POPULATION BIOLOGY AND LANDSCAPE ECOLOGY OF DIGENETIC
TREMATODE PARASITES IN THEIR GASTROPOD HOSTS, WITH SPECIAL
EMPHASIS ON ECHINOSTOMA SPP.
BY
MICHAEL R. ZIMMERMANN
A Dissertation Submitted to the Graduate Faculty of
WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCE
in Partial Fulfillment of the Requirements
for the Degree of
DOCTOR OF PHILOSOPHY
Biology
May 2014
Winston-Salem, North Carolina
Approved By:
Gerald W. Esch, Ph.D., Advisor
Michael V. K. Sukhdeo, Ph.D., Chair
Herman E. Eure, Ph.D.
Erik C. Johnson, Ph.D.
Miles Silman, Ph.D.
Clifford W. Zeyl, Ph.D. ACKNOWLEDGEMENTS
This dissertation could not have been completed without the help of a number of people along the way. First and foremost, this body of work would not have been the same without the collaboration and mentorship of Dr. Esch. His knowledge of parasitology, ecology, and sheer excitement for biology and discovering the unknown after nearly 50 years of mentorship were truly inspiring and helped shape me into the biologist, researcher, and person I am today. I will forever be grateful for his influence.
I also would like to thank my committee members, Drs Eure, Johnson, Silman,
Sukhdeo, and Zeyl for providing feedback, lab space , and endless banter that have greatly improved this dissertation. Additionally, I am also grateful to have spent my time at
Wake Forest during both my Master’s degree and Ph.D. with fellow lab mate Kyle Luth.
His input into data interpretation, statistical analyses, and writing has greatly improved my skills as a scientist. If you are going to spend 10 weeks in a van collecting snails, fishing, and camping with somebody, you better get along with them, and Kyle and I have become great friends as a result.
I must also thank several of my other fellow graduate students. Daniel Griffith was integral in helping to formulate and design statistical analyses and the members of the Johnson lab helped keep my sanity while failing at molecular work through discussing moral conundrums such as the ethics of stealing bitcoins and seemingly daily debates of whether viruses are ‘alive’. Additionally, I would like to thank Mike Rizzo, who despite his efforts to contribute to research in the Each Lab, has never successfully hunted an animal or caught a fish in spite of 25+ years of effort. Despite this, he has remained a good friend and sport about being the butt of many jokes.
ii There are also a few people to thank for inspiring my journey into biology and parasitology. First, I would like to thank my grandfather Joe Spenoso for influencing my foray into fishing and interest in aquatic life. Although I was unaware at the time, his guidance peaked my interest in all living things associated with aquatic environments and
I would have not have taken the path I am on now without his influence. I also want to acknowledge my 7 th grade biology teacher, Marlene Osborn. It was after taking her class that I knew I wanted to pursue a career in biology. Her enthusiasm and initiative in creating a stimulating learning environment peaked my interest, a sentiment that never wavered. I would also like to thank my undergraduate mentor, Dr. Danny Ingold. His bolstered enthusiasm for teaching and life motivated me to pursue a graduate degree, which ultimately led to the completion of this dissertation.
My family was also extremely supportive during my past 6 years at Wake Forest.
My parents, sisters, and mother-in-law Liz have been extremely supportive in the pursuit of my graduate degrees. Pretending to be interested in my research (although not so much toward the end when they knew degree completion was imminent) and encouraging me along the way was much appreciated. Even though my Dad ‘would have been fired 3 and a half ye ars ago for taking this long to write a report’, the support from him and my
Mom never faltered. Although I am the last of the Zimmermann children to earn their doctorate, I know my parents are proud and thank them for their encouragement and support.
I would also like to thank my wife Abby, because without her this degree would not have been possible. She moved down to Winston-Salem when I started this degree, after spending two years apart when we were both completing our Master’s degree,
iii probably the longest two years of my life. Her support and urging (although it was probably more like shoving at times) allowed me to complete this degree. Additionally, her taking care of the duties at home despite working a full-time job were much appreciated, even though I will probably have to wash the dishes for the next year.
