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MIAMI UNIVERSITY The Graduate School

CERTIFICATE FOR APPROVING THE DISSERTATION

We hereby approve the Dissertation Of Alberto Pilati Candidate for the Degree: Doctor of Philosophy

Director Dr. Michael J. Vanni

Reader Dr. María J. González

Reader Dr. Thomas O. Crist

Reader Dr. A. John Bailer

Graduate School Representative Dr. David L. Gorchov

ABSTRACT

STOICHIOMETRY AND THE RELATIVE IMPORTANCE OF AUTOCHTHONOUS AND ALLOCHTHONOUS FOOD SOURCES FOR A DOMINANT DETRITIVOROUS FISH By Alberto Pilati Many stoichiometric models assume that animals have ontogenetically constant body nutrient composition and excretion rates, but this assumption has not been adequately tested. I quantified ontogenetic variation in stoichiometry and diet in two fish, one with (gizzard shad) and one without (zebrafish) diet shift (Chapter 2). Both species showed considerable ontogenetic variation in body stoichiometry, driven by body P, which was associated with bone formation. Similar trends in both species suggest that ontogenetic variation in diet is not the main factor mediating fish body stoichiometry in larval and early juvenile stages. However, the N:P ratio of nutrient excretion also varied ontogenetically in gizzard shad, declining from larvae to juveniles, and this decline was apparently driven by changes in diet N:P rather than ontogenetic variation in fish body N:P. Diet instead, seemed to affect more growth and excretion rates. Adult gizzard shad feeds on lake sediments, which are composed by allochthonous and autochthonous detritus. Using experimental ponds, I manipulated the amount of phytodetritus (via dissolved nutrient addition) and allochthonous detritus (via sediment addition) and examined the response of two gizzard shad cohorts (Chapter 3). Shad spawned in all the ponds and young-of –the-year (YOY) recruitment appeared to be higher in conditions with elevated nutrients. Juvenile and adult shad grew better when sediments were added, probably because of their reliance on increased algal sedimentation through flocculation with clays. Yet, their biomass increased with the addition of either type of detritus as compared to the control. Mass specific N and P excretion rates by gizzard shad were higher in ponds with nutrients (phytodetritus) than in ponds with allochthonous detritus. This confirms the positive feedback to phytoplankton via nutrient translocation. To differentiate if gizzard shad is relying on allochthonous particulate organic matter (POM) or on the algae that flocculate with the sediments I labeled autochthonous and allochthonous detritus with stable isotopes and I offered them to the shad (Chapter 4). The use of a mixing model indicated that shad generally consumed algae, and not allochthonous POM. POM generally enters the food web via shad consumption because bacteria release nutrients which are later taken up by the algae, labeling algae with a terrestrial signal.

STOICHIOMETRY AND THE RELATIVE IMPORTANCE OF AUTOCHTHONOUS AND ALLOCHTHONOUS FOOD SOURCES FOR A DOMINANT DETRITIVOROUS FISH A DISSERTATION Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Zoology

by

Alberto Pilati Miami University Oxford, OH 2007 Dissertation Director: Dr. Michael J. Vanni

TABLE OF CONTENTS List of Tables List of Figures Acknowledgments

iii iv vi

Chapter 1. Introduction References

1 6

Chapter 2. Ontogeny, diet shifts and nutrient stoichiometry in fish. Abstract Introduction Methods Results Discussion Acknowledgments References

11 13 15 18 24 33 33

Chapter 3. The effect of agricultural subsidies of nutrients and detritus on a detritivorous fish population Abstract Introduction Methods Results Discussion Conclusion Acknowledgments References

38 39 43 50 78 87 88 88

Chapter 4. The relative importance of autochthonous and allochthonous subsidies for a detritivorous fish Abstract 94 Introduction 96 Methods 99 Results 105 Discussion 116 Acknowledgments 121 References 121 Chapter 5. Conclusions References

127 131

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LIST OF TABLES Chapter 3 Table 1. F statistics and p-values for repeated measures ANOVA for different limnological variables Table 2. F values for the comparison of interaction terms between dates Table 3. Elemental composition and molar ratios of bulk sediments

60 61 73

Chapter 4 Table 1. IsoSource results based on shad liver Table 2. IsoSource results based on shad white muscle

112 115

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LIST OF FIGURES Chapter 1 Fig. 1. Gizzard shad interactions with other components of the food web

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Chapter 2 Fig. 1. Ontogenetic changes in elements and ratios in gizzard shad and zebrafish Fig. 2. δ 15N and δ 13C changes with size in gizzard shad Fig. 3. Ontogenetic changes of Ca in gizzard shad Fig. 4. Ontogenetic variation in excretion N:P in gizzard shad Fig. 5. P variation for different freshwater fish families

