea urchin Heliocidaris erythrogramma. Aquaculture Nutrition, 11(3), 199-207. Shang, Y. C., 1986. Research on aquaculture economics: a review. Aquacultural Engineering, 5(2), 103-108. 84 Simpson, S. J., & Raubenheimer, D., 2012. The nature of nutrition: a unifying framework from animal adaptation to human obesity: Princeton University Press. 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 (30N, 85.5W) 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. 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Feeding preferences, seasonal gut repletion indices, and diel feeding patterns of the sea urchin Tripneustes gratilla (Echinodermata: Echinoidea) on a coastal habitat off Toliara (Madagascar). Marine Biology, 143(3), 451-458. 123 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(3), 699-708. doi: 10.1007/s10499-012-9604-7. 124 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 (30N, 85.5W) 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 LITERATURE CITED Agatsuma, Y., 2000. 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Protein‐leverage in Mice: The geometry of macronutrient balancing and consequences for fat deposition. Obesity, 16(3), 566-571. Taylor, A.M. 2006, Effects of dietary carbohydrate on weight gain and gonad production in juvenile sea urchins, Lytechinus variegatus. Masters Thesis. The University of Alabama at Birmingham. Birmingham, AL. USA. Unuma, T., Suzuki, T., Kurokawa, T., Yamamoto, T., & Akiyama, T., 1998. A protein identical to the yolk protein is stored in the testis in male red sea urchin, Pseudocentrotus depressus. Biological Bulletin, 194, 92- 97. Wallace, B. D., 2001. The effects of dietary protein concentration on feeding and growth of small Lytechinus variegatus (Echinodermata: Echinoidea). Masters Thesis. University of Alabama at Birmingham, Birmingham, Alabama, USA. 155 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 156 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 157 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. 158 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 159 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 160 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 161 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 (30N, 85.5W) 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 163 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. 164 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. 168 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 LITERATURE CITED Bishop, C., Watts, S.,1992. 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Journal of Insect Physiology, 41(7), 545-553. Simpson, S., Raubenhiemer, D., 1997. Geometric analysis of macronutrient selection in the rat. Appetite, 28, 201-213. Simpson, S., Raubenheimer, D., 2001. A framework for the study of macronutrient intake in fish. Aquaculture Research, 32(6), 421-432. Simpson, S., Raubenheimer, D., 2005. Obesity: the protein leverage hypothesis. Obesity Reviews, 6(2), 133-142. Simpson, S., Raubenheimer, D., Behmer, S., Whitworth, A., Wright, G., 2002. A comparison of nutritional regulation in solitarious-and gregarious-phase nymphs 207 of the desert locust Schistocerca gregaria. Journal of Experimental Biology, 205(1), 121-129. Simpson, S. J., Batley, R., 2003. Geometric analysis of macronutrient intake in humans: the power of protein? Appetite, 41(2), 123-140. Simpson, S. J., Raubenheimer, D., 2012. The nature of nutrition: a unifying framework from animal adaptation to human obesity: Princeton University Press. Sørensen, A., Mayntz, D., Raubenheimer, D., Simpson, S. J., 2008. Protein‐leverage in Mice: The Geometry of Macronutrient Balancing and Consequences for Fat Deposition. Obesity, 16(3), 566-571. Sterner, R. W., Elser, J.J., 2002. Ecological Stoichiometry: the biology of elements from molecules to the biosphere. Princeton University Press, Princeton, New Jersey, USA. 