Thank you again Abby for your love and support.
Finally, I would like to dedicate this dissertation to Grandma Esther. Although she passed away in the early stages of this degree, just prior to her 97 th birthday, nobody would have been more excited to see me complete this dissertation. Despite her having to overcome the fact that I am a Republican, she let me be her cribbage partner and always treated me as if I was her own grandchild. Her joy and zest for life, in the face of dealing with bouts of macular degeneration, were truly an inspiration and have made me a better person. I promise that someday she will finally get her rematch with my parents in that game of euchre.
iv TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS……………………………………………………………… ii
LIST OF TABLES.. …………………………………………………………………… ....ix
LIST OF FIGURES.. ……………………………………………………………………. .xi
ABSTRACT…………………………………………………………………………… .xvi
CHAPTER I
Introduction and study sites
BACKGROUND.. ………………………………………………………...1
STUDY SITES
MALL ARD LAKE……………………………………………...... 9
COON LAKE…………………………………………………….12
OTHER U. S. SAMPLE SITES………………………………… ..14
LITERAT URE CITED…………………………………………………..15
CHAPTER II
Digenetic trematode life cycles shape their infection patterns in their pulmonate
snail hosts
ABST RACT……………………………………………………………..47
INTR ODUCTION……………………………………………………….48
MATERIALS AND METHODS………………………………………...51
RESULTS………………………………………………………………..54
DIS CUSSION……………………………………………………………58
LITERATU RE CITED………………………………………………….. 69
v CHAPTER III
Infection pattern differences of digenetic trematode parasites across the North
American waterfowl flyways
ABSTRACT ……………………………………………………………..99
INTRODUCTION ……………………………………………………...100
MATERIALS AND METHODS ……………………………………….103
RESULTS ………………………………………………………………104
DISCUSSION …………………………………………………………..105
LITERATURE CITED ………………………………………………… 110
CHAPTER IV
Habitat disturbance affects the distribution of trematode parasites among a
pulmonate snail community
ABST RACT…………………………………………………………….134
INTR ODUCTION………………………………………………...……135
MATERIALS AND METHODS……………………………………….137
RESUL TS……………………………………………………………....1 39
DISCUS SION…………………………………………………………..141
LITERATURE CITED………………………………………………… 145
Submitted to the Journal of Limnology (In Review)
CHAPTER V
Differences in microhabitat affect parasite transmission in Helisoma anceps
ABS TRACT…………………………………………………………….156
INTROD UCTION……………………………………………………...157
vi MATERIAL S AND METHODS……………………………………….160
RE SULTS………………………………………………………………163
DISCUSSION…………………………………………………………..165
LITERA TURE CITED…………………………………………………169
Submitted to Freshwater Science (In Review)
CHAPTER VI
Transmission pattern differences of miracidia and cerceriae larval stages of
digenetic trematodes in relation to snail size and density
ABS TRACT…………………………………………………………….178
INTROD UCTION……………………………………………………...179
MATERIAL S AND METHODS……………………………………….182
RE SULTS………………………………………………………………184
DISCU SSION…………………………………………………………..186
LITERA TURE CITED…………………………………………………1 91
Submitted to the Comparative Parasitology (In Review)
CHAPTER VII
Auto-infection by Echinostoma spp. in nature
ABSTRACT ………………………………………………………….....204
INTROD UCTION……………………………………………………...205
MATERIAL S AND METHODS……………………………………….208
RE SULTS………………………………………………………………210
DISCUSSION…………………………………………………………..213
LITERA TURE CITED…………………………………………………218
vii CHAPTER VIII
Differential infection patterns of Echinostoma spp. in snails belonging to three