20 22 23 25 30

Chapter 3 Fig. 1. Interactions between agricultural activities and gizzard shad abundance Fig. 2. Final gizzard shad biomass Fig. 3. Condition factor of gizzard shad Fig. 4. Gizzard shad survival Fig. 5. Growth of individually marked gizzard shad Fig. 6. Bluegill final biomass, condition factor and survival Fig. 7. Time series of chlorophyll, primary production, total phosphorus, and total nitrogen Fig. 8. Time series of suspended solids and non-volatile suspended solids Fig. 9. Time series of Secchi depths and light extinction coefficient Fig. 10. Time series of sestonic particles molar ratios Fig. 11. Time series of total zooplankton biomass Fig. 12. Time series of main zooplankton groups Fig. 13. Carbon, nitrogen and phosphorus sediment fluxes Fig. 14. Molar ratios of the sediment fluxes Fig. 15. Time series of algal and non-algal carbon sediment flux Fig. 16. Time series of algal and non-algal nitrogen sediment flux Fig. 17. Time series of algal and non-algal phosphorus sediment flux Fig. 18. Nitrogen and phosphorus mass excretion and molar N:P excretion rate of gizzard shad in different treatments

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Chapter 4 Fig. 1. Temporal trends of chlorophyll and total phosphorus Fig. 2. Temporal isotopic trends for 4 food sources

106 108

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40 52 53 54 56 57 58 63 64 66 68 69 71 72 75 76 77

Fig. 3. Isotopic signatures in shad liver for the different treatments Fig. 4. Isotopic signatures in shad white muscle for the different treatments Fig. 5. Food source mixing polygon when algae were labeled Fig. 6. Food source mixing polygon when corn was labeled

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110 111 113 114

DEDICATION/ACKNOWLEDGEMENT First and foremost, I would like to acknowledge my major advisor, Mike Vanni, who I admire greatly as a scientist and a friend. I am also grateful for my committee members, both for their support and enthusiasm about science. I would like to recognize the help received from Michael Hughes for assistance with statistical analysis and for developing painful SAS codes for me, Lisette Torres for correcting the English of all the manuscripts, and all the amazing graduate and undergraduate students from the Vanni and the González laboratories, who helped me not only in the field but also processing million of samples. I am also indebted to the lab technicians who ran many nutrient samples, particularly to Annie Bowling and Peter Levi. And last, but not least, I would like to acknowledge my family who, in spite of the distance, encouraged me to move on with my studies. This research was funded by NSF, USDA, and Miami University through the Committee for Faculty Research and Summer Workshop grant (Dept. of Zoology).

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Chapter 1. Introduction For many years it was believed that heterotrophic lake production was only sustained by autochthonous primary production. This means that a great proportion of the algal production (33%) is transferred to and is sufficient to sustain upper trophic levels, while the unconsumed part (67%) is exported as sediments, a fraction of it enters the microbial loop (Hairston and Hairston 1993). Yet, recent studies show that if it were not for terrestrial subsidies, lakes would not be able to support as much life (Cole et al. 2002; Pace et al. 2004; Cole et al. 2006). The degree of allochthony in natural lakes tends to decrease with increasing lake productivity (Carpenter et al. 2005). When terrestrial subsidies enter a lake, they enter in the form of detritus. Detritus is any form of dead organic matter, dissolved or particulate (Swift et al. 1979). For a particular ecosystem, detritus can be classified as autochthonous or allochthonous detritus. Autochthonous detritus is the one produced within the same ecosystem (i.e. leaves and woody debris in terrestrial ecosystems and algal sedimentation (phytodetritus) in aquatic ecosystems). Allochthonous detritus are those that come from an adjacent ecosystem (e.g. terrestrial detritus from the watershed into a lake). Detrital pathways in an ecosystem can be very important because detritus is a more persistent reservoir of energy than primary producers (Moore et al. 2004). Actually, Hairston and Hairston (1993) and Shurin et al. (2006) showed that in terrestrial and aquatic ecosystems, there is more energy from net primary productivity flowing through the detrital pathway than the herbivore channel. Therefore, many species take advantage of this detrital pathway, using detritus as a main food source (detritivorous organisms). Gizzard shad (Dorosoma cepedianum) are very common detritivores in Midwestern bodies of water. These clupeids are geographically widespread and often dominate the fish biomass of warm-water lakes, rivers and reservoirs (lower than 45° latitude) of North America (Vanni et al. 2005). They have the potential to have unique effects on lake and reservoir ecosystems, owing to the fact that they are highly omnivorous in adult stages and very abundant. They have the ability to consume both zooplankton and sediments (Mundahl and Wissing, 1987; Yako et al. 1996; Schaus et al.

