Data collection for tracing the evolution of frugivory in characids (Ostariophysii: Characiformes)...

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BIOL 499L

Data collection for tracing the evolution of frugivory in characids (Ostariophysii:

Characiformes) using silver dollar (Metynnis sp.) dental microwear and digestive analysis

Harrison, D.

Department of Biological Sciences, California State University, Fullerton, 800 N. State College Blvd., Fullerton , CA 92834, USA

Article History:

Received January 1, 2010

Revised April 20, 2010

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Harrison, D.


Table of contents

I. Abstract

II. Introduction

Environmental influences/ diversity

Dental microwear/ digestive enzymes

Target species

III. Materials/Methods

Set up of aquaria and collection of specimens

Feeding treatments

Collection, storage, and transfer of tissue and bone

IV. Results/ Discussion

Mechanical/ physical degradation

Chemical breakdown / absorption

V. Conclusion

Future studies

VI. Acknowledgements

VII. References cited

VIII. Appendix

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Harrison, D.


Abstract

In this experiment we collected information for dental microwear and digestive enzyme analysis in silver

dollars (Metynnis sp.). Twelve fish in a ten tank set up (one (1) two-fish tank per sample) with centralized

circulation were raised on a hard (sinking granule, SG) and soft (flake, F) diet for twenty five (25) days. Average

ambient water conditions (26±0.8 °C) showed SG populations to have on average; murkier tanks, higher water

velocity and twice as much excretia than F. Foraging behavior varied within and between samples; F tending to

surface feed while demonstrating partitioning and SG aggressively catching falling granules then benthos feeding.

Although F sank slower than SG, the majority were consumed before touching the bottom of tanks. On average, SG

samples were 1.5mm longer and 1.2g heavier than F samples, suggesting more digestive activity in SG. Population

and individual metabolic activities such as mucus secretion, acid production and transport proteins influenced the

amount and rate of energy expenditure and nutrient absorption. At the end of feeding trials, fish were sacrificed and

portions of internal viscera tissue (intestine, liver and stomach) were collected and shipped for further analysis.

Remaining whole body specimens were stored in 70% ethanol for white light confocal microscopy on dental

microwear. Procedures conducted in this experiment can be applied to fossil specimens +100mya to better

understand past foraging behaviors. Additional research may include sediment and pollen examination, size and

number of seeds in fruit consumed, and biogeographic distribution of plant species. To better understand early

frugivores, experiments triggering the auditory system provide additional information on ostariophysiian feeding

behaviors.

Key words: Characiformes, Serrasalmidae, Metynnis spp., dental microwear, frugivory, granivory, dietary

specialization, feeding behavior

Introduction

Environmental influences/ diversity

Evolution of the freshwater Characiformes is of particular interest because complex

plant-animal interactions are involved. (Lundeberg et al., 1998; Monsch, 1998; Albert et al.,

2006 and Hubert et al., 2007). These and other animals including bats, birds, monkeys and some

teleosts have coevolved with their landscape to obtain nutritional content, redistribute and

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propagate plant species. In certain circumstances, different habitats as well as biodiversity allow

for unique evolutionary opportunities for animals to adapt to their surroundings.

The transatlantic distribution of some characid families, for example creates divergent

evolution, but suggests a shared common ancestor predating the split of South America and

Africa (>100 mya) (Calcagnotto et al. 2005; Javonillo et al. 2009 and Mirande et al., 2009).

Fossil remains from the late Cretaceous Maastrichtian age (+70mya) to early Paleocene (60-70

mya) in South America indicate characids had different ecological niches inferred from dentition

(Gayet et al., 2001; Machado-Allison and Garcia, 1986). More recently, from the Orinoco and

Amazon basins, jaw bones date back to the Miocene (10-15 mya) and are identified as extant

Colossoma sp. from the Serrasalmid family (Lundberg et al., 1986). Biogeographic distributions

allows for comparative research. High diversity in characids may be a consequence of vicariant

events from the Miocene to today, which led to isolation of South American populations in a

highly dynamic tropical rainforest (Lucas, 2008; Hubert et al., 2007; Albert et al., 2006;

Lundberg et al., 1998; and Monsch, 1998).

