Symbiosis and Nitrogen Cycling: 
Physiological Effects of Anemone Shrimps on Host Sea Anemones in the Caribbean Sea 
 
by 
 
Channing Cantrell 
 
 
A thesis submitted to the Graduate Faculty of 
Auburn University 
in partial fulfillment of the 
Requirements for the Degree of 
Master of Science 
 
Auburn, Alabama 
August 2, 2014 
 
 
Keywords: tropical marine ecology, nutrient cycling, sea anemone, shrimp, 
coral reef symbiosis, Caribbean Sea 
 
 
Approved by: 
 
Jack Feminella, Chair & Professor of Biological Sciences 
Nanette Chadwick, Associate Professor of Biological Sciences 
Raymond Henry, Professor of Biological Sciences 
 Holly Ellis, Associate Professor of Biochemistry 
ii 
 
Abstract 
 Symbioses are characteristic of coral reef communities and can involve networks of 
species interactions as well as specialized adaptations to reef conditions. Because coral reefs are 
surrounded by nutrient-poor water, mutualisms provide an opportunity for tight nutrient cycling 
among reef organisms. However, little is known about how mobile ectosymbionts, such as 
crustaceans, benefit their hosts, including how they contribute to nutrient cycling in reef 
cnidarians. I examined the benefits of ammonium contributions by 2 species of obligate 
ectosymbionts, the spotted cleaner shrimp Periclimenes yucatanicus, and the Pederson?s cleaner 
shrimp Ancylomenes pedersoni, to selected physiological processes in host Caribbean sea 
anemones Bartholomea annulata. Rates of ammonia excretion from 3 client fish species were 
also measured to estimate the potential nitrogen contributions from client fishes which pose and 
are cleaned by shrimps near host sea anemones. Starved sea anemones were subjected to 1of 3 
laboratory treatments for 6 weeks: 1) shrimps present, 2) ammonium present, or 3) neither 
shrimps nor ammonium present (control).  Changes in anemone body size and protein content 
were measured along with the density, mitotic index, chlorophyll a content and mycosporine-like 
amino acids of their endosymbiotic microalgae Symbiodinium. Shrimps excreted ammonium at 
whole-organismal rates that were much lower than those of reef fishes, while sea anemones 
absorbed ammonium at relatively high rates, indicating that shrimp presence alone cannot meet 
the needs of host nutrient budgets. The only clear impact of experimental nutrient supplements 
was enhancement of the mitotic index of Symbiodinium. Shrimp presence also caused a small but  
iii 
 
 statistically insignificant increase in the oral disc size of sea anemones. Future experiments may 
demonstrate whether shrimp presence causes significant increase in anemone size, resulting in 
more obvious visual cues for client fishes to locate cleaner shrimp stations on the reef. The 
ammonia excretion rates of client fishes reveal that they theoretically are able to meet the 
nitrogen needs of host sea anemones. Thus, a nutrient-driven positive feedback loop may occur 
among among cleaner shrimps, host sea anemones, their endosymbiotic microalgae, and the 
client fishes attracted by the shrimps to these cleaning stations.  The ectosymbiotic shrimp alone 
do not appear to provide enough ammonium to support the physiological needs of microalgae in 
host sea anemones, but the potential client fishes may.  These mutualistic interactions may 
facilitate tight nutrient cycling among multiple levels of species belonging to different phyla in 
this symbiotic system. Further research on the impacts of ammonia and other nutrients provided 
by client fishes is necessary to determine benefits from this mutualistic interaction to host sea 
anemones. 
 
iv 
 
Acknowledgments 
I would like to say a special thanks to my advisor, Dr. Nanette Chadwick, for giving me the 
opportunity to pursue my interests in the field of marine ecology and for her guidance and feedback in 
preparing and completing my thesis. Her input and ideas throughout this time have been a cornerstone in 
the formation of this thesis and she has always provided valuable and insightful feedback during my time 
here at Auburn. You have truly helped me cultivate a deeper understanding of marine ecology and to truly 
appreciate the complex interactions among organisms that inhabit the reefs we study. 
I would also like to thank Dr. Raymond Henry for his critiques, precise and constructive advice, 
and guidance in the study of physiology. I would also like to thank the members of the Ellis lab, 
particularly Dr. Holly Ellis for her guidance through part of this thesis. I want to give Dr. Tony Moss a 
special thank you for his guidance and generosity with use of lab materials during my time completing 
my experiments.   
I would like to thank my parents, Steve Cantrell and Leah Keith, and my grandparents for their 
support and encouragement throughout my life, and especially at my time at Auburn University. I would 
also like to thank my friends and labmates, Jess Gilpin, Mark Stuart, Matt McMay, Erin O?Reilly, and all 
the other Chadwick lab members for their patience and help in lab. Without your assistance and feedback, 
I don?t know how I would have completed my thesis. I want to also give a final thanks to all of my friends 
who have been there to help me along the way, through all of my frustrations and celebrations.  
 
 
v 
 
Table of Contents 
Abstract?????????????????????????... .........????????? ?i i 
Acknowledgments????? ????????????????????????????. ..iv 
List of Tables????????????????????? ??????????????? vi 
List of Figures?????????????????????????????????? .? .vii 
Chapter I. General Introduction????????????????????????????? .1 
Chapter II. Effects of Symbiotic Cleaner Shrimp on Caribbean Sea Anemone Physiology: Nitrogen 
Cycling in a Coral Reef Mutualism?????????????????????????? ? ...9 
 Introduction???????????????????????????????? ?...9  
 Methods????????????????????????????????? ?.. .17 
 Results?????????????????????????????????? ?.2 5 
 Discussion???????????????????????????????? ...? 33 
Cumulative References???????????????????????????? .??? .. 67 
 
 
 
 
vi 
 
 
List of Tables 
Table1 ? Selected physiological parameters of sea anemones and other cnidarians. .. . . . . .45 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
vii 
 
List of Figures 
Figure 1.A) Ammonium excretion by Periclimenes yucatanicus?????.?.???????.47  
Figure 1.B) Ammonium excretion by Ancylomenes pedersoni. ????? ...??????... .47 
Figure 2.) Ammonium excretion rates of anemoneshrimps. . . . . . . . . . . . . . . . . . . . . .  . . . . . . ..48 
Figure 3.A)  Excretion rates in relation to mass of P. yucatanicus individuals . . . . . . . . . . . . . . 49 
Figure 3.B) Excretion rates in relation to mass of A. pedersoni individuals???  ??..??.. 49 
Figure 4.A) Ammonium excretion of Stegastes partitus 24 hours starved????.. . . . . . . . . 50 
Figure 4.B) Ammonium excretion of S. partitus 48 hours starved. . . . . . . . . . . . . . . . .  . .. . . . .50 
Figure 5.A) Ammonium excretion of S. partitus 2 hours after last feeding. . . . . . . . . . . . . . . . . 51 
Figure 5.B) Ammonium excretion rates of S. partitus. . ??????????????. ?.. 51  
Figure 6.) Excretion rates in relation to mass of S. partitus individuals?????.. ????.. 52 
Figure 7.A) Ammonium excretion of M. chrysurus 48 hours starved??????  ? . . . . . . .53 
Figure 7.B) Ammonium excretion of M. chrysurus 24 hours starved???    ???????53  
Figure 7.C) Ammonium excretion of M. chrysurus 2 hours after feeding??  ??????...53  
Figure 8.) Ammonium excretion rates of M. chrysurus individuals???????????..54  
Figure 9.A) Ammonium excretion of S. diencaeus 48 hours starved??????? ????55  
Figure 9.B) Ammonium excretion of S. diencaeus 24 hours starved?????????? ? 55 
Figure 9.C) Ammonium excretion of S. diencaeus 2 hours after feeding???????? ?. 55 
Figure 10.) Ammonia excretion rates of S. diencaeus individuals ?????????? ?. ..56 
Figure 11.) Mass-specific ammonia uptake rates of B. annulata??????????? ?.. 57 
Figure 12.) Ammonia uptake rates relative to mass in B. annulata???????????...58  
Figure 13.) Wet mass vs. tentacle crown surface area (TCSA) in B. annulata . . .. . . . . . . . . .  . 59 
Figure 14.A) TCSA variation in B. annulata  . . . . . . . . . . . . . . . .  .  . . . . . . . . . . . . . . . . . . . . . .60 
Figure 14.B) ODSA variation in B. annulata before and after experimental treatments. . . . . .. .60  
 
viii 
 
Figure 15.A) Preliminary experiment - Symbiodinium density in B. annulata tentacles. . . . . . . 61 
Figure 15.B) Preliminary experiment - Mitotic index of in situ Symbiodinium of B. annulata. . 61 
Figure 16.A) Preliminary experiment - Chlorophyll a of in situ Symbiodinium of B. annulata. .62 
Figure 16.B) Preliminary experiment - Animal protein content of B. annulata????  . . . . . .62 
Figure 17.) Symbiodinium density in B. annulata before and after experimental treatments. . . ..63  
Figure 18.) Mitotic indices of Symbiodinium populations in B. annulata before and afer 
experimental treatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ?. . . . ???  . . .64  
Figure 19.) Chlorophyll a levels of Symbiodinium cells of B. annulata before and after 
experimental treatments? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. ? ..? . . 65 
Figure 20.) Animal protein levels of B.annulata before and after experimental treatments. . . . .66 
Figure 21.) Total MAA levels of Symbiodinium cells from B. annulata before and after 
experimental treatment?.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . ??. . 67 
Figure 22.) LC/MS peak of the MAA Mycosporine-Glycine ????????...?????6 8 
Figure 23.) LC/MS peak of the MAA Mycosporine-2 Glycine ????.???????.?69  
Figure 24.) LC/MS peak of the MAA Asterina-330 ????????...????????..70  
Figure 25.) LC/MS peak of the MAA Palythene ????????????????....?.71  
Figure 26.) LC/MS peak of the MAA Palythine-Threonine-Sulfate ??????????.??72  
 
  
 