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1992). They are also considered facultative detritivores (Yako et al. 1996) because they can feed on sediments when zooplankton is scarce. When they are in the larval stage, they feed exclusively on zooplankton, but when they leave the larval stage (~30 mm total length; 6-7 weeks old), they undergo a diet shift and consume lake sediments. The changing of mouth position (Heinrichs 1982), the appearance of the gizzard, and the lengthening of the intestine in juveniles (Heinrichs 1982; Smoot and Findlay 2000) are some of the morphological adaptations that indicate the switch from planktivory to detritivory. These changes result in higher growth rates (Smoot and Findlay 2000), which coupled with an early hatching, allow this species to outcompete other planktivorous fish species (Garvey and Stein 1998). At the ecosystem level, gizzard shad are important because they connect and impact various trophic levels in the aquatic ecosystem (Figure 1). For example, larvae (exclusively zooplanktivorous) and adults (omnivorous) can have strong impacts on zooplankton communities. It has been found that larval shad can practically decimate crustacean zooplankton communities (Dettmers and Stein 1992), and it has been suggested that gizzard shad can compromise the recruitment of other zooplanktivorous fish that hatch later in the season (Guest et al. 1990; DeVries et al. 1991; Miranda and Gu 1998). Gizzard shad are also important forage fish. Young-of-the-year (YOY) of this clupeid are the preferred prey for many piscivores (Carline et al. 1986; Johnson et al. 1988; Matthews et al. 1988; Wahl and Stein 1988). Nevertheless, in Ohio reservoirs, only 20-30% of gizzard shad production is consumed by predators (Carline et al. 1984; Johnson et al. 1988) because YOY grow quickly and reach a size refuge from predators by the end of the first year (Adams and DeAngelis 1987; Johnson et al. 1988). Another example of gizzard shad impacting aquatic ecosystems is found in the linkage of gizzard shad and phytoplankton via nutrient excretion. This fish has been shown to play an important role transporting nutrients from lake sediments to open water (Vanni and Headworth 2004). The consumption of sediments and subsequent excretion of nutrients ingested from sediments renders them 'nutrient pumps.' That is, they provide a source of nutrients (nitrogen and phosphorus) to the water column, which may stimulate algal production and decrease water quality. The importance of gizzard shad as a nutrient pump in Acton Lake has been well documented (Mather et al. 1995; Vanni 1996; Schaus

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et al. 1997; Schaus and Vanni 2000; Vanni et al. 2006). It is also known that gizzard shad abundance not only increases with lake productivity (Bachmann et al. 1996; DiCenzo et al. 1996; Michaletz 1997; Vanni et al. 2005), but the amount of primary production gizzard shad can support also increases as lake productivity increases (Vanni et al. 2006). The role in transporting nutrients depends not only on lake productivity but also on fish stoichiometry (Schindler and Eby 1997). Fish stoichiometry has been found to change with age and different fish groups (Sterner and George 2000; Vanni et al. 2002; Hendrixson et al. 2007). Stoichiometry, in this case, is the internal balance of multiple elements within the fish tissue (Sterner and Elser 2002). Gizzard shad has the ability to switch its diet from planktivorous (larvae) to detritivorous (adult). Therefore, as its nutrient requirements change along its life cycle, so may the amount of nitrogen and phosphorus excreted. Thus, depending on its dietary needs and age, this fish can exploit different food sources to maximize its fitness. There are no studies linking ontogeny and fish stoichiometry and nutrient excretion in species that are capable of changing feeding behavior, such as gizzard shad. If gizzard shad changes their diet as a result of changes in their internal stoichiometry, they should also excrete different amount of N and P along the life cycle (Chapter 2). Gizzard shad populations have been found to increase greatly with lake productivity (Bachman et al. 1996; Bremigan and Stein 2001; DiCenzo et al. 1996; Drenner et al. 1996, 1998; Michaletz 1997; Vanni and Headworth 2004) and agricultural activity of their watersheds (Vanni et al 2005, 2006; Sigler 2002). Agricultural activities increase the amount of nutrients being exported from the surrounding landscape (i.e. the watershed) because of the use of fertilizers. Some studies have shown that nitrogen (N) export to northeastern US rivers has increased 3-10 fold (Vitousek et al. 1997) and that 520% of the phosphorus (P) applied as fertilizers in agricultural watersheds goes to streams (Bennett et al 2001). These nutrients, once in the lake, stimulate the algal production and consequent sedimentation of algae (phytodetritus production). If conservation tillage is not employed, agriculture can also increase erosion processes during strong runoff (storm events). Runoff can wash away not only organic particles (particulate organic carbon, POC) but also other organic or inorganic substances that

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dissolve in water. Consequently, agricultural activities might be indirectly increasing shad populations (Chapter 3) by providing more allochthonous detritus (Chapter 4). This research attempts to shed light on how detritivorous fish change their stoichiometry when switching their diet from zooplankton (as larvae) to detritus (as adults). It will also explore the links between agriculturally impacted watersheds and the abundance of detritivorous gizzard shad in Midwestern reservoirs. To address these items, I have proposed the following objectives (Figure 1): 1) Explore how stoichiometric constraints in gizzard shad relate to the ontogenetic diet shift (Chapter 2), and changes in excretion rates before and after the diet shift. 2) Evaluate whether the elevated abundance of gizzard shad in agriculturally-impacted, productive reservoirs is due to subsidies of allochthonous detritus, increased deposition of phytodetritus, or both (Chapter 3). 3) Determine what kind of detritus gizzard shad utilizes to obtain energy (phytodetritus or allochthonous detritus) (Chapter 4).