Synapomorphic homologies defined from the 19th

and 20th

century makes efforts to

construct reciprocal monophyletic groups challenging because of its one dimensional

perspective. Phenotypic comparisons could be made between South American and African

characids but this data is limiting. Molecular sequencing and genetic expressions these past two

decades provide additional comparative information at the molecular and microscopic level.

(Hubert et al, 2005; and Orti et al., 1996). Proposals to classify groups at the subfamiliar level

continue today with the application of technology revealing complex natures of biodiversity.

(Mirande, 2009). Complete fossil specimens stored at museums are readily available for study

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however, not much change has been noticed in the serrasalmid lineage (Albert et al., 2006). The

absence of tissue on fossil specimens makes genetic and molecular comparisons difficult.

South American rainforests are abundant in plant biomass and water flow systems.

During the flood season, fallen plant materials serve as food sources for herbivorous fish (Horn,

1997, Drewe et al., 2004; Correa et al. 2007 and Lucas, 2008). Metabolic processes underlying

mechanical and chemical breakdown include mastication, production of enzymes and transport

proteins (Drewe et al., 2004; Correa et al., 2007; Lopez-Vasquez et al., 2009 and Vieira et al.

2005). Often times symbiotic relationships of bacteria exists in the gut lumen of animals aiding

in the further breakdown of food materials, especially herbivores (Roy et al. 2009). Flow speed,

temperature, and pH are a few factors influencing the rate of food metabolism. It is important to

measure digestive activity to better understand the acquisition and distribution of nutrient

absorption in organisms. Ecological interactions offer many opportunities for this research, and

so, in this paper we will examine the expressions of digestive enzymes, dental microwear and the

impact of dietary specialization (frugivorous and granivorous feeding behavior) in Neotropical

freshwater characins by collecting preliminary data.

Dental microwear/ digestive enzymes

Dental microwear is physical evidence an organism has been using its teeth (Scott, et al.

2005). Purnell et al. examined sticklebacks Gasterosteus aculeatus, a temperate North American

freshwater fish, and concluded that dental microware is sufficient to distinguish within species

diet variability (2006, 2007). Additional studies examined the production of digestive enzymes in

laboratory-fed and natural populations of Neotropical freshwater fish (Lopez-Vasquez et al.,

2009; Drewe et al., 2004; and Vieira et al., 2005). Many plant species contain toxins

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(Xenobiotics) to deter herbivores from consuming photosynthetic and reproductive structures

(Sorenson et al. 2006). Digestive enzymes which aide in the breakdown and absorption of

harmful chemicals include cytochrome P450 and p-glycoproteins. Interestingly, some genera of

characins crush seeds while others are viable dispersers (Horn, 1997; Correa et al., 2007; Lucas,

2008; Anderson et al., 2009) Seed size and number play an important role in resisting destruction

and chemical breakdown. Identifying when fish exhibited the production of detoxifying enzymes

associated with plant secondary metabolites necessitates a multidisciplinary approach.

Target species

Silver dollar (Metynnis sp.), a popular aquaria fish, serve as model organisms for

collecting preliminary data (Orti et al., 1996, 2008; and Freeman et al., 2007). Their omnivorous

behavior allows them to take advantage of the abundant near-shore and fallen plant material

during the flood season in their native South American habitat (Lucas, 2008). Being able to

utilize these food particles involves specialized mechanical and chemical processes. It is the

intent of this study to observe dental microwear and measure digestive enzymes. We predict that

hard-textured diets will show deeper feature density while soft-textured diets will show higher

feature length. Digestive enzymes will remain relatively constant between both hard and soft fed

populations because of similar nutritional content. The preliminary data collected in this

experiment will be applied to future studies involving feeding behavior in characid species.

Materials/ Methods

Set up of aquaria and collection of specimen

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Ten 10L aerated tanks set up by John Chappell ran at 21-23 C for two weeks to diminish

ammonia levels with a 12:12 photoperiod. Rearing methods follow Purnell et al. (2006) modified

with a hard and soft diet (Horn, 2009). Measures to control stress levels were taken by wet

transferring specimen in a dark, stabilized environment to McCarthy Hall in tanks with one

plastic plant to serve as a protective hiding place (Horn, 2009).