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. Unuma, T., Nakamura, A., Yamano, K., Yokota, Y., 2010. The sea urchin major yolk protein is synthesized mainly in the gut inner epithelium and the gonadal nutritive phagocytes before and during gametogenesis. Molecular Reproduction and Development, 77, 59-68. Watts, S.A., Lawrence, J.M. and Lawrence, A.L., 2010. Approaches to the study of sea urchin nutrition. In Harris, L., Bottger, S.A., Walker, C.W., Lesser, M.P. (eds.), Echinoderms: Durham, CRC Press, Boca Raton, FL, USA, pp. 331- 345. 208 Watts, S.A., Lawrence, A.L., Lawrence, J.M., 2013. Nutrition. In: J.M. Lawrence (ed), Sea urchins: biology and ecology. Third edition. Elsevier Science B.V., Amsterdam.155-169. Westoby, M., 1978. What are the biological bases of varied diets? American Naturalist, 112, 627-631. 209 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. 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J., Wood, J.T., Wallis, I.R., Lindenmayer, D.B., 2009. Protein content of diets dictates the daily energy intake of a free-ranging primate. Behavioral Ecology, arp021, 685-690. Gosby AK, Conigrave, A.D., Raubenheimer, D., Simpson, S.J., 2014. Protein leverage and energy intake. Obesity Reviews. 15, 183-191. Hammer, B. W., Hammer, H. S., Watts, S.A., Desmond, R., Lawrence, J.M. and Lawrence, A.L., 2004. The effects of dietary protein concentration on feeding and growth of small Lytechinus variegatus (Echinodermata: Echinoidea). Marine Biology, 145(6), 1143-1157. Hammer, H.S., 2006. Determination of dietary protein, carbohydrate, and lipid requirements for the sea urchin Lytechinus variegatus fed semi-purified feeds. Dissertation. University of Alabama at Birmingham, Birmingham, Alabama. Hammer, H.S., Hammer, B.W., Watts, S.A., Lawrence, A.L., and Lawrence, J.M., 2006. The effect of dietary protein and carbohydrate concentration on the biochemical composition and gametogenic condition of the sea urchin Lytechinus variegatus. Journal of Experimental Marine Biology and Ecology, 334(1), 109-121. 217 Hammer, H. S., Powell, M. L., Jones, W. T., Gibbs, V. K., Lawrence, A. L., Lawrence, J. M. and Watts, S. A., 2012. Effect of feed protein and carbohydrate levels on feed intake, growth and gonad production of the sea urchin Lytechinus variegatus. Journal of the World Aquaculture Society, 43(2), 145- 158. Heflin, L. E., Gibbs, V. K., Powell, M. L., Makowsky, R., Lawrence, J. M., Lawrence, A. L., & Watts, S. A. (2012) Effect of dietary protein and carbohydrate levels on weight gain and gonad production in the sea urchin Lytechinus variegatus. Aquaculture, 358, 253-261. Hewson-Hughes A. K., Hewson-Hughes, V. L., Miller, A.T., Hall, S. R., Simpson, S.J., Raubenheimer, D., 2011. Geometric analysis of macronutrient selection in the adult domestic cat, Felis catus. Journal of Experimental Biology, 214, 1039– 1051. Hewson-Hughes, A. K., Hewson-Hughes, V.L., Colyer, A., Miller, A.T., Hall, S.R., Raubenheimer, D. and Simpson, S.J., 2013. Consistent proportional macronutrient intake selected by adult domestic cats (Felis catus) despite variations in macronutrient and moisture content of foods offered. Journal of Comparative Physiology B, 183(4), 525-536. Hewson-Hughes, A. K., Hewson-Hughes, V.L., Colyer, A., Miller, A. T., McGrane, S. J., Hall, S.R., Butterwick, R.F., Simpson, S.J., Raubenheimer,D., 2012. Geometric analysis of macronutrient selection in breeds of the domestic dog, Canis lupus familiaris. Behavioral Ecology, ars168. 218 James, P., and Siikavuopio, S. I., 2012. The effect of continuous and intermittent feeding regimes on survival, somatic and gonadal growth of the sea urchin, Strongylocentrotus droebachiensis. Aquaculture. Lawrence, J., and Hughes-Games, L., 1972. The diurnal rhythm of feeding and passage of food through the gut of Diadema setosum (Echinodermata: Echinoidea). Israel Journal of Zoology, 21(1), 13-16. Lawrence, J. M., Lawrence, A.L. and Watts, S.A., 2013. Feeding, digestion and digestibility of sea urchins. pp 136-154. In J.M. Lawrence (ed.).Sea Urchins: Biology and Ecology, Third Edition, Elsevier Science B.V. Amsterdam. Lawrence, J. M., Plank, L. R., and Lawrence, A. L., 2003. The effect of feeding frequency on consumption of food, absorption efficiency, and gonad production in the sea urchin Lytechinus variegatus. Comparative Biochemistry and Physiology, 134, 69-75. Lee, K., Behmer, S. Simpson, S.J. and Raubenheimer, D., 2002. A geometric analysis of nutrient regulation in the generalist caterpillar Spodoptera littoralis (Boisduval). Journal of Insect Physiology, 48(6), 655-665. Lewis, J. B., 1964. Feeding and digestion in the tropical sea urchin Diadema antillarum Philippi. Canadian Journal of Zoology, 42(4), 549-557. Marsh, A.G., Powell, M.L. and Watts, S.A., 2013. Energy metabolism and gonad development. pp 35-50. In J.M. Lawrence (ed.).Sea Urchins: Biology and Ecology, Third Edition, Elsevier Science B.V. Amsterdam. 