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Figure 1: Model of the main interactions of gizzard shad with other components of the food web. Solid lines indicate fluxes of resources, dashed lines indicate growth, and dotted lines indicate excretion by gizzard shad. Nutrients from agricultural activities in the watershed stimulate algal production in the lake, and thus algal sedimentation (autochthonous detritus). Also, erosion from the watershed increases inputs of allochthonous detritus. Both detritus types form lake sediments (the main food source of adult shad). Larval shad, on the other hand, are exclusively zooplanktivorous. Both larvae and adults excrete nutrients and have the potential to regulate algal nutrient limitation. Larvae recycle nutrients within the water column, while adults translocate nutrients from the sediments back to the water column. POM: particulate organic matter, and YOY: young-of-the-year. The objectives of this research (see text) are indicated in the figure.

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References

Adams. S.M. and D. L. DeAngelis. 1987. Indirect effects of early bass-shad interactions on predator population structure and food web dynamics. Pages 103-117, in W.C. Kerfoot and A. Sih, editors. Predation: direct and indirect impacts on aquatic communities. University Press of New England. Hanover, New Hampshire. Bachman, R. W., B. L. Jones, D. D. Fox, M. Hoyer, L. A. Bull, and D. E. Canfield. 1996. Relations between trophic state indicators and fish in Florida (USA) lakes. Can. J. Fish. Aquat. Sci. 53:842-855. Bennett, E.M., S. R. Carpenter and N. F. Caraco. 2001. Human impact on erodable phosphorus and eutrophication: A global perspective. BioScience 51(3):227-234. Bremigan, M. T. and R. A. Stein. 2001. Variable gizzard shad recruitment with reservoir productivity: Causes and implications for classifying systems. Ecol. Appl. 11:14251437. Carline, R.F., B.L. Johnson, and T.J. Hall. 1984. Estimation and interpretation of proportional stock density for fish populations in Ohio impoundments. North Am. J. Fish. Manag. 4:139-154. Carline, R.F., R.A. Stein, and L.M. Riley. 1986. Effects of size at stocking, season, largemouth bass predation, and forage abundance on survival of tiger muskellunge. Am. Fish. Soc. Special Pub. 15:151-167. Carpenter, S.R., J.J. Cole, M.L. Pace, M. Van de Bogert, D.L. Bade, D. Bastviken, C.M. Gille, J.R. Hogson, J. F. Kitchell and E. S. Kritzberg. 2005. Ecosystem subsidies: terrestrial support of aquatic food webs from 13C addition to contrasting lakes. Ecology 86(10):2737-2750. Cole J.J., S.R. Carpenter, J.F. Kitchell and M.L. Pace. 2002. Pathways of organic carbon utilization in small lakes: results from a whole-lake 13C addition and coupled model. Limnol. Oceanogr 47(6):1664-1675. Cole, J.J., S.R. Carpenter, M.L. Pace, M.C. Van de Bogert, J.L. Kitchell, and J.R. Hodgson. 2006. Differential support of lake food webs by three types of terrestrial organic carbon. Ecol. Letters 9:558-568.

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Dettmers J.M. and R.A. Stein. 1992. Food consumption by larval gizzard shad: zooplankton effects and implications for reservoir communities. Trans. Am. Fish. Soc. 121:494-507. DeVries, D.R., R.A. Stein, J.G. Miner, and G.G. Mittelbach. 1991. Stocking threadfin shad: consequences for young-of-year fishes. Trans. Am. Fish. Soc. 120:368-381. DiCenzo, V. J., M. J. Maceina, and M. R. Stimpert. 1996. Relations between reservoir trohpic state and gizzard shad population characteristis in Alabama reservoirs. North Am. J. Fish. Manag. 16:888-895. Drenner, R. W., K. L. Gallo, R. M. Baca, and J. D. Smith. 1998. Synergistic effects of nutrient loading and omnivorous fish on phytoplankton biomass. Can. J. Fish. Aquat. Sci. 55:2087-2096. Drenner, R. W., J. D. Smith, and S. T. Threldeld. 1996. Lake trophic state and the limnological effects of omnivorous fish. Hydrobiol. 319:213-223. Garvey, J. E. and R. A. Stein. 1998. Competition between larval fishes in reservoirs: The role of relative timing of appearance. Trans. Am. Fish. Soc. 127(6):1021-1039. Guest. W.C., R.W. Drenner, S.T. Threlkeld, F.D. Martin, and J.D. Smith. 1990. Effects of gizzard shad and threadfin shad on zooplankton and young-of-the-year white crappie production. Trans. Am. Fish. Soc. 119:529-536. Hairston NG Jr. and N.G. Hairston Sr. 1993. Cause effect relationships in energy flow, trophic structure and interspecific interactions. Am. Nat. 142(3):379-411. Heinrichs, S. M. 1982. Ontogenetic changes in the digestive tract of the larval gizzard shad, Dorosoma cepedianum. Trans. Am. Microsc. Soc. 101(3):262-275. Hendrixson H.A., R.W. Sterner, and A.D. Kay. 2007. Elemental stoichiometry of freshwater fishes in relation to phylogeny, allometry and ecology. J. Fish Biol. 70:121-140. Johnson B.M., R.S. Stein, and R.F. Carline. 1988. Use of a quadrat rotenone technique and bionenergetics modeling to evaluate prey availability to stocked piscivores. Trans. Am. Fish. Soc. 117:127-141. Mather, M. A., M. J. Vanni, T. E. Wissing, S. A. Davis, and M. H. Schaus. 1995. Regeneration of nitrogen and phosphorus by bluegill and gizzard shad: Effects of feeding history. Can. J. Fish Aquat Sci 52(11):2327-2338).