Feeding treatments

Fish (40-50 mm SL; <5g) obtained from Discount Tropical Fish Store in La Habra, were

raised on a standardized hard (Tetramin sinking granules) or soft (TetraMin flakes) diet.

Collection, storage, and transfer of tissue and bone

Collection of tissue (<10min after death) follow Forbey’s protocol (2009). Immediately

following cold shock, fish were decapitated. Using disposable razorblades, 5 mm of the proximal

gastrointestinal tract (stomach and intestine), and 5 mm3 of liver were collected, stored in micro

centrifuge vials with RNA later 7021, and refrigerated overnight. Saturated tissue was then

stored at -20 to -80 C and shipped for digestive enzyme analysis.

Jaw bones were detached from skull using scalpel and forceps with minimal contact to

teeth preventing additional dental microwear. Remaining whole body specimen were stored in

70% ethyl alcohol ready to be shipped and have dentition analyzed (Horn, 2009).

Results/ Discussion

Mechanical/ physical degradation

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Feeding behavior varied within and between diets. Once fish were acclimated to their

environment, feeding commenced immediately as soon as food was administered. Both samples

relied heavily on visual cues to obtain food. The size and shape of flakes versus granules

influenced the location in water column with flakes floating before becoming saturated, and

granules having a faster sinking rate than flakes (SG>F). Granules were also noted to

dissociation after ~15 min whereas flakes lasted > 24hrs. These factors undoubtedly influenced

foraging behaviors of Metynnis sp. in this experiment with other considerations taking place.

Flake populations tended to surface feed more often than SG because of the floating

flakes. Some F fish developed a unique behavior to slap the surface of the water to disturb the

flakes causing them to sink. Whether this behavior is learned or inherited, neighboring fish had

the ability to observe each other.

In instances where fish were paired (two in one tank), the flake population exhibited

niche partitioning. A slowly sinking flake was pursued by both fish when one decided to turn

away. Sinking granule populations fed more aggressively than flakes because they chased

granules as they sank, catching them before they touched the bottom. As soon as all the granules

sank, benthos feeding began.

Observations suggest evolution of dentition co-evolves with the texture, density and

buoyancy of available food resources. Different food texture and nutritional content likely

influenced the need to pick or scrape fleshy fruit from a large seed while small seeds may have

been consumed and crushed by molariform teeth. These complex factors play important roles in

the redistribution and dispersal of select plant species (Horn, 1997; Anderson, et al., 2009). The

impact of food on dental morphology provides clues to past foraging behavior.

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Chemical breakdown / absorption

Quantity and rate of metabolism, which was apparent within and between diets, also

varied (Graphs 1 & 2). Both food types had similar compositional makeup (see Appendix A3 &

A7) but nutritional content differed with flake diets having added vitamins and minerals. On

average, SG were 1.5mm longer and 1.2g heavier than F.

Graph 1: Average silver dollar body mass (g) before removal of internal viscera. Y-error bars

represent 95% confidence intervals.

Graph 2: Average silver dollar standard length (SL). Y-error bars represent 95% confidence

intervals.

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

Av

era

ge

Bo

dy

Ma

ss (

g)

SD Body Mass (g)

Control

Flakes

Sinking

Granules

0.0

10.0

20.0

30.0

40.0

50.0

60.0

Av

era

ge

Sta

nd

ard

Len

gth

(SL

) m

m

SD Standard Length (SL) mm

Control

Flakes

Sinking

Granules

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These data suggest SG excited enzyme activity or F inhibited enzyme activity. Ingested

food is further broken down by mucous, bile salts, acid and enzymes produced by mucous

secreting cells, liver, gall bladder and pancreas (Helfman et al., 1997). Secretion of endogenous

digestive enzymes is facilitated by parasympathetic nerve cells signaling gastrointestinal

hormone release. Neural and hormonal responses involve cranial nerve and endocrine glands