1 
 
Chapter One 
General Introduction 
 Coral reefs are some of the most productive ecosystems in the world and supply tropical 
coastal nations with food and other resources that contribute to economic development.  Reef 
systems are also a foundation for many tourist industries worldwide, often in countries with 
struggling economies (Sinclair 1998). These ecosystems have levels of biodiversity comparable 
to tropical rainforests (Hubbell 1997), but questions remain about how they achieve this in a 
nutrient-poor environment with low available nitrogen in the surrounding water column (<1 
?M/L, Muscatine and Porter, 1977). The intricate cycling of nitrogen in these vast communities 
is a key to understanding their productivity, as are many of the adaptations, symbioses and 
behaviors of reef organisms.      
On coral reefs, many benthic organisms form intimate symbioses, partly in response to a 
highly competitive environment where space and nutrients are limiting, particularly nitrogen 
(Trench, 1979). A major example of this relationship is the association between corals, the 
organisms that provide the physical structure of reef environments, and their dinoflagellate 
endosymbionts Symbiodinium. These dinoflagellates are symbiotic with corals and other benthic 
invertebrates such as sea anemones, clams and nudibranchs (Muscatine  and Porter 1977, Trench 
1979, Kempf 1984). The algae benefit from the association by obtaining protection from 
predation and from the host providing a physically and chemically stable environment inside 
another animal. In return, the animal host receives organic carbon skeletons produced from 
photosynthesis by the algal cells (Cates and McLaughlin 1976, Muscatine 1980, Battey and 
Patton 1987, Cleveland et al. 2011). When bleaching occurs, in which symbiotic corals and 
 
2 
 
anemones lose their microalgae, the hosts cannot survive for more than a few weeks on the 
particulate matter they capture from the water column. If the Symbiodinium algae are unable to 
recolonize the host, the result is a mass die-off of corals and other bleached cnidarians, 
threatening the structure and survival of entire reefs and the organisms which depend on them 
(Baker et al. 2008). Although one could argue that this specific symbiosis is the most integral to 
coral reef communities, it is certainly not the only symbiosis found in reef systems. The 
complexity and frequency of organismal interactions, including symbioses, are bioindicators of 
community-wide reef health, and can serve as proxies to estimate levels of biodiversity on 
regional and local scales (Linton and Warner 2003).  Examination of various symbioses in reef 
communities helps to tease apart the factors that allow such nutrient-barren environments to 
support high levels of biodiversity. Central to nutrient conservation is the formation of 
mutualistic symbioses that allow tight and efficient nutrient cycling between organisms, and 
contribute to the high productivity of reef ecosystems.  
Research on the flow of nutrients in these complex ecosystems is necessary to help us 
understand changes in organismal adaptations (like symbiosis) on reefs in response to 
environmental change, how these adaptations vary, and how they impact ecological function. In 
a reef system, each type of resource is often utilized by more than one species for a number of 
services. For example, a benthic invertebrate such as a sea anemone can be used by many 
organisms including fishes, crabs and shrimps for shelter and habitat (Chadwick et al. 2008). In 
the Red Sea, the bulb-tentacle sea anemone Entacmea quadricolor is used by the two-banded 
anemonefish Amphiprion bicinctus as a refuge and nesting site (Huebner et al. 2012). These fish 
use the stinging qualities of the anemone as a means of protection from predation as well as for 
provision of a suitable long-term habitat for a mating pair to spawn (Mariscal 1970). Benefits to 
 
3 
 
the anemone from the fish symbionts include nutrient enrichment (specifically nitrogen, Roopin 
and Chadwick 2008) and a reduction of hypoxic areas nearest the host due to ventilation 
behaviors of the fish (Szczebak et al. 2013).  A positive feedback loop of benefits to both 
partners  perpetuates the existence of the symbiosis, in which anemones hosting fish survive 
longer and grow larger than those without fish symbionts, thus allowing more and larger fish to 
inhabit them (Porat and Chadwick-Furman 2004).  Other types of symbioses between motile 
ectosymbionts and cnidarian hosts include the association between trapeziid crabs and reef 
building corals of the genera Acropora and Pocillopora. The crabs cause increases in the growth 
and survival rates of these coral colonies, in part because they remove sediment from among the 
coral branches (Stewart et al. 2006), an invaluable asset with increasing sediment loads being 
deposited on coral colonies worldwide. Trapeziid crabs also protect their branching coral hosts 
from predation by crown of thorns sea stars (Pratchett 2001). 
On Caribbean Sea coral reefs, the ubiquitous corkscrew sea anemone Bartholomea 
annulata hosts several species of small crustaceans (Knowlton and Keller 1985, Briones-Forzan 
et al. 2012). The shrimp Alpheus armatus is an obligate symbiont of these anemones and protects 
the host anemone from predatory bristle worms (Smith 1977, McCammon 2010). This alpheid 
shrimp also appears to prevent burial of the anemone in sand, by actively removing sand from 
around the host (S. Ratchford and N. Chadwick, unpublished data).  Other crustacean associates 
of this anemone include two species of paleomonid shrimps ? the spotted shrimp Periclimenes 
yucatanicus, and Pederson?s cleaner shrimp Ancylomenes pedersoni. The Pederson?s shrimp 
provides an ecosystem service which is imperative for reef fish populations ? ectoparasite 
control.  Cleaner organisms occur on reefs worldwide, and they are often either crustacean or 
fish species.  They function as organisms which keep ectoparasite loads in check on reef fish 
 
4 
 
populations, by consuming parasitic isopods and gnathiids (Bunkley-Williams and Williams 
1998, Sikkel et al. 2004). Although no experimental studies of the benefits of long-term cleaning 
over the lifetime of a fish are available, in situ work in the Caribbean on the longfin damselfish 
Stegastes diencaeus found that diel variation in gnathiid loads on fish positively correlated with 
how long clients spent at cleaning stations (Sikkel et al. 2004). The Pederson?s cleaner shrimp 
attracts client fishes from at least 16 families (Wicksten 1998, Hubebner and Chadwick 2012a), 
indicating that their cleaning stations provide services with widespread effects throughout the 
reef community. By increasing the health and well-being of resident fish populations, overall 
biodiversity of the reef system may be  increased. Effect of cleaners on client populations have 
been documented in Egypt and Australia, where the removal of cleaners results in a decline in 
fish biodiversity over the long-term (Bshary 2003, Grutter et al. 2003, Waldie et al. 2011).   
Without healthy populations of fish in reef systems, the trophic flow of energy can 
become easily disrupted by keeping most nutrients locked in primary and lower trophic levels. A 
loss of higher trophic levels of fishes may lead to decreasing efficiency and productivity yields 
on reefs, resulting in a decrease in species diversity and richness, and a decrease in overall 
ecosystem function (Roberts 1995). This has been observed in the Caribbean as the reefs of 
Jamaica and other nations have seen decades of degradation, pollution, nutrient loading and 
overfishing. The reef fish populations around Jamaica have steadily decreased over generations, 
resulting in a loss of total fish biomass (Hughes 1994, Holmlund and Hammer 1999).  Other 
areas in the Caribbean are facing similar degradation of their reef systems, which is jeopardizing 
the economic stability of many developing countries. With nearly two-thirds of all human 
populations living within 100 miles of a coastline, these countries often depend on the reefs as a 
constant resource for food and other materials necessary for sustaining their growing human 
 
5 
 
populations (Cesar et al. 2003). Overfishing is an increasing problem in marine ecosystems, with 
populations of once numerous fish species declining. The economic importance of fisheries in 
these regions of the world cannot be understated. As a human resource, in 2009 the Caribbean 
and South American region captured a total of 17.1 million tons of live fishes (FAO 2009), with 
most of the catch being kept and sold within the country.  
  Fish populations serve many ecological functions, such as linking different ecosystems 
(benthic vs. pelagic), providing services for other species (Roopin et al. 2008), preserving 
ecological memory and genetic diversity, and serving as major vehicles that drive nutrient cycles 
between communities worldwide, while  providing ecosystem resilience (Holmund and Hammer 
1999, Allgier et al. 2014).  In the Caribbean Sea, determination of physiological processes in 
marine organisms, such as their rates of photosynthesis, primary productivity, and growth, is 
often an overlooked component of coastal management, and cannot remain unaccounted for if 
integrated management strategies are to be effective (Linton and Warner 2003).  
Client fishes in the Caribbean Sea use the corkscrew sea anemone B. annulata as a visual 
cue to find shrimp cleaning stations on reefs (Huebner and Chadwick 2012b), highlighting the 
importance of the sea anemone host to cleaning interactions in this region. If anemone 
abundances decline, interspecies aggression can occur on sea anemones shared by more than one 
species of shrimp, indicating that cleaner dynamics may be susceptible to host anemone 
population declines (Silbiger and Childress 2008).  The same study also observed that densities 
of anemone and cleaner shrimp populations appear to be higher in areas frequented by potential 
fish clients, indicating a relationship between abundance of cleaners and clients. Focusing on 
potential interactions between these partners on a physiological level can help us understand the 
 
6 
 
underpinnings of how this relationship evolved, a process which remains unclear in many 
interspecies cleaning interactions (Poulin and Grutter 1996).  
 Other studies on the potential benefits to cnidarian hosts from their association with 
ectosymbionts have concluded, for example, that the close association of anemonefishes 
increases sea anemone fitness by increasing host surface area, oxygenation/respiration, and  
Symbiodinium densities, as well as chlorophyll a levels of the endoalgal symbionts (reviewed in 
Roopin et al. 2008, Szczeback et al. 2013). Nitrogen cycling from the excretion products of these 
fishes explains in part how such symbioses can evolve in an environment that is a nutrient desert. 
In symbiotic clams, an increase in available nitrogen in the form of ammonium under light 
conditions stimulates photosynthesis in in situ algal populations (Summons et al. 1986). Similar 
results were found in the scleractinian coral Pocillopora damicornis and the hydroid Myrionema 
amboinense (Fitt and Cook 2001, Hoegh-Guldberg 2006).  Other studies have demonstrated that 
association with an ectosymbiont can provide phosphate and nitrogen nutrient enrichment which 
is utilized for sea anemone growth, reproduction, regeneration, biosynthesis of metabolites, and 
stimulation of symbiotic algal growth (Muller-Parker et al. 1994, Godinot and Chadwick 2009, 
Cleveland et al. 2011).  While ammonium enrichment coupled with phosphate enrichment yields 
greater densities of Symbiodinium, nitrate and ammonium enrichment alone has shown lesser 
effects on algal populations, and phosphate enrichment alone showed no significant effects (Fitt 
and Cook 2001). These data are consistent with the results of nutrient enrichment in Red Sea 
coral populations, which indicate that corals and related species are nitrogen limited (Muscatine 
et al. 1989) and other work that indicates that this pattern is evident in other symbiotic marine 
organisms (Lee 1994).  While some research suggests that nitrogen could be supplied to hosts via 
shrimp symbionts (Spotte 1996), no definitive evidence is available concerning the physiological 
 
7 
 
benefits that cleaner shrimp may deliver to host anemones.  In the Indo-Pacific, anemonefish 
provide excess nutrients in the form of nitrogenous wastes that enhance host anemone fitness, 
but on Caribbean reefs it remains unclear how the symbiosis between sea anemones B. annulata 
and the shrimps P. yucatanicus and P. pedersoni may increase anemone fitness. Inorganic 
nitrogen can be utilized by the anemone?s symbiotic algae as a fertilizer, causing an increase in 
abundance and in organic compounds transferred to the host (Muscatine et al. 1984). These 
nutrients can include sugars, amino acids, and other nitrogenous compounds such as 
mycosporine-like amino acids (MAAs).  The idea that shrimp excretions may have positive 
effects on host anemones is a largely unexplored aspect of this symbiosis (Spotte 1996). The 
transfer of nutrients from shrimps to host anemones could have implications for the dynamics 
and evolution of their populations, as well as the conservation and management of these species.  
The objective of this thesis is to investigate potential physiological benefits of shrimp 
nitrogenous wastes to host sea anemones, by assessing how host body size, protein content, 
Symbiodinium populations, chlorophyll a and MAA levels are affected when shrimp presence 
and nutrient supplies are manipulated.  
 