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Matthews, W.J., L.G. Hill, D.R. Edds, J.J. Hoover, and T.G. Heger. 1988. Trophic ecology of striped bass, Morone saxatilis, in a freshwater reservoir (Lake Texoma, U.S.A.) J. Fish Biol. 33:273-288. Michaletz, P. H. 1997. Factors affecting abundance, growth, and survival of age-0 gizzard shad. Trans. Am. Fish. Soc. 126-:84-100. Miranda, L.E. amd H. Gu. 1998. Dietary shifts of a dominant reservoir planktivore during early life stages. Hydrobiol. 377:73-83. Moore, J.C., E.L. Berlow, D.C. Coleman, P.C. de Ruiter, Q. Dong, A. Hastings, N. Collins Johnson, K.S. McCann, K. Melville, P.J. Morin, K. Nadelhoffer, A.D. Rosemond, D.M. Post, J.L. Sabo, K.M. Scow, M.J. Vanni and D.H. Wall. 2004. Detritus, trophic dynamics and biodiversity. Ecol. Letters 7:584-600. Mundahl, N. D. and T. Wissing. 1987. Nutritional importance of detritivory in the growth and condition of gizzard shad in an Ohio reservoir. Environ. Biol. Fish. 20(2):129142. Pace, M. L., J. J. Cole, S. R. Carpenter, J. F. Kitchell, J. R. Hodgson, M. C. Van der Bogert, D. L. Bade, E. S. Kritzberg and D. Bastviken. 2004. Whole-lake carbon-13 additions reveal terrestrial support of aquatic food webs. Nature 427:240-243. Schaus. M. H. and M. J. Vanni. 2000. Effects of gizzard shad on phytoplankton and nutrient dynamics: Role of ediment feeding and fish size. Ecology 81(6):17011719. Schaus, M. H., M. J. Vanni, and T. E. Wissing. 1997. Nitrogen and phosphorus excretion by detritivorous gizzard shad in a reservoir ecosystem. Limnol. Oceanogr. 42(6):1386-1397. Schaus, M. H., M. J. Vanni, and T. E. Wissing. 2002. Biomass dependent shifts in omnivorous gizzard sahd: Implications for growth, food webs and ecosystem effects. Trans. Am. Fish. Soc. 131(1):40-54). Schindler, D. E. and L. A. Eby. 1997. Stoichiometry of fishes and their prey: Implications for nutrient recycling. Ecology. 78(6):1816-1831. Shurin, J.B., D. S. Gruner, and H. Hillebrand. 2006. All wet or dried up? Real differences between aquatic and terrestrial food webs. Proc. R. Soc. B 273:1-9.

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Sigler, K. 2002. Interaction strength via nutrient cycling by omnivores in reservoirs along a productivity gradient. Masters thesis. Miami University, Oxford, OH. Smoot, J. C. and R. H. Findlay. 2000. Digestive enzyme and gut surfactant activity of detritivorous gizzard shad (Dorosoma cepedianum). Can. J. Fish. Aquat. Sci. 57:1113-1119. Sterner, R.W. and J.J. Elser. 2002. Ecological stoichiometry: The biology of elements from molecules to the biosphere. Princeton University Press. New Jersey. 439 pp. Sterner, R. W. and N. B. George. 2000. Carbon, nitrogen and phosphorus stoichiometry of cyprinid fishes. Ecology 81:127-140 Swift, M. J., O. W. Heal, and J. M. Anderson. 1979. Decomposition in terrestrial ecosystems. University of California Press. Berkeley, CA. Vanni, M. J. 1996. , in: Polis, G.A. and K.O. Winemiller, editors, Food webs: integration of patterns and dynamics, Chapman and Hall Vanni, M.J., A.M. Bowling, E.M. Dickman, R.S. Hale, K.A. Higgins, M.J. Horgan, L.B. Knoll, W.H. Renwick, and R.A. Stein. 2006. Nutrient cycling by fish supports relatively more primary production as lake productivity increases. Ecology 87(7):1696-1709. Vanni, M. J., A. S. Flecker, J. M. Hood, and J. L. Headworth. 2002. Stoichiometry of nutrient recycling by vertebrates in a tropical stream: Linking biodiversity and ecosystem function. Ecol. Lett. 5:285-293. Vanni, M. J, and J. L. Headworth. 2004. Cross-habitat transport of nutrients by omnivorous fish along a productivity gradient: Integrating watershed and reservoir food webs. In: Food webs at the landscape level. G. A. Polis, M. E. Power and G. R. Huxel (eds.). The University of Chicago Press Vanni, MJ, K. K. Arend, M. T. Bremigan, D. B. Bunnell, J. E. Garey, M. J. Gonzalez, W. H. Renwick, P. A. Soranno, and R. A. Stein. 2005. Linking landscapes and food webs: effects of omnivorous fish and watersheds on reservoir ecosystems. BioScience 55(2):155-167. Vitousek, P. M. et al. 1997. Human alteration of the global nitrogen cycle: sources and consequences. Eccol. Appl. 7(3):737-750.