influenced by chemo-receptors, osmo-receptors and mechanoreceptors. Other enzymes likely to

occur in characid lumen are amylase, maltase, lipase, and alkaline protease which facilitate the

breakdown of starch, cellulose, glycogen, amino and fatty acids. Recent studies indicate

symbiotic relationships of bacteria producing exogenous enzymes in fresh water fish hind and

foregut (Mondal et al., 2008; Roy et al. 2009). These relationships are complex but offer

interesting research opportunities for bacterial species such as distribution of, and aerobic as

wells as anaerobic metabolism (Mondal et al., 2008). Further examination is needed to determine

if digestive enzymes of the omnivorous Metynnis sp. are associated with symbiotic bacterial

relationships in plant secondary metabolism (Boucher et al., 2003; Korneva, 2008; Evans, et al.,

2009).

The evolution of plant defensive mechanisms produce aromatic compounds (xenobiotics)

which are known to be toxic at high levels (Sorenson et al. 2006). Absorption and excretion

occurs in the intestine, liver and kidneys and traverses cellular tissue via transport and efflux

proteins (P-glycoprotein and cytochrome P450) (Hemmer et al.,1995; Hemmer et al., 1998; Lee

et al., 2001; Trambas et al., 2001; Da Silva et al., 2004; Shuilleabhaim et al. 2005; Damare et al.

2009). It has been shown by Drewe et al. 2004 and Lopez-Vasquez et al. 2009, that food types

alter genetic expression by influencing the levels of digestive enzymes which are age and species

dependent. The co-evolution of fish-plant interactions elucidate these properties to where it is

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advantageous for fruit to be eaten and redistributed (Horn, 1997; Anderson et al., 2008; Lucas,

2008) Investigating early herbivores, frugivores and granivores are likely to express symbiotic

relationships, however internal tissues are no longer present in many fossilized samples.

Conclusion

The ecological origin of feeding on fallen fruit and plant materials by characids may date

back >100 mya when early angiosperms first arrived in the Cretaceous. This complex aquatic-

terrestrial interaction has co-evolved in characid lineages (and other animals) to aid in the

breakdown and absorption of secondary plant metabolites. Symbiotic relationships may help

determine when such behavior was exhibited.

Niche differentiation and trophic ecology have evolved in numerous forms, some unique

to the order Characiformes. Species are often classified by their ecological niche which is

elucidated by biogeographic distributions, phenotypic makeup and chemical secretions. Such

high diversity and divergence is characterized by genetic expression, influenced by physical

factors, foraging, and social behavior. The history of the Amazon is evident in fossilized remains

which provide clues to past foraging behavior and environmental conditions. Understanding the

genetic expressions associated with foraging is described by analyzing tissue and observing

dental microwear, however it is less understood how the impact of food influences diversity

between and within families. Dentition and digestive enzymes allow evolutionary biologists

exciting opportunities to compare fossilized remains and infer habitat and foraging behavior.

Metabolism involves the breakdown and digestion of food particles regulated by neural

responses and sometimes symbiotic relationships. With this data, the impact of diet and

metabolism in growth and development can be quantified. Genetic expression in the form of

metabolic processes gives clues to past foraging behavior while additional biogeographical

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research will be necessary to recreate past distributions and physical environments affecting

evolutionary development of fish.

Applications to future studies

Opportunities to incorporate multiple disciplines should be considered for future studies.

Looking at historical distributions of characid species along with terrestrial plants and

sedimentology (e.g pH and pollen) will better describe the climates and possible food sources

encountered by ancestral characid lineages. Foraging behaviors affecting physical and chemical

expressions may even describe early Ostariophysi development and distribution (Briggs, 2005).

In order to observe additional feeding behaviors, experiments testing auditory cues and

eliminated visual cues may provide useful information for revealing early frugivorous fish. This

project provides biogeographers data for tracing the evolution and origin of frugivory in characid

fish lineages.