 
 
 
 
 
 
 
 
8 
 
Chapter Two 
Effects of Symbiotic Cleaner Shrimp on Caribbean Sea Anemone Physiology:  
Nitrogen Cycling in a Coral Reef Mutualism 
 
Introduction 
 The metabolism of proteins and amino acid complexes by animals results in the 
formation of nitrogenous waste products in the body. In animal tissues, these occur primarily as 
forms of ammonia, which may be separated into dissolved gaseous ammonia (NH3) or the 
positively charged ion ammonium (NH4+). Many marine organisms excrete these products 
directly into the surrounding water column (Weihrauch et al. 2004). Mechanisms of total 
ammonia excretion have been a topic of debate, and often vary among environments and species. 
Rates of ammonia excretion among crustaceans vary up to 10x among species within genera 
(Dall and Smith 1986), and may be linked to specific activity levels and dietary factors. 
Excretion rates also varies with external conditions such as temperature, salinity, and time of 
day, as well as individual physiological state (starved vs. well-fed, healthy vs. injured), with fed 
organisms normally excreting more total ammonia than starved conspecifics (Rychly and Marina 
1977). Whether the organism is primarily aquatic or terrestrial can also have an effect on 
excretion rates and mechanisms, with terrestrial crustaceans often converting ammonia to less 
volatile compounds such as urea or purines, and then excreting them via specialized excretory 
organs. In contrast, amphibious crustaceans often retain their nitrogenous waste in the form of 
ammonia until they reach a body of water, and then release their wastes at high rates in a manner 
similar to fully aquatic species (Greenaway 1991, Weihrauch et al. 2004). External 
environmental levels of ammonia dissolved in seawater can exceed physiological concentrations, 
 
9 
 
thus impeding the process of passive diffusion of ammonia. This occurs, for example, when high 
rates of organic matter decomposition take place in low water flow or, in the case of lungfish, 
during drought season when external ammonia levels are concentrated in small volumes of 
surrounding water (Lohnse et al. 1993). This fact highlights the importance of ammonia 
excretion and mechanisms which work against potential and partial gradients under such 
conditions.  
Because ammonia quickly becomes protonated in the hemolymph or blood to form the 
lipid-insoluble ion ammonium, most early research on aquatic organism excretion focused on the 
discovery and explanation of mechanisms by which ammonium ions are expelled from the body 
in fish species (Evans 1977, Maetz 1973, Maetz and Garcia-Romeu 1964). Later though, it was 
realized that the proper measurements to determine acid/base balance in these experiments were 
not accounted for, and that Na+ uptake that appeared to be stimulated by ammonium injection 
could also be explained by H+ exchange. Once these experiments were repeated it was noted that 
a dramatic drop in pH occurred when NH4Cl was injected into catfish and trout species. The drop 
in pH in response to ammonium salt addition could only be explained when the permeability of 
gaseous ammonia and ionic ammonium were taken into account. This suggests that there is a 
cellular equilibrium of NH3 and NH4+ within the gill epithelium that allows for the passive 
diffusion of NH3. This is also supported by a small (~65 ?torr) partial pressure gradient which 
favors the passive diffusion of NH3 from the gills to the external aquatic environment. In order 
for this dramatic pH drop to occur, the permeability ratios must be greatly in favor of NH3 
diffusion, with over 50% of excreted excess total ammonia occurring in the form of NH3 
(Cameron and Heisler 1983). The ionic exchange of NH4+ with Na+ or H+ occurs in the gills of 
various crustaceans and invertebrates (Mangum 1978, Claiborne et al. 1982, Greenaway 1991). 
 
10 
 
In contrast, this mechanism in fishes may serve to maintain acid-base equilibrium and potential 
gradients which favor NH3 diffusion, and not direct NH4+ excretion.  
Another pathway by which NH4+ can enter the gill epithelium is by utilizing Na+/K+- 
ATPase.   In blue crabs, excretion rates correlated with Na+ uptake rates and depend on ATP 
availability. NH4+ can replace K+ ions due to their similar ionic properties, supporting the 
hypothesis that a Na+/K+- ATPase may facilitate excretion by allowing substitution of NH4+ in 
ion exchange (Pressley et al. 1981, Towle and Holleland 1987).  Other methods of ammonium 
excretion have been found in other marine species. In green shore crabs, H+- ATPase acidified 
vesicles collect gaseous ammonia and transform it into ammonium, trapping it until microtubules 
transport the vesicles to the apical cell membrane of the gill epithelium and release the 
ammonium via exocytosis (Weihrauch et al. 2002).  
Other potential mechanisms or facilitators of total ammonia excretion in crustaceans  
include  Na+/K+/2Cl- co-transporters and carbonic anhydrase (CA) activity (Henry 1988, Rankin 
and Jensen 1993). CA may maintain concentration gradients in favor of ammonium release in 
freshwater organisms, by creating an acidic environment at the gill epithelia which traps the ion 
ammonium in the water column, keeping the partial pressure gradient in favor of NH3 diffusion 
to the outside. This has yet to be demonstrated for marine species due to the confounding effects 
of ionic differences between fresh and saltwater and the higher buffering capacity of seawater. 
More recently, Rhesus (Rh) proteins are glycoproteins have been found in the gill tissue of some 
species of fish.  Rh protein gene sequences have similarities with another group of proteins 
demonstrated to function as ammonia transporters in yeast and plants (Huang and Peng 2005) 
and their function in fish and other marine species, is speculated to transport ammonia. They are 
speculated to function as ammonia transporters in some animals, because gene expression of this 
 
11 
 
class of glycoproteins occurs in various mammals and fishes (Weihrauch 2004, Nakada et al. 
2007, Nawata et al. 2007). They are thought to be a mechanism embedded in the gill epithelium 
for the transport of either NH3 or NH4+ through the gill epithelial layer to the external 
environment. This mechanism may function one of three ways; direct facilitated diffusion of 
NH3, transport of NH4+, or by coupling with Na+/H+ exchange, but this has yet to be determined. 
Gene expression of Rh proteins increased in response to elevated external ammonia levels 
(Wood and Wright 2009) supporting the theory that they function in ammonia excretion for these 
organisms.  
 Some fishes also convert internal ammonia to urea via uricolysis and excrete less toxic 
nitrogenous products (Smith 1929, Gregory 1977, Randall and Wright 1987), however this 
process is not common with most marine fishes due to the disadvantages of excreting urea as 
nitrogenous waste in seawater compared to using passive diffusion and partial pressure gradients.  
Some fish species, mainly elasmobranchs,  synthesize  and store urea, primarily as an osmolyte 
in order to maintain osmotic homeostasis in response to external osmotic changes (Ip and Chew 
2010).  
 In coral reef environments, symbiotic interactions facilitate tight nutrient exchange 
among organisms (Muscatine and Porter 1977, Sorokin 1993, Roopin and Chadwick 2009) and 
help organisms to cope with low nutrient levels in the surrounding water column. Nutrient 
cycling thus is likely to occur between mobile ectosymbionts (fishes and crustaceans) and their 
coral reef cnidarian hosts that harbor endosymbiotic microalgae. Fish ectosymbionts are known 
to provide essential nutrients to giant sea anemones in clownfish symbioses on coral reefs. Host 
anemones become larger, absorb more nitrogen, and withstand starvation longer when associated 
with symbiotic pomacentrid fishes (Holbrook and Schmitt 2005, Porat and Chadwick-Furman 
 
12 
 
2005, Roopin and Chadwick 2009). Anemonefishes also enhance oxygen exchange in host 
anemones (Szczebak et al. 2013), and supply other nutrients such as carbon and phosphate that 
may be used for host growth and metabolism (Godinot and Chadwick 2009, Cleveland et al. 
2011). In turn, anemonefishes are larger, older and have higher reproductive success when 
associated with larger anemones (Fautin 1992), thus creating a positive feedback loop of 
nutritional and other benefits among partners in this mutualism (Roopin et al. 2011).  Likewise, 
in the Caribbean Sea, excess nitrogen from crustacean ectosymbionts such as Pederson?s cleaner 
shrimp,  Ancylomenes pedersoni and spotted cleaner shrimp, Periclimenes yucantanicus, could 
provide valuable nitrogen and other nutrients for endosymbiotic mircoalgae, Symbiodinium spp. 
(also known as zooxanthellae) within the  tentacles of host corkscrew sea anemones, 
Bartholomea annulata. Benefits from this process are expected to cause physiological changes in 
both the host and symbiotic microalgal populations. 
Zooxanthella cells occur inside vacuoles within host cnidarian endothelial cells, the 
cytoplasm of which harbors high concentrations of nitrogen (5-50?M for ammonium) and 
phosphorous compared to the surrounding water column outside the host (<1?M, Wilkerson and 
Muscatine 1984). This relatively nutrient rich environment provides a suitable habitat for 
microalgal growth (Hoegh-Guldberg 2006). However, excessive nutrient levels have been shown 
to lower growth rates in the stony coral Stylophora pistillata, presumably due to inhibition of 
calcification reactions by excess available phosphorous, and decreased photosynthesis (Ferrier-
Pag?s et al. 2000). Nitrogen supplement experiments in B. annulata, reveal that nitrogen is stored 
in host anemone tissues surrounding the microalgal cells, as well as in  low molecular weight 
molecules (LMWM) pools in both host and algal cell bodies (Lipshultz and Cook 2000).  In the 
sea anemone Aiptasia pallida, exposure to high nitrogen levels (20-50?M) results in enhanced 
 