different families
ABS TRACT…………………………………………………………….230
INTROD UCTION……………………………………………………...231
MATERIALS AND METHODS………………………………………. 235
RESUL TS……………………………………………………………....237
DISCUSSION………………………………………………………… ..240
LITERATURE CITED………………………………………………… 246
Submitted to Acta Parasitologica (In Press)
CHAPTER IX
The dilution effect in Echinostoma spp.: Examples from field collected data
ABS TRACT…………………………………………………………….257
INTROD UCTION……………………………………………………...2 58
MATERIALS AND METHODS……………………………………….262
RE SULTS………………………………………………………………264
DISCUSSION…………………………………………………………..266
LITERA TURE CITED…………………………………………………273
CHAPTER X
Summary ………………………………………………………………………..290
APPENDIX …………………………………………………………………………… ..298
CURRICULUM VITAE……………………………………………………………….. 341
viii LIST OF TABLES
Table I-1. Fish, amphibian, and reptile species observed in Mallard Lake ……………..22
Table I-2. Bird species observed visiti ng Mallard Lake………………………………...23
Table I-3. Parasite species, the hosts from which they were recovered, and the
parasitic stage recovered from Mallard Lake ……………………………..……...24
Table I-4. Vertebrate species observed in Coon Lake in the summer of 2013 ………….25
Table I-5. Parasite species, the hosts from which they were recovered, and the parasitic
stage recovered from Coon Lake ………………………………………………...26
Table I-6. Snail species and the number collected from sample sites across t he U.S.….27
Table II-1. Trematode parasite species found in Mallard Lake…..……………………..77
Table II-2. Trematode parasites infecting H. anceps in Charlie’s Pond………………...78
Table II-3. Trematode parasites infecting P. gyrina in Charlie’s Pond…………………79
Table III-1. Bodies of water sampled with permanent year round residents of
aquatic waterfowl ………………………………………………………………. 120
Table IV-1. Trematode parasite species f rom Coon Lake……………………………..148
Table VIII-1. Shell length and prevalence of Echinostoma sp. metacercariae from
5 pulmonate snail species……………………………………………………….251
Table AI-1. Other U.S. samples sites collected from in the summers of 2012
and 2013……………………………………………………………………… ...299
Table AII-1. P-values and AIC values for regression models of trematode
prevalence in Mallard Lake…………………………………………………… .301
Table AII-2. P-values and AIC values for regression models of trematode
prevalence in H. anceps from Charlie’s Pond……………………………… .....303
ix Table AII-3. P-values and AIC values for regression models of trematode
prevalence in P. gyrina from Charlie’s Pond………………………………… ...304
x LIST OF FIGURES
Figure I-1. Echinostome cercariae recovered from L. columella in Coon Lake………...29
Figure I-2. Adult echinostome recovered from an American Herring Gull…………….31
Figure I-3. Echinostoma spp. metacercariae in the kidney of H. anceps ………………..3 3
Figure I-4. Map of U.S. waterf owl flyways……………………………………………..35
Figure I-5. Satellite image of Mall ard Lake………………………………………...…...37
Figure I-6. Map of Mallard Lake transects and substrat um……………………………..39
Figure I-7. Satellite image of Coon Lake ……………………………………………….41
Figure I-8. Map of Coon Lake transects………………………………………………...43
Figure I-9. Map of other U.S. sample sites in the U.S. waterfowl flyways and the
presence/absence of Echinostoma spp……………………………………………...45
Figure II-1. Seasonal abundance of Canada Geese and Mallard in Mallard Lake……...81
Figure II-2. Seasonal prevalence of allogenic pa rasites from Mallard Lake…………… 83
Figure II-3. Seasonal prevalence of autogenic para sites from Mallard Lake…………...85