Acknowledgements

I would like to thank Dr. Dahdul, Dr. Forbey, and Dr. Ungar for analyzing our fish

populations. John Chappell, the animal facilities director at CSUF for setting up the aquaria and

constantly maintaining aquaria environment, and the members of the conservation, fish and

seabird feeding ecology laboratory, thank you for your thoughtful contributions during meetings,

and persistent work in the field and lab. And a special thanks to Dr. Horn for introducing the lab

at California State University, Fullerton to Neotropical characids.

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References cited

Albert, J., Lovejoy, N., Crampton, W. 2006. Miocene tectonism and the separation of cis- trans-

Andean river basins: evidence from Neotropical fishes. Journal of South American Earth

Sciences. 21:14-27.

Anderson, J., Rojas, J., and Flecker, A. 2009. High-quality seed dispersal by fruit-eating fishes in

Amazonian floodplain habitats. Oecologica. 161: 279-290.

Boucher, I., Vadeboncoeur, C., and Moineau, S. 2003. Characterization of genes invovled in the

metabolism of α-galactosides by Lactococcus raffinolactis. Applied and Environmental

Microbiology. 69: 4049-4056.

Briggs, J. 2005. The biogeography of otophysan fishes (Ostariophysi: Otophysi): a new

appraisal. Jounral of Biogeography. 32:287-294.

Calcagnotto, D., Schaefer, S., and Desalle, R. 2005. Relationships among characiform fishes

inferred from analysis of nuclear and mitochondrial gene sequences. Molecular Phylogenetics

and Evolution. 36:135-153.

Correa, S., Winemiller, K., Lopez-Fernandez, H., and Galetti, M. 2007. Evolutionary

perspectives on seed consumption and dispersal by fishes. Bioscience. 57:748-756.

de Merona, B., and Rankin-de-Merona, J. 2004. Food resource partitioning in a fish community

of the central Amazon floodplain. Neotropical Ichthyology. 2:75-84.

Da Silva, M., Silva, J., Marangoni, S., Novello, J., and Meirelles, N. 2004. A new method to

purify hepatic CYP1A of Prochilodus scrofa, a Brazilian freshwater fish. Comparative

Biochemistry and Phsyiology. 138: 67-74.

Damre, C., Kaddoumi, A., and Baer, K. 2009. Investigation of the multixenobiotic resistance

mechanism in the freshwater fishes western mosquitofish, Gambusia affinis, and the blusgill

sunfish Lepomis macrochirus. Bulletin of Environmental Contamination and Toxicology. 83:

640-643.

Drewe, K., Horn, M., Dickson, K., and Gawlicka, A. 2004. Insectivore to frugivore: ontogenetic

changes in gut morphology and digestive enzyme activity in the characid fish Brycon

guatamalensis from the Costa Rican rain forest streams. Journal of Fish Biology. 64:890-902.

Eastwood, M. 2001. A molecular biological basis for the nutritional and pharmacological

benefits of dietary plants. Association of Physicians. 94:45-48.

Evans, J., Klesius, P., and Shoemaker, C. 2009. First isolation and characterization of Lactoccus

garvieae from Brazilian Nile tilapia, Oreochromis niloticus (L.), and pintado,

Pseudoplathystoma corruscans (Spix & Agassiz). Journal of Fish Diseases. 32: 943-951.

14

Forbey, J. 2009. Fish Tissue Collection Protocol Detox Enzymes.

Freeman, B., Nico, L., Osentoski, M., and Jelks, H. 2007. Molecular systematics of

Serrasalmidae: deciphering the identities of piranha species and unraveling their evolutionary

histories. Zootaxa. 1484:1-38.

Gayet, M., Marshall, L., Sempere, T., Meunier, F., Cappetta, H., and Rage, J. 2001. Middle

Maastrichtian vertebrates (fishes, amphibians, dinosaurs and other reptiles, mammals) from

Pajcha Pata (Bolivia). Biostratigraphic, palaeoecologic and palaeobiogeographic implications.

Palaeogeography, Palaeoclimatology, Palaeoecology. 169:50-53.

Helfman, G., Collete, B., and Facey, D. 1997. The diversity of fishes. Blackwell Sciences, Inc.

43-45, 65-66.