13 
 
rates of dark C fixation in microalgal cells, with light C fixation rates similar to those of well-fed 
individuals. In contrast, individual anemones starved for 30 days rapidly increase their uptake of 
carbon over time when incubated in nitrogen rich water (Cook et al. 1992). Other studies on the 
hermatypic coral Pocillopora damicornis and the hydroid Myrionema ambionense concluded 
that an increase in cell densities and mitotic indices occurs when available levels of ammonium 
in solution are increased (Fitt and Cook 2001, Hoegh-Guldberg 2006). 
 Generally, fertilizing endosymbiotic microalgal cells with inorganic nutrients leads to an 
abundance of sugars and nutrients in the algal cell body that are translocated to the host.  
Microalgal transfer of fixed carbon to their hosts primarily in the form of glycerol (Muscatine 
1967) and LMWMs that form amino acids. These molecules are then transformed and stored in 
host cells as lipids, wax esters and protein. All are used for anabolic activity and maintenance as 
well as cellular functions like osmoregulation (Battey and Patton 1987, Mayfield and Gates 
2007, Dubinsky and Stambler 2011).  Research conducted with B. annulata using 15N tracers in 
NH4+ revealed a rapid accumulation of label in internal host ammonium concentrations, with the 
amount of labeling increasing in zooxanthella LMWM pools over time. This continued even 
after host ammonium concentrations began to decline and turnover rates slowed (Lipshultz and 
Cook 2002). The data suggest that symbiotic algae growth and metabolism is controlled by the 
amount of ammonium made available within host cnidarian tissues. Host feeding rates are 
directly linked with continual algal growth and division also supports the theory that nitrogen 
obtained by the host regulates algal populations (Fitt and Cook 2001). Laboratory experiments 
on giant sea anemones have revealed that ammonium supplements significantly enhanced the 
abundance and mitotic index of their microalgae, resulting in prevention of shrinkage in starved 
anemones (Roopin and Chadwick 2009). 
 
14 
 
 Coral reef habitats are considered oceanic oases in a desert of clear, tropical waters that 
contain very little suspended organic matter or dissolved nutrients. Corals and other reef 
invertebrates are able to absorb these limited nutrients directly from the water column.  
Microalgal cells facilitate host absorption of dissolved ammonium, and assimilate ammonium via 
the glutamine synthetase/2-oxoglutamate animotranferase (GS/GOGAT) pathway (Anderson and 
Burris 1987, Wilkerson and Muscatine 1984), as first revealed in 1970 (Meers et al.) with the 
discovery of the enzyme glutamate synthase (GOGAT). Discovery of this new enzyme coupled 
with glutamine synthetase (GS) solved the question of which alternate pathway allowed 
ammonium uptake without utilizing glutamate dehydrogenase (GDH), when Km values for GDH 
in cnidarians exceeded intracellular NH4+ levels by an order of magnitude or more (Wilkerson 
and Muscatine 1987). Other data have demonstrated that the uptake and assimilation of NH4+ is 
concentrated in the tentacles of sea anemones, which suggests that algal populations are a strong 
influence on  host metabolism and nutrient uptake (Lipshultz and Cook 2002).  
 The regulation of Symbiodinium populations within host tissues is of great importance 
when considering host metabolism and dietary needs. These algal cells provide a source of 
energy for the surrounding animal cells, and can supplement up to 90% of host dietary needs, an 
especially important fact in an oceanic desert such as the tropical waters around coral reefs 
(Muscatine et al. 1984, Rahav et al 1989). Other major molecules absorbed and utilized by host 
cnidarian cells include amino acids and mycosporine-like amino acids (MAAs), which can only 
be found in various plants, fungi and protozoans (Haslam 1974) with many animals obtaining 
them from their diet. These molecules have photoprotective properties, with an absorbency 
spectrum of ~310-360 nm (Bandaranayake 1998, Stochaj et al. 1994). This class of molecules 
can confer advantages to cnidarian species living in the upper photic zone with daily exposure to 
 
15 
 
high levels of UVR (ultraviolet radiation) by combating the potentially detrimental effects of the 
UVR extremes that are characteristic of coral reef environments. These molecules have been 
shown to increase in macroalgae when they are subjected to high levels of nutrients (Korbee et 
al. 2004, Korbee et al. 2005), and thus also may be enhanced in endosymbiotic microalgae by 
nutrient enrichment to host sea anemones. 
This study quantifies rates of ammonia excretion by anemoneshrimps and client fishes of 
host sea anemones, as well as ammonia uptake rates of the anemones. It also determines whether 
nitrogen excreted by ectosymbiotic shrimps leads to enhanced levels of fitness-related 
characteristics in host sea anemones and their associated microalgae. The fitness characters 
examined here are host body size and protein content, and microalgal Symbiodinium abundance, 
mitotic index, growth rate, chlorophyll a and MAA content. Changes in these parameters are 
examined in response to experimental manipulation of shrimp presence and nutrient exposure.  
 
Methods 
 
Animal Collection and Maintenance  
 
 Individuals of the Corkscrew sea anemone (Bartholomea annulata), the spotted 
anemoneshrimp Periclimenes yucatanicus, and Pederson?s cleaner shrimp Ancylomenes 
pedersoni were collected by hand from hard bottom and rubble habitats at 0-3m depth on the 
bayside of the upper and middle Florida Keys. They were placed in plastic bags of seawater, 
 
16 
 
transferred to coolers with aerated seawater changed approximately every 12 hours, and 
transported to Auburn University within 1-3 days of collection. Individuals of bicolor damselfish 
Stegastes partitus, yellowtail damselfish Microspathodon chrysurus, and longfin damselfish 
Stegastes dienceaous were ordered from a commercial collector (Dynasty Marine, Marathon, 
FL) and shipped live to Auburn University. These 3 fishes were selected for study due to their 
small size (5-15 cm length) and omnivorous diet (Humann and Deloach 1989), which make them 
easy to culture in closed-system aquaria, and because damselfishes are common clients of 
Pederson?s cleaner shrimp (Cheney and Cot? 2001, Huebner and Chadwick 2012b)  
 For at least one month prior to laboratory experiments, all organisms were maintained in 
40 gallon saltwater aquarium tanks (77cm x 33cm x 32cm) at Auburn University under 
controlled conditions. Lights were kept on 12:12 hour timers and salinity (34-36 ppt) and 
temperature (25-26?C) were monitored daily and kept stable. Tanks were illuminated with 400W 
Radium Metal Halide Lamps (Ocean Light 250, AquaMedic). PAR (photosynthetically active 
radiation) levels in all tanks averaged 66-73 ?E/m2/sec; within the range of recorded irradiance 
levels in natural reef habitats occupied by these anemones (Nelson 2008), as measured using a 
QSL-2001 Scalar PAR Sensor (Biospherical Instruments, San Diego, CA, USA; for details on 
culture regime see Roopin and Chadwick 2009, Huebner et al. 2012). Nutrient levels were 
monitored throughout the experimental period, and levels of NH4+, NO2- and NO3?2 were 
maintained at low levels to mimic coral reef conditions.  All animals were fed regularly a diet of 
frozen shrimps (San Francisco Bay Brand Newark, CA, USA) and a commercial pellet food 
(Ocean Nutrition San Diego, CA, USA). Fish and shrimps were fed daily, while anemones were 
fed weekly.  All organisms were measured regularly, with most maintaining their original body 
sizes or growing during regular laboratory culture.  
 
17 
 
 
Nitrogen excretion and uptake rates 
 
 To quantify ammonium excretion rates by anemoneshrimps, a phenolhypochlorite 
method and methods adjusted from Solorzano (1969) and Spotte (1996) were used. Glass 
beakers (Fischer Scientific) for all analyses were rinsed first with 10% HCl solution, and then 
rinsed again with microfiltered saltwater (MFSW- 0.45?m). Beakers of MFSW were placed in a 
polystyrene tub with heated water (25-26?C) and a water circulation pump. To measure shrimp 
excretion rates, each individual shrimp (A. pedersoni: 0.06-0.25 grams wet mass, N = 12; P. 
yucatanicus: 0.12-0.36 grams wet mass N = 10) was placed in 250 mL of MFSW water for 
excretion studies. All shrimp excretions are daytime rates at 2 hours after feeding. All 
experiments included an empty vessel of MFSW as a control, in which the change in ammonium 
level was <1.0% of maximum ammonium levels recorded in experimental vessels  thus 
adjustments were not made to compensate for atmospheric loss or bacteria utilization of 
ammonium.  Light levels in sea anemone ammonium uptake experiments were maintained using 
halogen lamps, at natural levels described above.  Water samples (5mL) were taken from the 
beakers every 40 minutes for 2 hours, and immediately prepared for ammonium analysis (for 
methods see Solorzano 1969, Roopin et al. 2009).  
To quantify rates of ammonium excreted by potential client fishes of these 
anemoneshrimps, individuals of S. partitus (5.8-7.2 grams wet mass, N = 8), M. chrysurus (4.3-
5.9 grams, N = 4), and S. dienceaous (4.2 ? 5.7 grams, N = 3) each were incubated separately in 
750 mL of MFSW, with water samples taken every 20 minutes for 1 hour. Sampling intervals for 
 
18 
 
fishes were more frequent than for shrimps because the fish are larger vertebrates, so they 
produced more ammonium per unit time compared to shrimps. Beakers containing fishes 
received airstone bubblers to ensure high dissolved oxygen levels during excretion 
measurements of ammonium. Excretion rates were measured for fishes both when starved (24 
and 48 hours without food) and fed (2 hours after feeding, after Roopin et al. 2008).  
 Daytime rates of ammonium uptake were measured for host sea anemones B. annulata 
because they contain Symbiodinium microalgae, and thus were able to absorb inorganic forms of 
nitrogen from the surrounding water column during the daytime (Muscatine and D?Elia 1978, 
Wilkerson and Muscatine 1984, Lipschultz and Cook 2002). Each anemone used in treatment 
groups for laboratory experiments (see details below, N = 39) also was examined for variation in 
ammonium uptake rates among individuals, within 1 week after the experiments ended. Sea 
anemones were placed in 1L of MFSW in beakers under florescent light bulbs at the above 
natural irradiance levels. Due to their sessile nature, the anemones were allowed 20-30 minutes 
for acclimation from the disturbance of moving to the beakers, so they could attach their basal 
discs to the beaker and expand their tentacles. 5mL water samples then were taken from each 
beaker every 40 minutes for 2 hours, and immediately processed for quantification of NH4+  
(Solorzano 1969).  
 