Figure II-4. Seasonal prevalence of allogenic parasites from Charl ie’s Pond…………..87
Figure II-5. Seasonal prevalence of autogenic para sites from Charlie’s Pond………….89
Figure II-6. Seasonal prevalence of Echinostoma sp. and Cotylurus sp. and the
monthly abundance of waterfow l in Mallard Lake…………………………………91
Figure II-7. Seasonal prevalence of H. occidualis in H. anceps and seasonal
abundance of H. occidualis in R. clamitans ………………………………………...93
Figure II-8. Seasonal prevalence of H. occidualis and M. temperatus in H. anceps
from Charlie’s Pond………………………………………………………………...95
xi Figure II-9. Seasonal prevalence of H. eccentricus and G. quieta in P. gyrina
from Charlie’s Pond………………………………………………………………...97
Figure III-1. Map of the ponds and lakes sampled across the U.S. with the 4 waterfowl
flyways ……………………………………………………………………….……122
Figure III-2. Prevalence and mean abundance of Echinostoma spp. metacercariae
infection in pulmonate snails in 2 yr of collection ………………………………...124
Figure III-3. Prevalence and mean abundance of Echinostoma spp. metacercariae
infection in pulmonate snails across waterfo wl flyways…………………...... 126
Figure III-4. Prevalence and mean abundance of Echinostoma spp. metacercariae
in collection sites with and without permanent waterfowl r esidents………………128
Figure III-5. Average precipitation in 3 waterfowl flyways between May and July
in 2012 and 2013…………………………………………………………………..130
Figure III- 6. Winter waterfowl abundance in Louisiana coastal marshlands………….132
Figure IV-1. Prevalence of autogenic and allogenic parasites in different habitat
types...... 150
Figure IV-2. Prevalence of autogenic and allogenic parasites and the distance from
the point of maximum disturbance …………………………………………… ..152
Figure IV-3. Generalized additive models of trematode prevalence and distance
from disturbance…………………………………………………………………...154
Figure V-1. Prevalence of parasites infecting H. anceps from Mallard Lake in 3
different substratum types…… ... ……………………………………………….174
Figure V-2. Intensity of parasites infecting H. anceps from Mallard Lake in 3
different substrata types……………………………………………………….. .176
xii Figure VI-1. Prevalence of Echinostoma sp. miracidia and cercariae infection and
mean intensity of metacercariae in sn ail size classes…………………………...196
Figure VI-2. Prevalence of Echinostoma sp. miracidia infection in size classes of 5
pulmonate snail specie s…..……..……………………………………………....198
Figure VI-3. Prevalence of Echinostoma sp. cercariae infection in size classes of 5
pulmonate snail species…………………………………………………………201
Figure VI-4. Mean intensity of Echinostoma sp. metacercariae infection in size
classes of 5 pulmonate snail species……………………… ... ………………….203
Figure VII-1. Prevalence and mean intensity of Echinostoma sp. metacercariae in
Mallard L ake……………………………………………………………………224
Figure VII-2. Mean shell length of H. anceps and infection of Echinostoma sp. in
Mallard L ake……………………………………………………………………226
Figure VII-3. Prevalence and mean intensity of Echinostoma sp. metacercariae in
the presence of re diae…………………………………………………………...228
Figure VIII-1. Prevalence and mean intensity of Echinostoma spp. metacercariae in
3 pulmonate snail genera……………………………………………………….253
Figure VIII-2. The prevalence and intensity of Echinostoma spp. metacercariae in
the presence of other pulmonate snail families…………………………………255
Figure IX-1. Frequency, density, and abundance of H. anceps across 139 U.S.