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Wahl D.H. and R.A. Stein. 1988. Selective predation by three esocids: the role of prey behavior and morphology. Trans. Am. Fish. Soc. 117-142-151. Yako, L. A., J. M. Dettmers, and R. A. Stein. 1996. Feeding preferences of omnivorous gizzard shad as influenced by fish size and zooplankton density. Trans. Am. Fish. Soc. 125(5):753-759.

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Chapter 2. Ontogeny, diet shifts, and nutrient stoichiometry in fish

Abstract Stoichiometry is an important tool in linking the proportion of different elements from molecular to ecosystem levels. Many stoichiometric models assume that animals have ontogenetically constant body nutrient composition (and hence nutrient excretion rates and ratios), even though many animals undergo ontogenetic shifts in diet and in the relative allocation of structural nutrients. To explore the relationship between ontogenetic variation in stoichiometry and diet shifts, I analyzed two fish species: a natural population of gizzard shad (Dorosoma cepedianum), a numerically dominant species which undergoes a pronounced ontogenetic diet shift; and zebrafish (Danio rerio) grown in the lab with no diet shift. In both species, body stoichiometry varied considerably along the life cycle. Larval gizzard shad and zebrafish had higher molar C:P and N:P ratios than larger fish. Variation in body nutrient ratios was driven mainly by body P, which increased with size while nitrogen (N) content, C:P and N:P ratios decreased with size. Shad body calcium content was highly correlated with P content, indicating that ontogenetic P variation is associated with bone formation. Similar trends in body stoichiometry of zebrafish, grown under constant diet in the laboratory, suggest that ontogeny (e.g. bone formation) and not diet shift is the main factor affecting fish body stoichiometry in larval and juvenile stages. The N:P ratio of nutrient excretion also varied ontogenetically in gizzard shad, but the decline in excretion N:P from ~35:1 in small, zooplanktivorous larvae to ~13:1 in detritivorous juveniles, was driven by variation in the N:P of alternative food resources (zooplankton vs. detritus) rather than by fish body N:P. My results thus show that fish exhibit considerable ontogenetic variation in body stoichiometry, comparable to interspecific variation among adults, that is driven by an inherent increase in the relative allocation of P to bones, whereas ontogenetic variation in excretion N:P ratio of gizzard shad is driven more by variation in food N:P than by body N:P. Therefore, the dominance

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of larval shad might not only release phytoplankton from grazing pressure but also might change the nutrient limitation through nutrient cycling.

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Introduction Many ecological stoichiometric models assume that consumer species are homeostatic with respect to body nutrients (Sterner 1990, Hanson et al. 1997, Schindler and Eby 1997). However, some species show ontogenetic changes in, or at least some variation in, body nutrients. For example, in copepods Villar Argaiz et al. (2000) found a gradual decrease in body P as individuals grew, while the C and N increase observed in copepodite stages was mainly affected by growth rate. Hessen (1990) found that juvenile Daphnia magna had more body P than did adults, probably due to the high growth rate experienced by small bodied organisms (the growth rate hypothesis; Elser et al. 2000). Diet might also have an important effect on the organism’s body composition. For example, DeMott et al. (1998), DeMott et al. (2004), and DeMott and Pape (2005) demonstrated that P deficient diets can affect growth rate and body P content of daphniids. In insects, Frost and Elser (2002) found a negative relationship between body size and body P content, and this relationship was, as in daphniids, affected by the amount of P in food. Diet shifts might also have an important effect in the body elemental composition of higher organisms. In vertebrates, a different diet during juvenile stages tends to maximize growth and survival during the most vulnerable stages of the life cycle. Most studies of diet shifts have been done from an energetics, rather than a stoichiometric, perspective (e.g. Bouchard and Bjorndal 2006). To date, only one study analyzed the effect of a diet shift on the body composition. In a clupeid fish, Deegan (1986) reported a decrease in body N content, and an increase in body P, after it switched its diet from zooplanktivory to detritivory (mainly phytodetritus). Also, no studies have linked diet shifts, ontogenetic changes in body composition, and nutrient excretion. Among aquatic animals, body elemental ontogenetic changes may be particularly important in fish due to bone formation during larval-juvenile development. As vertebrates invest relatively more P for bone ossification during maturation, whole-body P content may increase and body N:P ratio may decline, as individuals increase in size (Elser et al. 1996). This relationship between size and changes in body elemental composition was reported for adult fish (Davis and Boyd 1978, Sterner and George 2000) but studies are needed to examine changes in body nutrients over the whole life cycle from larvae to adults, and the consequences of this ontogenetic variation for nutrient cycling. 13