Effects of shrimp nitrogen excretion on host anemones 
 
To determine potential impacts of nitrogen excretion by anemoneshrimps on host sea 
anemones, a preliminary laboratory experiment was conducted, in which 4-5 sea anemones were 
 
19 
 
randomly assigned to each of 9 tanks (N = 32 sea anemones total), and each tank then was 
randomly assigned to 1 of 3 treatments for a period of 6 weeks: (1) shrimp present: at least 3 
individuals of  A. pedersoni and 1 P. yucatanicus, (2) nutrient supplement of NH4Cl spiked 
seawater (see details below), or (3) neither shrimps nor added nutrients (control, modified after 
Roopin and Chadwick 2009). Thus, 3-4 anemones per tank x 3 tanks per treatment = 10-11 
anemones per treatment x 3 treatments, with 32 anemones total. Due to their symbiotic 
relationship with dinoflagellate Symbiodinium, these anemones are able to withstand periods of 
starvation (Battey and Patton 1987). Thus, to ensure that the anemones began the experiment 
without large energy or nutrient reserves, and were in a similar basal metabolic state so they 
potentially would respond to nutrient supplements, all anemones were subject to a month of 
starvation prior to the beginning of treatments. This starvation period was based on previous 
growth experiments on symbiotic anemone species in the same culture system (Godinot and 
Chadwick 2009, Roopin and Chadwick 2009).  
The treatment of NH4+ enrichment (~30?M) was administered 3x per week (every other 
day) for the duration for the experiment (6 weeks), with each enrichment period lasting 1.5-2 
hours. This differs from methods in Roopin and Chadwick (2009), in which individuals of the 
coral reef sea anemone Entacmea quadricolor were placed in nutrient treatments (~10?M) every 
day, because individuals of B. annulata are more delicate in body form, with more slender, 
longer columns and delicate tentacles. Thus, nutrient treatments were reduced in frequency to 
every other day, to avoid damage to the delicate body structure of this anemones through 
frequent moving and disturbance of individuals. Ammonium supplement concentration was 
increased in the present study, to compensate for the infrequency of nutrient baths compared to 
previous studies (Roopin et al. 2008). To control for effects of the physical disturbance of 
 
20 
 
moving anemones to and from the nutrient enrichment containers, every other day all sea 
anemones in all treatments were carefully extracted from their tanks and each placed a 1L plastic 
container which fit inside the experimental tank. Sea anemones in  shrimp and control treatments 
were exposed to micro filtered saltwater (MFSW, 0.45?) with no added nutrients, while nutrient 
enrichment anemones were placed in MFSW spiked to a concentration of 30 ?M/L. MFSW was 
used to ensure that no microorganisms utilized the inorganic nitrogen in the nutrient treatment 
water. 
The above preliminary experiment was conducted during June to August 2012 (N = 32) 
with all anemone parameters measured only at the end of the experimental treatments. Then a 
more complete experiment was run during March to May 2013 (N = 39), with all anemone 
parameters measured both before the start of treatments, and after they had ended. In both 
experiments, the following parameters were measured for each anemone: body size, protein 
content, chlorophyll a content, Symbiodinium density and mitotic index (after Wilkerson et al. 
1983, Kuguru et al. 2007, Godinot and Chadwick 2009, Roopin and Chadwick 2009).  
Mycosporine-like amino acid (MAA) content was analyzed only in the second experiment  
 
Measurement of physiological parameters in sea anemones 
 
To assess changes in anemone body size, tentacle crown surface area (TCSA) and oral 
disc surface area (ODSA) were measured. Calipers were used for all measurements, and 
calculations were made using the equation for the elliptical shape of the oral disc of sea 
anemones (Hattori 1991, Mitchell and Dill 2005, Huebner et al. 2012). 
 
21 
 
TCSA = ?
????? ?? ?22 WL
 
 To determine physiological parameters in tentacles of sea anemones, 5 tentacle clippings 
were obtained from each individual sea anemone, each 1-2 cm in length and weighing 0.025-
0.0120 grams. Clippings were made from long tentacles attached to the inner oral disc, avoiding 
the smaller outer tentacles, with no single tentacle sampled twice. Tentacle selection was based 
on length of tentacle, ease of collection and minimization of harm to the anemone. Clippings 
were taken at midday, both before and after 6 weeks of treatments, in the case of the second 
experiment. Each tentacles sample was homogenized in 1mL of MFSW and centrifuged at 5g for 
5 minutes at ~24?C, following a standard protocol for cnidarian tissue and algal separation. 
Supernatant containing homogenized animal tissue from samples was saved for immediate 
protein analysis. The remaining pellet of microalgal cells was vortexed thoroughly before being 
diluted for microalgal cell counts, mitotic index and chlorophyll analysis (after Porat and 
Chadwick-Furman 2005, Roopin et al. 2011).  
 Analyses of protein content in the animal fraction of tentacle samples were performed 
using the Bradford Method (BioRAD Quick start, after Roopin et al. 2011). Serial dilutions were 
made for the standard curve according to instructions provided with the BioRAD kit. Protein 
samples were analyzed immediately after tentacle homogenization (5000rpm for 5 minutes) at 
590nm using a Genesys 5 spectrophotometer.   
To assess microalgal cell abundances in anemone tentacles, five subsamples of each 
diluted algal slurry were taken and Symbiodinium cells counted using a haemocytometer under 
400x magnification with a confocal microscope. Preliminary 24 hour studies of B. annulata 
revealed no discernible diel pattern of microcell division in this species, similar to many 
 
22 
 
Caribbean corals (Wilkerson et al. 1983, Wilkerson et al. 1988), but unlike E.quadricolor and 
some other sea anemone species previously investigated (Wilkerson et al. 1988, Roopin et al. 
2011). Environmental influences, habitat or climate could be a possible explanation for this 
deviation from other documented anemone species. Therefore, tentacles clippings were taken 
haphazardly between the hours of 10:00 am and 4:00 pm.  Mitotic index was calculated as a 
percentage of doubling cells per 1000 cells. Doubling times and growth rates were calculated 
using methods and equations described in Wilkerson et al. (1983).    
 Chlorophyll analysis was performed on homogenized and centrifuged tentacle samples, 
also using the algal pellet portion that was re-suspended. Chlorophyll a was extracted from the 
re-suspended algal pellet using 90% acetone solution chilled to 4?C for 24hrs. The resulting 
slurry was later centrifuged at 5g for a minimum of 5 minutes, or until all debris was pelleted 
out. The resulting chlorophyll containing supernatant extracted and analyzed using a Genesys 5 
spectrophotometer at 630nm, 664nm, 690nm and 750nm (after Chadwick and Roopin 2009). An 
acetone blank was used to zero the spectrophotometer before each measurement was taken. 
Chlorophyll a content was quantified using equations from Jeffery and Humphries (1975). 
 To analyze anemone tentacles for MAA content, a different set of 5 tentacles was clipped 
from each anemone, in order to accurately quantify MAA amounts and normalize them to host 
tissue protein content.  Due to retraction of tentacles immediately after clipping, it is unlikely 
that any tentacles were sampled twice in this study. Tentacle clippings were blotted dry and 
weighed before being manually homogenized using a Potter-Elvehjem tissue grinder at room 
temperature. Samples were centrifuged for 5 minutes at 5000rpm. Protein portions were set aside 
for immediate analysis (see above to normalize MAA content to protein content. Algal pellets 
were concentrated using an Eppendorf Vacufuge for 1 hour or until dry. Pellets were frozen for 
 
23 
 
later analysis at -10?C for up to 2 weeks. Frozen pellets were thawed and  later suspended in 25% 
(v:v) methanol and sonicated using a Branson Sonifier 250 (Branson Ultrasonics Corporation, 
Danbury, CT) at 30W for 60 seconds, after which samples were heated at 45?C for 2 hrs. This 
extraction method was chosen due to the high extraction rate reported in Tartarotti and 
Sommaruga (2002). The solution was centrifuged at 10000rpm for 10 minutes. The resulting 
pellet was discarded and the supernatant filtered through a Millipore (Millex GP) 0.22?m filter. 
This supernatant was then stored at -80?C until LC/MS and spectrophotometric analysis.  
 
Liquid Chromatography /Mass Spectrometry 
 
 Extraction samples for MAA content were analyzed using an Ultra Performance LC 
Systems (ACQUITY, Waters Corp., Milford, MA, USA) coupled with a quadruple time-of-flight 
mass spectrometer (Q-Tof Premier, Waters) with electrospray ionization (ESI) in ESI+-MS mode 
operated by the Masslynx software (V4.1). Each sample, in aqueous solution, was injected into a 
C18 column (ACQUITY UPLC? BEH C18, 1.7 ?m, 2.1 x 50 mm, Waters) with a 150 ?L/min 
flow rate of mobile phase of solution A (95% H2O, 5% acetonitrile, 0.1% formic acid) and 
solution B (95% acetonitrile, 5% H2O,  0.1% formic acid) in a 10 min gradient starting at 95% A 
to 5% A in 6 min and back to 95%  in 8 min. 
The ion source voltages were set at 3 KV, sampling cone at 37 V and the extraction cone 
at 3 V. The TOF MS scan was from 200 to 800 m/z at 1 s with 0.1 s inter-scan delay. For s 
calibration, sodium formate solution (10% formic acid/0.1M NaOH/isopropanol at a ratio of 
1:1:8) at 1 sec/10 sec to ion source at 1?L/min was used.   
 
24 
 
LC/MS conditions were tuned so that metabolites were separated from each other in a 
linear gradient while maintaining optimal sensitivity. The instrument was calibrated at the time 
of data acquisition in addition to real time calibration by the lockmass. Mass accuracy at 5 ppm 
or less was the key for assuring the presence of target molecules from abundant noise molecules 
from the biological samples that could increase false positive results. Ion source parameters such 
as the source temperature (gas and sample cone), mobile phase flow rate, and cone voltage were 
fixed throughout analysis. 
Ions of interest were analyzed for mass accuracy, elemental composition (using accurate 
mass measurement of less than 5 ppm error) and isotope modeling to identify the formula.  
Identification of unknowns were confirmed by having the same retention times and molecular 
weight. 
Extracted MAAs were taken to the Mass Spectrometry Laboratory at Auburn University 
for acquisition and data analysis. Correspondence with published experts on MAA analysis in 
cnidarians revealed a lack of either individually prepared or commercially produced standards 
for MAA assessment in corals and sea anemones (personal communication, J.I. Carreto [South 
America], W.C. Dunlap [Australia], U. Karsten [Europe], and J.M. Shick [North America]). As 
such, after identification via LC/MS, samples were quantified using published maximum UV 
absorbencies and extinction coefficients (Bandaranayake 1998) and the Beer-Lambert equation. 
This method has been used recently as an accepted means to quantify MAAs in a Caribbean 
coral species (Torres-Perez and Armstrong 2012). 
 