sample sites………………………………………………………………….….281
Figure IX-1. Prevalence of Echinostoma spp. miracidia infection in H. anceps in
the presence of varying numbers of snail species………… ..... ……………….. .283
xiii Figure IX-2. Prevalence of Echinostoma spp. miracidia infection in H. anceps in
the presence of other pulmonate snail species…………………...... ………….285
Figure IX-3. Prevalence and mean intensity of Echinostoma spp. metacercariae
in H. anceps in the presence of varying numbers of snail species ……………...287
Figure IX-4. Prevalence and mean intensity of Echinostoma spp. metacercariae
in H. anceps in the presence of other pulmonate snail species…………………289
Figure AI-1. Three species of trematode cercariae from L. columella in
Coon Lake………………………………………………………………………306
Figure AI-2. Echinoparyphium recurvatum cercariae from L. columella in
Coon Lake……………………………………………………………………… 308
Figure AI-3. Three species of trematode cercariae from P. gyrina in Coon Lake…… .310
Figure AI-4. Notocotylus attenuatus larval stages from P. gyrina in Coon Lake…… ..312
Figure AI-5. Unknown strigeid from M. dilatus in Coon Lake……………………… ..314
Figure AI-6. Chaetogaster limnaei limnaei from snails in Coon Lake……………… ..316
Figure AII-1. Seasonal patterns of the prevalence of E. trivolvis infections in
H. anceps from various years of collection in Charlie’s Pond………………… 318
Figure AII-2. Seasonal patterns of the prevalence of D. scheuringi infections in
H. anceps from various years of collection in Charlie’s Pond………………… 320
Figure AII-3. Seasonal patterns of the prevalence of E. trivolvis infections in
P. gyrina from Charlie’s Pond………………………………………………… .322
Figure AII-4. Seasonal patterns of the prevalence of E. parvum infections in
P. gyrina from Charlie’s Pond………………………………………………… .324
xiv Figure AII-5. Seasonal patterns of the prevalence of P. minimum infections in
P. gyrina from various years of collection in Charlie’s Pond. ……………… ....326
Figure AII-6. Seasonal patterns of the prevalence of an unknown strigeid infection
in P. gyrina from Charlie’s Pond……………………………………………….328
Figure AII-7. Seasonal patterns of the prevalence of H. occidualis infections in
H. anceps from various years of collection in Charlie’s Pond…………………330
Figure AII-8. Seasonal patterns of the prevalence of H. eccentricus infections
in P. gyrina from various years of collection in Charlie’s Pond…………….. ...332
Figure AII-9. Seasonal patterns of the prevalence of H. complexus infections in
P. gyrina from various years of collection in Charlie’s Pond………………… ..334
Figure AII-10. Seasonal patterns of the prevalence of M. temperatus infections in
H. anceps from various years of collection in Charlie’s Pond……………… ....336
Figure AII-11. Seasonal patterns of the prevalence of M. temperatus infections in
P. gyrina from various years of collection in Charlie’s Pond……………….….338
Figure AII-12. Seasonal patterns of the prevalence of G. quieta infections in
P. gyrina from various years of collection in Charlie’s Pond……………….….340
xv ABSTRACT
Michael R. Zimmermann
POPULATION BIOLOGY AND LANDSCAPE ECOLOGY OF DIGENETIC
TREMATODE PARASITES IN THEIR GASTROPOD HOSTS, WITH SPECIAL
EMPHASIS ON ECHINOSTOMA SPP.
Dissertation under the direction of
Gerald W. Esch, Ph.D., Charles M. Allen Professor of Biology
Digenetic trematode parasites are characterized by having complex life cycles involving both vertebrate and invertebrate hosts, and are grouped into 1 of 2 categories.
Allogenic parasites have at least one host capable of moving between aquatic habitats, while autogenic species have all hosts restricted to a single body of water. These life cycle categories help shape the transmission dynamics of trematode larval stages in their gastropod hosts. Allogenic parasites, particularly those cycling through waterfowl, have distinct seasonal trends in their infection patterns in gastropod first intermediate hosts due to their ephemeral nature, while autogenic species exhibit continuous recruitment due to the consistent presence of definitive hosts. Additionally, the ecology and abundance of the definitive hosts and snail size were intimately associated with the infection patterns of miracidia in their gastropod first intermediate hosts.
Changes in the environmental landscape resulting from natural and anthropogenic disturbance affect the abundance and distribution of trematode larval stages in gastropod first intermediate hosts. Differences in microhabitat associated with variations in the
xvi substratum resulted in infection pattern differences in trematode miracidia, cercariae, and the parasitic nematode Daubaylia potomaca . Bare clay/silt substratum had significantly greater parasite infection compared to substratum covered in leaves and other debris due to decreased obstruction in the former substratum type. Additionally, alterations in the habitat surrounding the pond affected not only host distribution, but the spatial arrangement of their parasites as well. Allogenic species using waterfowl as definitive hosts were found in the open habitats associated with disturbance, while autogenic species, cycling through frogs, infected snails adjacent to the densely wooded habitat.