Fish can be important in nutrient cycling (Vanni 2002), and in some cases excretion by young of the year is particularly important (e.g. Kraft 1992). Changes in fish body nutrient content can influence nutrient excretion and recycling to primary producers. In the case of the omnivorous gizzard shad (Dorosoma cepedianum), nutrient excretion by fish supports up to 50% of summer primary production in productive reservoirs (Vanni et al. 2006a). Gizzard shad often dominates the fish biomass of Eastern North America warmwater eutrophic and hypereutrophic lakes, rivers, and reservoirs lower than 45° latitude (Bremigan and Stein 1999, Vanni et al. 2005). It undergoes a drastic diet shift when approximately 30 mm total length (TL). Larval gizzard shad (< 20 mm) feed mainly on zooplankton, while juveniles become facultative detritivores (Yako et al. 1996). In the reservoir ecosystems I study, non-larval gizzard shad rely almost entirely on sediment detritus as a food source, as evidenced by extensive gut analyses and stable isotope analyses (Schaus et al 2002; Higgins et al. 2006). It is not easy to predict how the N:P ratio excreted by gizzard shad should vary ontogenetically. Larvae likely have a high body N:P compared to adults; all else being equal, stoichiometry theory would thus predict that larvae excrete at a lower N:P than adults. On the other hand, per unit N, larvae need to allocate relatively more P to growth per unit mass (compared to adults), and larvae feed on zooplankton, which have a higher N:P (~25:1; Sterner and Elser 2002) than the sediment detritus consumed by adult gizzard shad (~12:1; Higgins et al. 2006). Ontogenetic differences in food N:P (and perhaps in the relative allocation of N vs. P to growth) lead to the prediction that larvae excrete at a higher N:P than adults. In this study I explored variation in body nutrient content during ontogeny, in two fish species, one with and one without an ontogenetic diet shift. I analyzed body nutrient content and excretion rates in gizzard shad (Dorosoma cepedianum) over the course of its ontogenetic diet shift so I could determine how excretion N:P varies ontogenetically. I also analyzed ontogenetic variation in body elemental composition in zebrafish (Danio rerio) grown in the laboratory under a constant diet. This species provides a “control” for the amount of variation in body nutrients in the absence of a diet shift. I also analyzed if body elemental variations are related to diet or to bone development. To my knowledge, this is the first study in which ontogenetic variation in body nutrients is explicitly linked with

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variation in nutrient excretion, and the first study comparing elemental body composition of species with and without a diet shift.

Methods Ontogenetic variation in body nutrient composition of gizzard shad. I quantified ontogenetic changes in gizzard shad body nutrients and nutrient excretion over the first summer of their life cycle in Acton Lake, a hypereutrophic reservoir located in Butler and Preble Counties, Ohio, where gizzard shad is by far the most abundant fish species (Schaus et al. 1997; Vanni et al. 2005). Total length (TL) varied from 7 mm to 135 mm, comprising larvae and young of the year (YOY) juveniles. I assessed the extent to which variation in gizzard shad body stoichiometry is correlated with size and the diet shift using stable isotope analysis. Furthermore, I measured body calcium to explore the role of bone formation on P stoichiometry. Finally, I incorporated results of previous studies that quantified diets, body nutrients and excretion rates of adult gizzard shad in Acton Lake (Schaus 1998, Schaus et al. 1997, 2002, Higgins et al. 2006) to produce a complete picture of the ontogeny of nutrient stoichiometry in this species. In summer 2003, I sampled gizzard shad by following the cohort for the whole growing season (June to September) in Acton Lake. Small larvae (7 to 20 mm) were sampled with an ichthyoplankton net with 500 µm mesh. Small young-of-the-year (YOY) (20 to 55 mm) were sampled with an ichthyoplankton net with 4 mm mesh. YOY >55 mm were collected with an electroshocker. All fish were sorted into size classes of 5 mm for individuals 50 mm. This sorting resulted in more detailed information for larvae and juveniles undergoing the diet shift (which usually occurs at sizes of 20-40 mm) than for fish after the diet shift. For the smallest size classes, I pooled up to 30 individuals in each replicate to get enough tissue for nutrient content analysis (C, N and P). For size classes larger than 30 mm, I used only one individual per sample and I analyzed 1-3 replicates (most often 2) per sampling date. To explore the interannual stoichiometric variability, this sampling protocol was repeated during the summer of 2004 but less intensively. I did not sample individuals beyond their first year of