219 Mayntz, D., Nielsen, V.H., Sørensen, A. Toft, S., Raubenheimer, D., Hejlesen, C. and Simpson,S.J., 2009. Balancing of protein and lipid intake by a mammalian carnivore, the mink, Mustela vison. Animal Behaviour, 77(2), 349-355. Minor, M., and Scheibling, R., 1997. Effects of food ration and feeding regime on growth and reproduction of the sea urchin Strongylocentrotus droebachiensis. Marine Biology, 129(1), 159-167. Nelson, B., and Vance, R., 1979. Diel foraging patterns of the sea urchin Centrostephanus coronatus as a predator avoidance strategy. Marine Biology, 51(3), 251-258. Nelson, W., Scheving, L., and Halberg, F., 1975. Circadian rhythms in mice fed a single daily meal at different stages of lighting regimen. The Journal of Nutrition, 105(2), 171-184. Noeske, T. A., and Spieler, R. E., 1984. Circadian feeding time affects growth of fish. Transactions of the American Fisheries Society, 113(4), 540-544. Ogden, J. C., Brown, R. A., and Salesky, N., 1973. Grazing by the echinoid Diadema antillarum Philippi: formation of halos around West Indian patch reefs. Science, 182(4113), 715-717. Perez, J., Zanuy, S., and Carrillo, M., 1988. Effects of diet and feeding time on daily variations in plasma insulin, hepatic c-AMP and other metabolites in a teleost fish, Dicentrarchus labrax L. Fish Physiology and Biochemistry, 5(4), 191-197. Philippens, K., Von Mayersbach, H., and Scheving, L. E., 1977. Effects of the scheduling of meal-feeding at different phases of the circadian system in rats. The Journal of Nutrition, 107(2), 176-193. 220 Raubenheimer, D., and Simpson, S. J., 1997. Integrative models of nutrient balancing: application to insects and vertebrates. Nutrition research reviews, 10(01), 151179. Simpson, S. J. and Raubenheimer, D., 1993. A multi-level analysis of feeding behaviour: the geometry of nutritional decisions. Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences, 342(1302), 381-402. Simpson, S. J. and Raubenheimer, D., 1995. The geometric analysis of feeding and nutrition: a user's guide. Journal of Insect Physiology, 41(7), 545-553. Simpson, S., & Raubenheimer, D., 2005. Obesity: the protein leverage hypothesis. Obesity Reviews, 6(2), 133-142. Simpson, S. J. and Raubenheimer,D., 2012. The nature of nutrition: a unifying framework from animal adaptation to human obesity: Princeton University Press, Princeton, New Jersey, USA. Sørensen, A., Mayntz, D., Raubenheimer, D., and Simpson, S. J., 2008. Protein‐leverage in Mice: The Geometry of Macronutrient Balancing and Consequences for Fat Deposition. Obesity, 16(3), 566-571. Sundararaj, B. I., Nath, P., and Halberg, F., 1982. Circadian meal timing in relation to lighting schedule optimizes catfish body weight gain. The Journal of Nutrition, 112(6), 1085. Tacon, A. G., 1995. Feed formulation and on-farm feed management. FAO Fisheries Technical Paper, 61-74. 221 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. Vaïtilingon, D., Rasolofonirina, R., and Jangoux, M., 2003. Feeding preferences, seasonal gut repletion indices, and diel feeding patterns of the sea urchin Tripneustes gratilla (Echinodermata: Echinoidea) on a coastal habitat off Toliara (Madagascar). Marine Biology, 143(3), 451-458. Webster, A., 1993. Energy partitioning, tissue growth and appetite control. Proceedings of the Nutrition Society, 52(01), 69-76. Zhao, C., Zhang, W., Chang, Y., Zhou, H., Song, J., and 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(3), 699-708. 222 APPENDIX A INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE APPROVAL 223