Statistical Analyses  
 
 
25 
 
 All statistical analyses were conducted using R 3.0.2 For Statistical Computing. Variation 
in excretion rates between the two shrinp species examined was analyzed using nonparametric 
procedures (Mann-Whitney-Wilcoxon tests), while fish excretion rates were analyzed using 
repeated measures ANOVA with time as a repeated variable, because they were assessed at 
several times since feeding. Data from the preliminary experiment were analyzed using 
nonparametric methods (Kruskal-Wallis tests), while results from the second experiment were 
analyzed using repeated measures ANOVA with time as a repeated variable for all treatments. 
Tukey post-hoc testing was utilized to identify which groups differed from each other. 
Differences among groups were considered significant at p < 0.05 for all statistical tests. All 
results are presented as means +/- one standard error unless otherwise noted.  
 
Results 
 
Ammonium excretion by shrimps and fishes  
 
 Fed individuals of the spotted anemone shrimp Periclimenes yucatanicus (N =10) 
excreted ammonium at a rate of 3.44 ? 0.61 ?M g-1 hr-1, while fed individuals of Pederson?s 
cleaner shrimp Ancylomenes pedersoni (N = 12) excreted ammonium at 1.16 ? 0.12 ?M g-1 hr-1 
(Figure 1), significantly less than P. yucatanicus (Mann-Whitney U-test, U = 8.81, p < 0.01, 
Figure 2). Excretion rates for P. yucatanicus ranged from 1.03 to 5.96 ?M NH4+ g-1 hr-1, while 
those for A. pedersoni ranged from only 0.74 to 1.72 ?M g-1 hr-1. Rates of mass specific 
excretion for A. pedersoni increased with body mass, while no clear pattern was visible for P. 
 
26 
 
yucatanicus (Figure 3). Thus, whole-individual rates of excretion by P. yucatanicus (mean body 
mass 0.27 grams) and A. pedersoni (0.13 grams) were approximately 0.87 ?M NH4+ hr -1 (0.014 
?M NH4+ min-1) and 0.17 ?M NH4+  hr -1  (0.003 ?M NH4+ min-1), respectively.   
Rates of excretion by bicolor damselfish Stegastes partitus (N = 8) at 2 hours after feeding 
were 3.75 ? 0.17 ?M NH4+ g-1 hr-1, at 24 hours after feeding 2.42 ? 0.21 ?M NH4+ g-1 hr-1, and at 
48 hours after feeding 3.66 ? 0.31 ?M NH4+ g-1 hr-1(Figures 4 & 5).  These rates did not vary 
significantly with time since feeding (repeated measures ANOVA, F = 0.012, p = 0.916). Mass-
specific excretion rates decreased with increasing body mass (Figure 6). The total amount of 
ammonium excreted per individual was approximately 0.21 ?M NH4+ min-1 for individuals 
below 5 grams body mass, to about 0.41 ?M NH4+ min-1 for individuals above 5 grams (using 
basal excretion rate after 48 hours starvation). 
 Rates of excretion by the yellowtail damselfish Microspathodon chrysurus (N = 4) at 2 
hours after feeding were 4.85 ? 0.42 ?M NH4+ g-1 hr-1. Starved rates of excretion were 2.35 ?  
0.16 ?M NH4+ g-1 hr-1  after 24 hours of fasting, and 1.65 ? 0.1 ?M NH4+ g-1 hr-1  after 48 hours of 
fasting (Figure 7). Excretion rate varied significantly with time since feeding (Repeated 
measures ANOVA, F = 40.67, p < 0.001, Figure 8), with all pair-wise comparisons being 
significantly different (Tukey post-hoc tests, p < 0.05), except between 24 and 48 hours since 
feeding (p = 0.20). Rates of excretion in the fed state resulted in 2x to 4x higher rates of 
excretion when compared to 24 and 48 hours starved, respectively. Whole-animal excretion rates 
ranged from about 0.14 ?M NH4+ min-1 (for individuals < 5.1 grams) to 0.44 ?M NH4+ min-1 for 
individuals > 5.5 grams.  
 
27 
 
Rates of excretion in the longfin damselfish (N = 3) Stegastes diencaeus were 3.89 ? 0.13 
?M NH4+ g-1 hr-1 at 2 hours after feeding and 1.58 ? 0.26 ?M NH4+ g-1 hr-1 at 24 hours after 
feeding, and 1.60 ? 0.36 ?M NH4+ g-1 hr-1 at 48 hours (Figure 9). They varied significantly with 
time since feeding (repeated measures ANOVA, F =12.62, p < 0.05), except for between the 2 
starved states (Tukey post-hoc test, p = 0.99, Figure 10).  Whole-animal excretion for these 
juvenile fish was about 0.13 ?M NH4+ min-1 (<4.9 grams body mass) to 0.33 ?M NH4+ min-1 
(>5.1 grams).  
Ammonium uptake by sea anemones 
 
 The wet mass of individuals of B. annulata correlated positively with tentacle crown 
surface area (TCSA, Figure 13). Thus, all uptake rates were converted to mass-specific values 
based on TCSA?body mass conversions. Sea anemones that had been subjected to experimental 
treatments (shrimp, nutrients or neither, see Methods) had similar nitrogen uptake rates, with no 
significant difference among groups (ANOVA, F = 0.098, p = 0.907). Rate of ammonium uptake 
by B.annulata in all treatments pooled together (N = 32) was 3.47 ? 0.03 ?M L-1hr-1. Sea 
anemone ammonium uptake rates declined significantly with increasing mass (Figure 12). Mass 
specific rates were calculated via linear regression based upon individuals weighed and measured 
in lab (Figure 13). Ammonium uptake rates for individuals that had been cultured with 
anemoneshrimps (0.40 ? 0.07, ?M g-1 hr1), ammonium supplements (0.37 ? 0.12 ?M g-1 hr-1), or 
neither (0.23 ? 0.05 ?M g-1 hr-1) for the preceding 6 weeks were similar to those for the whole 
group together (Figure 12). The whole-animal nitrogen uptake rate for individuals of B. annulata 
 
28 
 
was around 5.05 ?M NH4+ hr -1 or 0.08 ?M NH4+ min -1 , based on a mean uptake rate of 0.34 ?M 
NH4+ g -1 hr -1 and 14.9 grams body mass on average. 
 
 
 
Effects of shrimp and nutrient treatments on sea anemones  
 
In the preliminary experiment, none of the examined parameters varied significantly 
among anemones after exposure to the 3 types of 6-week treatment groups. Anemone body sizes 
did not exhibit a clear pattern of overall increase or decrease in any treatment group. The mitotic 
indices of endosymbiotic microalgae also did not vary among the 3 treatment groups  (Kruskal-
Wallis test, ?2 = 3.47 , df = 2, p = 0.18) with indices for the shrimp, nutrient, and control 
treatments being 3.57 ? 0.75 %, 5.78 ? 0.93 % and 4.67 ? 0.71 %, respectively (Figure 15 B). 
Thus, at any given time, 3-6% of the microalgae cells in all of the sea anemones were 
undergoing mitotic division (Figure 15B). The T, or doubling time of groups also did not differ 
significantly between the three treatments (Kruskal Wallis test, ?2 = 3.31, p = 0.19) at 15.94 ? 
3.65 days for sea anemones exposed to symbiotic shrimps, 9.76 ? 3.56 days for sea anemones 
exposed to nutrient -rich water, and 8.25?0.98 days for control anemones exposed to neither 
treatment.   
 
29 
 
 The growth rate (day-1) of Symbiodinium, or ?, was 0.08 ? 0.02 in the shrimp 
treatment, 0.12 ? 0.02 in the nutrient treatment, and 0.10 ? 0.01 in the  control treatment, and did 
not vary significantly among the groups (Kruskal Wallis test, ?2 = 3.31, p = 0.19). 
The abundance of Symbiodinium cells varied widely among individual anemones and did 
not very significantly among treatments (Kruskal-Wallis test, ?2 = 1.37 p = 0.50); measured 
levels were 1.38 x 108? 8.65 x 106, 1.53 x 108 ? 1.36 x 107, and 1.99 x 108 ? 3.43 x 107 cells 
gwm-1 in the shrimp, nutrient and control groups, respectively (Figure 15A). 
Chlorophyll a level per Symbiodinium cell likewise did not vary significantly with 
treatment (Kruskal-Wallis test, ?2 = 5.23,  p = 0.07), and was 1.26 ? 0.43, 0.35 ? 0.11, and 0.71 
? 0.45 pg cell-1 in the shrimp, nutrient and control  treatments, respectively (Figure 16A). 
However, the p-value for this comparison was almost significant, and the mean values above 
show that chlorophyll in the shrimp treatment was almost 3x that in the nutrient treatment and 
almost twice that in the control.   
Finally, the protein content of host anemone tissues did not vary significantly (Kruskal-
Wallis test, ?2 = 0.63 p = 0.73) among groups (Figure 16B). Anemone tissue in each group had a 
narrow range of protein content between 30.0 -35.00 mg g-1. Shrimp treatment protein levels 
were 34.56 ? 3.26 mg g-1,  nutrient treatment levels were slightly lower at 32.45 ? 2.80 mg g-1, 
and control sea anemones had the lowest protein content of 31.35 ? 3.30 mg g-1 (Figure 8B).  
 In the second experiment, similar but slightly different trends were observed. Anemone 
body size measurements (TCSA) varied widely among individuals, but did not vary significantly 
with treatment (repeated measures ANOVA, F = 1.14, p = 0.33). The average change in TCSA 
for the nutrient group averaging -0.5%, -9.7% in the control group, and +17% in the shrimp 
 