Hemmer, M., Courtney, L., and Benson, W. 1998. Comparison of three histological fixative on

the immunoreactivity of mammalian P-glycoprotein antibodies in the sheepshead minnow,

Cyprinodon variegatus. The Journal of Experimental Zoology. 281: 251-259.

Hemmer, M., Courtney, L., and Ortego, L. 1995. Immunohistochemical detection of P-

glycoprotein in teleost tissue using mammalian polyclonal and monoclonal antibodies. The

Journal of Experimental Zoology. 272: 69-77.

Horn, M. 1997. Evidence for dispersal of fig seeds by the fruit-eating characid fish Brycon

guatamalensis Regan in Costa Rican tropical rain forest. Oecologia. 190:259-264.

Horn, M. 2009. Request for approval of animal care and use. IACUC. California State

University, Fullerton.

Hubert, N., Duponchelle, F., Nunez, J., Garcia-Davila, C., Paugy, D., and Renno, J. 2007.

Phylogeography of the piranha genera Serrasalmus and Pygocentrus: implications for the

diversity of the Neotropical ichthyofauna. Molecular Ecology. 16:2115-2136.

Hubert, N. Bonillo, C., and Paugy, D., 2005. Does elision account for molecular saturation: case

study based on mitochondrial ribosomal DNA among Characiform fishes (Teleostei:

Ostariophysii). Molecular Phylogenetics and Evolution. 35:300-308.

Javonillo, R., Malabarba, L., Weitzman, S., and Burns, J. 2010. Relationships among major

lineages of characid fishes (Teleostei: Ostariophysi: Characiformes), based on molecular

sequence data. Molecular Phylogenetics and Evolution.54:498-511.

Korneva, J. 2008. Nanobacteria associated with mucous intestines of freshwater fishes and

tegument of their parasites (Cestoda). Acta Parasitologica. 53:312-314.

15

Lee, S. Hedstrom, O., Fischer, K., Wang-Buhler, J., Sen, A., Cok, I., and Buhler, D. 2001.

Immunohistochemical localization and differential expression of cytochrome P450 3A27 in the

gastrointestinal tract of rainbow trout. Toxicology and Applied Pharmacology. 177: 94-102.

Lucas, C. 2008. Within flood season variation in fruit consumption and seed dispersal by two

characin fishes of the Amazon. 2008. Biotropica. 40:581-589.

Lundberg, J., Machado-Allison, and A., Kay, R. 1986. Miocene characid fishes from Colombia:

Evolutionary stasis and extirpation. Science. 234:208-209.

Lundberg, J., Marshall, L., Guerrero, J., Horton, B., Malabarba, M., and Wesselingh, F. 1998.

The stage for Neotropical diversification: a history of tropical South American rivers. Phylogeny

and Classification of Neotropical Fishes. 13-48.

Machado-Allison, A., and Garcia, C. 1986. Food habits and morphological changes during

ontogeny in three serrasalmin fish species of the Venezuelan floodplains. Copeia. 1:193-196.

Mirande, J. 2009. Weighted parsimony phylogeny of the family Characidae (Teleostei:

Characiformes). Cladistics. 25:1-40.

Mondal, S., Roy, T., Sen, S. and Ray, A. 2008. Distribution of enzyme-producing bacteria in the

digestive tracts of some freshwater fish. Acta Ichthyologica et Piscatoria. 38:1-8.

Monsch, K. 1998. Miocene fish faunas from the northwestern Amazonia basin (Columbia, Peru,

Brazil) with evidence of marine incursions. Palaeogeography, Paleoclimatology, Palaeoecology.

143:31-50.

Orti, G., Petry, P., Porto, J., Jegu, M., and Meyer, A. 1996. Patterns of nucleotide change in

mitochondrial ribosomal RNA genes and the phylogeny of piranhas. Journal of Molecular

Evolution. 45:169-182.

Orti, G., Sivasundar, A., Dietz, K., and Jegu, M. 2008. Phylogeny of the Serrasalmidae

(Characiformes) based on mitochondrial DNA sequences. Genetics and Molecular Biology.