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life because prior studies provide data on both body nutrients and excretion rates of these size classes in Acton Lake (Schaus et al. 1997, Higgins et al. 2006). Fish of all size classes were placed immediately on ice and taken to the lab where they were measured, gutted, and dried at 60 ºC until a constant weight was obtained. Depending on fish size, the whole fish was ground to a powder using either a mortar and pestle or an ultracentrifugal grinding mill (Restch ZM 100). Body C and N contents were obtained with a Perkin Elmer 2400 Series II CHN analyzer, and P content was measured with a Lachat QuickChem 8000+ FIA autoanalyzer following digestion of tissue with HCl (Higgins et al. 2006). Stable isotopes were measured at the University of California-Davis Stable Isotope Facility with a PDZ Europa 20-20 isotope ratio mass spectrometer coupled with a PDZ Europa ANCA elemental analyzer. An average of two replicate fish samples were analyzed per date, and only one analytical replicate was obtained from each stable isotope sample. I also took 2 zooplankton samples for isotope analysis during the period shad rely exclusively on zooplankton (July 2003). One sample included the whole zooplankton community on a GF/F filter, while the other one was comprised of only calanoids (the most abundant and the most preferred prey item by the larvae at the time) (A. Pilati, personal observation). Sediment isotope data were obtained from Schaus 1998 and Schaus et al. 2002, and I found no need to run more samples due to the observed small variability in δ 15N and δ 13C in these previous studies. For whole body Ca analysis, the samples were ashed in a muffle furnace at 550 ºC for 6 hrs. The ashes were dissolved with concentrated nitric acid, and then diluted with deionized water to reach a concentration of ~1% HNO3. Ca data were obtained by emission spectroscopy (Leeman Labs Inductively Coupled Plasma/Profile) at the Institute of Ecosystem Studies Analytical Laboratory, Millbrook, New York. Ontogenetic variation in body nutrient composition of zebrafish. To gain insight into the importance of a diet shift on the ontogeny of fish stoichiometry, I compared temporal changes in the body nutrients of gizzard shad caught in the wild with those of zebrafish reared in the laboratory with a constant diet following protocols by Westerfield (2000) and Matthews et al. (2002). I kept ~20 individuals per 100 ml during embryonic stage, 400 ml during the larval period or 3 L during the juvenile stage. Larvae and juveniles were maintained in continuous flow through aquaria kept at 28 ºC. Zebrafish reached 30

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mm total length in 7 months. The diet consisted of dried brine shrimp (Artemia sp. δ 15N: 0.87‰ ± 0.69), ground to edible sizes. Fish were fed three times a day during larval stages and once a day during adult stages. Zebrafish samples were processed following the same methodology as for gizzard shad, but Ca was not analyzed due to the small mass of tissue available. Ontogenetic variation in nutrient excretion by gizzard shad. Stoichiometry models predict that variation in both body nutrients and diet can lead to significant variation in nutrient excretion rates or ratios (Sterner 1990; Elser and Urabe 1999). To assess these potential links, during June and July of 2005 I analyzed excretion rates of larval and juvenile gizzard shad collected from Acton Lake. Detailed description of the procedure is found elsewhere (Schaus et al. 1997, Higgins et al. 2006). Briefly, gizzard shad were collected with ichthyoplankton nets. Fish were immediately transferred to small acid washed beakers containing from 15 to 50 ml of pre-filtered lake water (0.7 µm pore glass fiber filters) depending on fish size. For larvae smaller than 10 mm I kept up to 3 larvae per container (so that enough nutrients were excreted for analysis), but for larger sizes I kept only one individual per container. Beakers were kept at epilimnetic temperatures for approximately 15 min. Water samples (before the addition of fish and after the incubation) were filtered using a Gelman A/E glass fiber filter, preserved with sulfuric acid, and analyzed for ammonia and soluble reactive phosphorus with a Lachat 8000 autoanalyzer. To standardize the molar N:P excretion with fish size, I calculated the mass-specific excretion rate. For this purpose the length of each fish was used to estimate mass using the following formula: Mass (g) = 0.0035 * Length (cm)3.3749 (A. Pilati, unpublished data). Statistical analysis: To compare the elemental composition and ratios of the 2003 and 2004 gizzard shad cohorts, I used analysis of covariance (ANCOVA; Ott and Longnecker 2001) (proc glm, SAS version 9.1), with body nutrient contents and ratios as the dependent variables, cohort as the categorical variable, and size (total length) as a covariate. Inspection of the body nutrient and ratio data showed that these parameters did not vary linearly with fish size over the entire first year. Rather, nutrient contents and ratios changed more during the early stages of growth and then remained relatively constant. Therefore, I split the data series using a threshold value of 40 mm to ensure linearity below and above that threshold. The threshold was estimated using a segmented regression

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(Netter et al. 1996, Kutner 2005, Littell 1991), and allowing SAS to calculate the “knot” (proc nlin SAS version 9.1). Then, separate ANCOVAs were run for each portion of the data. To compare trends in elements and ratios between gizzard shad and zebrafish, I ran ANCOVAs after standardizing body size and nutrient content/ratio variables. As the range in absolute body size and elemental composition is different for these two species (gizzard shad attain much larger adult size; see Fig. 1), I transformed fish size to percent maximum reported size, and element or ratio as percent of maximum observed value, to bring all variables to comparable scales. I then used ANCOVA to compare species, using species as the categorical variable.

Results Gizzard shad body nutrients. Gizzard shad body C, P and N varied considerably throughout development. Percent C decreased to a threshold size of ~40 mm (fish