30 
 
group TCSA declined in all 3  groups, from 1003.01 ? 272.59 cm2 to 968.37 ? 241.49 cm2 when 
cultured with shrimps . from 970.61 ? 148.18 cm2 to 689.00 ? 90.80cm2 when exposed to 
nutrients, and from 1031.66  ? 146.24 cm2  to 610.18 ? 88.49cm2 when exposed to neither 
(Figure 14A). 
Oral disc surface area (ODSA) also did not vary significantly with treatment (repeated  
measures ANOVA, F = 0.73, p = 0.49), but did not decline in  all groups.. Sea anemones 
subjected to nutrient treatments declined from 70.82 ? 18.00 cm2 to 46.97 ? 6.07 cm2 and control 
sea anemones declined from 69.96 ? 13.02 cm2  to 53.36 ? 11.77 cm 2, while  anemones cultured 
with shrimps increased slightly (but not significantly) in OSDA from 60.42?12.33 cm2 to 
76.67?14.78 cm2 (Figure 14B). 
Mitotic indices of Symbiodinium were significantly different among treatment groups 
(repeated measures ANOVA, F = 7.47 p ? 0.001), with a significant difference between 
anemones given nutrient supplements and control anemones (Tukey post-hoc tests, p < 0.001).  
The shrimp treatment group did not vary significantly from those given nutrient supplements 
(p>0.05). The algal mitotic indices for nutrient treatments almost doubled from 6.14 ? 0.86%  to 
11.8 ? 0.77% after 6 weeks of experimental treatment. Anemoneshrimp treatment indices also 
increased slightly from 5.97 ? 0.56% to 7.90 ? 1.38%. In contrast, for the control anemones 
exposed to neither anemoneshrimp nor nutrients, indices remained stable averaging 5.16 ? 
0.64% at the beginning of the experimental period to 5.80 ? 1.0% at the end of experimental 
period (Figure 18). Thus, algal division rates almost doubled with exposure to increased levels of 
nutrient treatments and became significantly higher than the control treatments.  
 
31 
 
The doubling time, or T, of each group also varied significantly among treatments over 
time (repeated measures ANOVA, F = 5.35, p ? 0.01), with a significant difference between only 
the nutrient and control groups (Tukey post-hoc, p < 0.05). Doubling times at the beginning of 
the experiment were 6.00?0.48 , 6.18?0.89  and 7.73?1.10 days for shrimp, nutrient and control 
treatment groups, respectively.  After 6 weeks of treatments the values for the shrimp group rose 
to 12.48?2.80 days, while nutrient treatments stayed stable with an average of 6.17?0.43 days. 
Control sea anemones saw the highest increase in doubling time with an average of 19.79?4.58 
days at the end of the experimental period.  
The growth rate (day-1) of Symbiodinium, or ?, before the experiment was 0.13?0.01 for 
the shrimp treatments, 0.13?0.02 for the nutrient treatments, and 0.11?0.01 for the control sea 
anemones. After the experimental treatments, microalgal growth rates of anemones exposed to 
anemoneshrimp rose to 0.16?0.03, while nutrient treatments saw the highest growth rate of 
0.24?0.02. Control sea anemones also saw a slight increase in growth rates with an average of 
0.122?0.02. These data reveal statistically significant variation in microalgal growth rates among 
the three groups over time (repeated measures ANOVA, F = 7.40, p ? 0.001), with differences 
only between the nutrient and control groups (Tukey post-hoc, p<0.05). 
Abundance of Symbiodinium cells in host tentacles did not vary significantly with 
treatments (repeated measures ANOVA, F = 0.202, p = 0.82, Figure 17). The abundance of 
microalgae in shrimp treatments increased slightly from 1.41x108 ? 0.15x108 cells gwm-1 to 2.0 
x108 ? 0.24x108 cells gwm-1. Nutrient treatments also saw a slight increase in density from 
1.60x108 ? 0.16x108  to  1.87x108  ? 0.16x108 cells gwm-1. Those anemones which were neither 
subject to anemoneshrimp nor nutrient treatments also saw a slight increase from their beginning 
density of 1.28x108 ? 0.14x108 to 2.0x108 ? 0.23x108 cells gwm-1 at the end. 
 
32 
 
Chlorophyll a levels additionally did not vary significantly between experimental groups 
(repeated Measures ANOVA, F = 1.54, p = 0.22, Figure 19). Average chlorophyll a levels in the 
shrimp treatment groups were 1.01 ? 0.22 pg cell-1 before the experiment and decreased to 0.22 ? 
0.11 pg cell-1. Anemones which had received dissolved nutrients also saw a decline in 
chlorophyll per algal cell from 0.71 ? 0.16 pg cell-1 to 0.20 ? 0.07 pg  cell-1. Anemones which 
received neither treatment saw a decline in chlorophyll a from 0.78? 0.17 to  0.31?0.14 pg cell-1. 
Animal protein levels in all groups remained stable with no significant difference over 
time among treatment groups (repeated measures ANOVA, F = 0.08 p = 0.93, Figure 20).  
Anemones exposed to shrimp had concentrations of 36.45?2.3 mg protein mg-1in the beginning 
and 36.51 ?3.8 mg protein g-1at the end. Protein content in the nutrient treatments declined 
slightly from 37.20?1.74 mg protein g-1 to 35.60?3.10 mg protein g-1. Anemones which received 
neither anemoneshrimp nor nutrient treatments averaged 36.80?1.78 mg protein g-1 in the 
beginning of the experiment and 36.67?2.92 mg protein g-1 at the end. 
  Total MAA levels did not vary significantly with treatment over time (repeated 
measures ANOVA, F = 0.27, p = 0.77, Figure 21).  MAAs found in B. annulata were 
Mycosporine-glycine (?max= 310 nm), Asterina-330 (?max = 300 nm), Palythinol (?max = 332nm), 
Palythene(?max = 360), and Palythine-threonine-sulfate (?max = 320nm, Figures 22-26). Total 
MAA levels declined in all treatments by almost half. Shrimp treatments declined from  822.50 ? 
49.82 to 476.00 ? 21.23 nmole mg protein -1, while nutrients treated anemones declined from 
960.26  ? 35.76 to 448.92 ? 22.17 nmols mg protein-1. Control anemones saw a similar decline 
from 620.48 ? 35.01 in the beginning of the experiment to 397.01 ? 20.17 nmols mg protein-1 
(Figure 21). 
 
33 
 
 
Discussion 
 
The whole-organism nitrogen budget for B. annulata on a per minute basis reveals a 
demand for nitrogen at a rate that outstrips the ammonia supplied by ectosymbiotic cleaner 
shrimps. With each shrimp weighing on average less than 0.5 grams, their contribution is small.  
In contrast, potential client fishes for the cleaner shrimps on this anemone had much higher 
nitrogen contributions. The damselfishes used in the present study were small juveniles, but large 
adult reef fishes such as groupers can contribute much greater amounts of ammonia at an average 
rate of ~8.75 ?M NH4+ g -1 hr-1 or  0.146 ?M NH4+ g -1 min-1   (Leung et al. 1999). As such, 
large client fishes could be selected by cleaner shrimps over smaller ones due to their potential 
for greater parasite loads, which is a direct benefit to the shrimp, but also possibly because they 
provide indirect benefits through nitrogen excretion to host sea anemones. The trophic levels of 
client fishes can also have varying impacts on host sea anemones. Larger fishes such as those in 
the family Serranidae (groupers) and could impact microalgal population patterns in host sea 
anemones through providing different N:P ratios, with piscivorous species potentially delivering 
higher phosphorous loads (Allgiers et al. 2014). The combination of N and P contributions 
impacts Symbiodinium populations, as well as potentially providing cleaner shrimp with a major 
food source, more so than do the nutrient ratios excreted by smaller-bodied herbivorous fishes 
(Poulin and Grutter 1996). Cleaner stations could be especially negatively impacted by 
population declines in large client fishes, which also tend to be overfished due to their economic 
value. In general, fish biomass and population dynamics contribute significantly to N:P ratios 
 
34 
 
that sustain coral reefs and their adjacent environments (Allgiers et al. 2014). Thus, overfishing 
of large client fishes populations could have unforeseen impacts on coral reef health, and should 
be of special concern to coral reef managers in the Caribbean Sea. 
Results from the preliminary laboratory experiment on benefits to host anemones from 
cleaner shrimps and nutrients were inconclusive. However, to accurately compare group 
changes, information from both before and after the treatments was needed. While the 
preliminary data did not support the idea that symbiotic shrimps contribute to anemone 
metabolism, they provided useful information on ranges of physiological parameters for this sea 
anemone, which is lacking for many tropical actiniarians. This is valuable information due to the 
abundance of B. annulata in the Florida Keys, with a recent census (Miller et al. 2009) revealing 
B. annulata as 90% of the total anemone populations surveyed from the Upper Keys to Key 
West.  
Symbiodinium counts were similar to those reported in previous publications on sea 
anemones that used equivalent methodology (Stambler and Dubinsky 1987, Spotte 1996, Roopin 
and Chadwick 2009), as were mitotic indices (Wilkerson et al. 1983, Table 1). Additionally, 
specific growth rate (?) and doubling times (T) were all within the ranges found in previous 
publications on other species of actiniarian anemones (Wilkerson et al. 1983, Porat and 
Chadwick 2005, Roopin and Chadwick 2009).  Animal protein content was slightly higher in B. 
annulata compared to some previous publications (Muller-Parker 1987), but still within the 
range of known protein content for sea anemones (Table 1).  The information in Table 1 
underlines the necessity for the use of uniform methodologies by future researchers, to provide 
accurate species to species comparisons for sea anemones.  
 