31:343-351.

Purnell, M., Bell, M., Baines, D., Hart, and P., Travis, M. 2007. Correlated evolution and dietary

change in fossil stickleback. Science. 317.

Purnell, M., Hart, P., Baines, D. and Bell, M. 2006. Quantitative analysis of dental microwear in

threespine stickleback: a new approach to analysis of trophic ecology in aquatic vertebrates.

Journal of Animal Ecology. 75:967-977.

Roy, T., Mondal, S. and Ray, A. 2009. Phytase-producing bacteria in the digestive tracts of some

freshwater fish. Aquaculture Research. 40:344-353.

16

Scott, R., Ungar, P., Bergstrom, T., Brown, C., Grine, F., Teaford, M. and Walker, A. 2005.

Dental microwear texture analysis shows within species diet variability in fossil hominins.

Nature. 436.

Shuilleabhain, S., Davoren, M., Mothersill, C., Sheehan, D., Hartl, M., Kilemade, M., O’Brien,

N., O’Halloran, J., Van Pelt, F., and Lyng, F. 2005. Identification of a multixenobiotic resistance

mechanism in primary cultured epidermal complex mixtures on its activity. Aquatic Toxicology.

73: 115-127.

Sorenson, J., Skopec, M., and Dearing, M. 2006. Application of pharmacological approaches to

plant-animal interactions. Journal of Chemical Ecology. 32: 1229-1246.

Trambas, C., Wang, Z., Cianfriglia, M., and Woods, G. 2001. Evidence that natural killer cells

express mini P-glycoproteins but not classic 170 kDa P-glycoproteins. British Journal of

Haematology. 144: 177-184.

Vieira, V., Inoue, L., and Moraes, G. 2005. Metabolic responses of matrinxa (Brycon cephalus)

to dietary protein level. Comparative Biochemistry and physiology. 140:337-342.

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Appendix

Table A-1: Silver dollar standard length (mm), body mass (g), water quality, and key notes for

control, flake and sinking granule populations.

Table A-2: Key Notes for Silver Dollar (SD) feeding experiment.

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Table A-3: Temperature profile for twenty five (25) day feeding trials.

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Graph A-1: Temperature fluctuation throughout the 25 day feeding trials.

Graph A-2: Temperature range for the twenty five (25) day feeding trial varied by ±3.05 C. Y-

error bars represent 95% confidence intervals.

23.223.423.623.8

2424.224.424.624.8

2525.225.425.625.8

2626.226.426.626.8

2727.227.427.627.8

2828.2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Av

era

ge

Wa

ter

Tem

per

atu

re (°C

)

Day

Temperature (°C) Fluctuation over 25 Days

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23

24

25

26

27

28

29

30

31

Av

era

ge

Wa

ter T

emp

era

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(°C

)

Temperature Range ±3.05 °C

Min

Max

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Table A-4: Nutritional content in TetraMin flakes.

Graph A-3: Percent (%) composition of protein, fat and fiber in TetraMin flakes.

% Composition Flakes

Protein

Fat

Fiber

700 1400

2100

1

8000

390

0

2,000

4,000

6,000

8,000

10,000

mg

/kg

Flake Make up

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Graph A-5: Niacin, Inositol, Choline, Biotin, Omega-3 Fatty acid and Ascorbic acid make up in

TetraMin flakes.

Graph A-6: Vitamin A, D3 and E make up of TetraMin flake diets.

Table A-5: Nutritional content in TetraMin sinking granules.

15000

1400 140

0

5,000

10,000

15,000

20,000

Vitamin A Vitamin D3 Vitamin E

IU/k

g

Flake Make up

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Graph A-7: Percent (%) composition of protein, fat, and fiber in TetraMin sinking granules.

Graph A-8: Ascorbic acid makeup in TetraMin sinking granules.

% Composition Sinking

Granules

Protein

Fat

Fiber

400

0

100

200

300

400

500

Ascorbic Acid

mg

/kg

Sinking Granule Make up

Data collection for tracing the evolution of frugivory in characids (Ostariophysii: Characiformes)...

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