35 
 
In the second experiment, clearer patterns emerged, the most obvious being increased 
microalgal mitotic indices with the addition of nitrogen supplements to the water column, 
resulting in different patterns of algal replication and growth. Although no apparent effects of 
nutrient enrichment were seen in sea anemone size or protein content, the effect on in situ 
Symbiodinium populations was significant. This indicates that while excess nutrients do not 
make an observable effect on the sea anemone exclusively, the impacts on microalgal 
populations within anemone tentacles are clear. This pattern is seen in other cnidarian microalgal 
populations subjected to nutrient supplements (Fitt and Cook 2001, Hoegh-Guldburg 2006, 
Roopin and Chadwick 2009).  This, along with the fact that no significant change in algal density 
was detected in the present study, could suggest a high turnover rate for microalgal populations 
in this anemone species, with a potential to expel unwanted microalgae. Expulsion rates of 
microalgae have been demonstrated in some species of sea anemones, but not for corals. Rapid 
expulsion rates have been linked to high temperatures, with bleaching being the extreme end of 
microalgal regulation.  Various cnidarian species have exhibited a positive linear relationship 
between division rates and expulsion rates of Symbiodinium. The physiological mechanism of 
this expulsion is still unknown, but it is believed that some algal cells are selected for expulsion 
in order to keep the holobiont at a ?steady state? equilibrium.  Expelled algal cells also retain 
higher levels of 3 H-Thymidine, a chemical involved in the regulation of cell cycles, than algal 
cells kept in the host. This also supports the hypothesis that certain cells are selected and 
preferred for expulsion, with cell cycle phase as an indicator (Baghdasarian and Muscatine 
2000). Algal density patterns seen in this study could be an effect of this sea anemone species 
regulating in situ algal populations. Microalgae in starved Aiptasia exhibited a significant 
 
36 
 
decrease in chlorophyll and growth rates, and an increase in the carbon content of algal cells, 
consistent with the findings of the present study.  
MAAs found here in B. annulata are also consistent with previous publications that have 
reported MAA content of cnidarians. MAAs in the stony coral Porites furcata that were also 
detected here in B. annulata include Mycosporine-glycine and asterina-330 (Torres-Perez and 
Armstrong 2012), with asterina-330 also found in the macroalga Porphyra. Other research has 
revealed mycosporine-glycine and palythinol as primary MAAs found in the stony coral 
Acropora formosa, similar to MAAs found in this study (Dunlap and Chalker 1986). Although 
MAAs in another actiniarian sea anemone species, Anthopleura elegantissima, were not found in 
B. annulata, this could be due to differences in environment, as A. elegantissima is a temperate 
sea anemone in nutrient rich waters of the Pacific Ocean along the western coast of North 
America (Stochaj et al. 1994). 
Although, the decrease seen sea anemone size was unexpected and the results indicate 
that host anemones were not significantly impacted by shrimp presence alone, the data show a 
pattern of increasing mean oral disc size in anemones cultured with shrimps, compared to a mean 
decrease in the size of the oral disc in sea anemones cultured without shrimp or nutrients and 
those supplemented with nutrients, but no shrimps. This supports the hypothesis that sea 
anemones which are inhabited by shrimp may be more visible to client fishes (Huebner and 
Chadwick 2012b).  The results of this experiment indicate that shrimp presence alone may not 
provide enough nutrients to significantly alter the aspects of host anemone physiology or growth 
measured here. It is probable that the behaviors of obligate cleaner shrimps that draw client 
fishes close to host anemones also are of major importance in nutrient cycling among the 
symbiotic partners.  
 
37 
 
Figure Legends 
 
Figure 1.A) Ammonium buildup inside experimental vessels with Periclimenes yucatanicus  . . . 
?????????????????????????????????????? 41 
Figure 1.B) Ammonium buildup inside experimental vessels with Ancylomenes pedersoni. . . . . . 
. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41 
Figure 2.) Ammonium excretion rates of anemoneshrimps. . . . . . . . . . . . . . . . . . . . . .  . . . . . . ..42 
Figure 3.A) Ammonium excretion rates plotted in relation to mass of P. yucatanicus individuals . 
. . . . . . . . . . . . . . ??????????????????????????????...  43 
Figure 3.B) Ammonium excretion rates plotted in relation to mass of A. pedersoni 
individuals.????...........?????????????????????????.....  43 
Figure 4.A) Ammonium buildup inside experimental vessels with Stegastes partitus 24 hours 
after last feeding. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  44 
Figure 4.B) Ammonium buildup inside experimental vessels with S. partitus 48 hours after last 
feeding. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .44 
Figure 5.A) Ammonium buildup inside experimental vessels with S. partitus 2 hours after last 
feeding. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ????.... 45 
Figure 5.B) Ammonium excretion rates of S. partitus at 2, 24 and 48 hours after last feeding  . . 
?????????????????????????????????????. ...45  
Figure 6.) Ammonium excretion rates plotted in relation to mass of S. partitus individuals. . . ..46 
Figure 7.A.) Ammonium buildup inside experimental vessels with M. chrysurus individuals after 
48 hours of fasting? ???????????????????????????? ?47  
Figure 7.B)  Ammonium buildup inside experimental vessels with M. chrysurus individuals after 
24 hours of fasting? ?????????????????????????????.47  
Figure 7.C) Ammonium buildup inside experimental vessels with M. chrysurus individuals 2 
hours after feeding? ?????????????????????????????.47  
Figure 8.) Ammonium excretion rates of M. chrysurus  at 48, 24, and 2 hours after last 
feeding??????????????????????????????????. 48 
Figure 9.A) Ammonium buildup inside experimental vessels with S. diencaeus individuals after 
48 hours of fasting ??????????????????????????????49  
Figure 9.B) Ammonium buildup inside experimental vessels with S. diencaeus individuals after 
24 hours of fasting? ?????????????????????????????.49  
 
38 
 
Figure 9.C) Ammonium buildup inside experimental vessels with S. diencaeus individuals 2 
hours after feeding? ?????????????????????????????.49  
Figure 10.) Ammonia excretion rates of S. diencaeus individuals at 2, 24, and 48 after last 
feeding............................................................................................................................................50 
Figure11.) Ammonium level decline in experimental vessels with Bartholomea annulata 
immediately following 6 weeks of experimental treatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . .51 
Figure 12.) Mass specific uptake rates of B. annulata individuals?????????? ??52  
Figure 13.) Wet mass in relative to tentacle crown surface area (TCSA) in the sea anemone B. 
annulata . . . . . . . . . . . . . .  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ?????????? 53 
Figure 14.A) Tentacle crown surface area variation in B. annulata before and after 6 weeks of 
experimental treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .54 
Figure 14.B) Oral disc surface area variation in B. annulata before and after 6 weeks of 
experimental treatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .54  
Figure 15.A) Preliminary experiment - Symbiodinium density variation in B. annulata tentacles 
after 6 weeks of experimental treatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ?????. .55 
Figure 15.B) Preliminary experiment - Mitotic index variation of in situ Symbiodinium 
populations in B. annulata. . . . . . . . . . . .  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 
Figure 16.A) Preliminary experiment - Chlorophyll a variation of in situ Symbiodinium cells in 
tentacles of B. annulata. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..?.. 56 
Figure 16.B) Preliminary experiment - Animal protein content of B. annulata tentacles. . . . . . .56 
Figure 17.) In situ Symbiodinium density variation in B. annulata before and after 6 weeks of 
experimental treatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .57  
Figure 18.) Mitotic indices of in situ Symbiodinium populations in B. annulata before and after 6 
weeks of experimental treatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .58 
Figure 19.) Chlorophyll a levels of in situ Symbiodinium cells of B. annulata before and after 6 
weeks of experimental treatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59  
Figure 20.) Animal protein levels in tentacles of B.annulata before and after 6 weeks of 
experimental treatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .60 
Figure 21.) Total MAA levels of in situ Symbiodinium cells from B. annulata before and after 6 
weeks of experimental treatment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 
Figure 22.) LC/MS peak of the MAA Mycosporine-Glycine found in the Symbiodinium cells in 
the tentacles of B. annulata??????????????????????????62  
Figure 23.) LC/MS peak of the MAA Mycosporine-2 Glycine found in the Symbiodinium cells in 
the tentacles of B. annulata?????????????????????????.?63  
 
39 
 
Figure 24.) LC/MS peak of the MAA Asterina-330 found in the Symbiondium cells in the 
tentacles of B. annulata???????????????????????????..64  
Figure 25.) LC/MS peak of the MAA Palythene found in the Symbiodinium cells in the tentacles 
of B. annulata?? ?????????????????????????????.65  
Figure 26.) LC/MS peak of the MAA Palythine-Threonine-Sulfate found in the Symbiodinium 
cells in the tentacles of B. annulata??????????????????????????66  
 
 
40 
 
 
          Table 1 
Al
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                                                                               Figure 8 
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                                                                                                         Figure 9 
 
50 
 
                                      
                                                                                   Figure 10 
   
 
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51 
 
              
 
                
                                                                                             Figure 11  
0
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52 
 
       
                                                                         Figure 12 
 
 
 
 
 
y = 4.3388x-1.149 
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0
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0 10 20 30 40 50 60
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 g 
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-1 
 
Mass (g) 
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Shrimp
Nutrients
Control
 
53 
 
  
 
                                                                       Figure 13 
 
                                                                                       
 
              
y = 5.42e0.0011x 
R? = 0.34 
0
5
10
15
20
25
30
35
40
0 200 400 600 800 1000 1200 1400
We
t Mass
 (g
) 
Tentacle crown surface area (cm2) 
 
54 
 
                                   
             
                                                                          Figure 14 
0
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S h r i m p N u tr i e n ts N e i th e r
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ize
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          Figure 15 
 
0
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 Alg
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dance 
 
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 8 
cell
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0
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Shrimp  Nutrients  Control
Mit
otic 
ind
ex 
 
B 
 
56 
 
                                        
 
      Figure 16 
 
0
0.5
1
1.5
2
Ch
lor
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hyll
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(pg 
cell
 -1 )
 
 
A 
0
10
20
30
40
50
Shrimp Nutrients Neither
Prot
ein
 (mg g 
-1 )
 
B 
 
57 
 
            
                                                                                 Figure 17 
 
 
 
 
 
0
1
2
3
Shrimp Nutrients Neither
Alg
al Ab
un
danc
e  
 
(10
8  ce
lls 
g -
1 )
 
Before
After
 
 
58 
 
 
                       Figure 18 
 
 
 
 
 
0
0.05
0.1
0.15
Shrimp Nutrients Neither
Mit
otic
 in
de
x 
Before
After
 
59 
 
 
                          Figure 19 
 
 
 
 
0
0.5
1
1.5
2
Shrimp Nutrients Neither
Chlo
rop
hyll 
a  
(pg 
ce
ll-1
 ) 
Before
After
 
60 
 
 
          Figure 20 
 
 
 
 
 
 
0
10
20
30
40
50
Shrimp Nutrients Neither
Prot
ein
 ( mg 
g-1
) 
Before
After
 
61 
 
 
                        Figure 21 
 
 
 
 
 
0
200
400
600
800
1000
1200
Shrimp Nutrients Neither
(nmol mg
 pr
otei
n -
1 )
 
Tot
al 
MAAs
 
Before
After
 
62 
 
 
Figure 22 
 
 
63 
 
 
Figure 23 
 
 
64 
 
 
Figure 24 
 
65 
 
 
Figure 25 
 
 
66 
 
 
Figure 26 
 
 
 
67 
 
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