Furthermore, increased trematode infection was observed in ranges associated with the edge of disturbance due to overlapping habitat of the avian and amphibian hosts.
Echinostoma spp. offer an interesting model system for studying trematode parasite transmission because they use aquatic gastropods as both first and second intermediate hosts, allowing the transmission of multiple larval stages to be monitored simultaneously. Transmission of miracidia and cercariae larval stages of Echinostoma spp. is influenced by different factors even though they both infect snails. Miracidia have high specificity for their snail hosts and are more successful in infecting larger gastropods that produce more distinct chemical cues. However, the prevalence of Echinostoma spp. cercariae infection is influenced more by host density than snail size, while the intensity of infection results in greater accumulation of metacercariae in the larger, older gastropod hosts. As a result of using snails as both first and second intermediate hosts, auto- infection can occur in these gastropods. Snails possessing first intermediate larval stages of the parasite had significantly higher levels of infection in comparison to gastropods
xvii lacking sporocysts and rediae, indicating that auto-infection occurs in nature, particularly in single snail populations.
Echinostoma spp. is a useful model because their larval stages, particularly metacercariae, are easy to track in their gastropod hosts. This allows cercariae infection patterns to be easily deciphered and monitored in nature. The ecology of second intermediate and definitive hosts played a large role in the recruitment patterns of metacercariae. The prevalence and abundance of Echinostoma spp. in gastropod intermediate hosts were associated with alterations in waterfowl distribution in recent yr.
Habitat degradation, climate change, and other abiotic factors have led to decreased parasitism in the southeastern U.S. and increases in permanent waterfowl likely led to increased parasitism by Echinostoma spp. in gastropods in the mid-Atlantic region.
Abiotic factors, namely precipitation, may have also led to differences between yr and flyways sampled.
Distinct differences in the infection pattern of snail second intermediate hosts were also observed. Gastropod species that were more active and had higher metabolic needs were more likely to recruit and accumulate metacercariae than more sedentary species that did not forage extensively. Additionally, the presence of other potential snail hosts created a dilution effect of parasitism in that as species richness increased, parasitism in a focal species decreased. Snail species that were not capable of harboring first intermediate larval stages had stronger diluting properties than snails that were compatible with Echinostoma spp. miracidia. Although often overlooked, the role of gastropods in trematode life cycles are integral for the understanding and assimilation of
xviii factors regarding host ecology and behavior, small and large scale changes in the environmental landscape, and disease transmission.
xix CHAPTER I
INTRODUCTION AND STUDY SITES
1 BACKGROUND
Digenetic trematodes are a group of parasitic flatworms characterized by having complex life cycles, oftentimes utilizing both invertebrate and vertebrate hosts. Most trematode life cycles incorporate 3 hosts, an invertebrate first intermediate host, typically a gastropod, and a wide array of second intermediate and definitive hosts. These parasites frequently utilize natural trophic dynamics (Lafferty, 2006; Lafferty et al., 2006;
Kuris et al., 2008), manipulate host behavior (Dobson, 1984; Poulin, 2010), or have lasting residual effects to aid in the completion of their life cycles. In the typical trematode life cycle, eggs are passed into an aquatic ecosystem where they hatch, releasing a free-swimming miracidium. Miracidia infect a molluscan first intermediate host and develop into asexually reproducing larval stages called sporocysts and rediae.
These stages produce free-swimming clones, called cercariae, which leave the first intermediate host, locate a second intermediate host, and penetrate it. These cercariae then encyst in the tissue of their second intermediate hosts and develop into a stage called the metacercariae. Metacercariae remain encysted until they are consumed by a definitive host, where they excyst and migrate to a final infection site.
Digenetic trematode life cycles can be divided into 2 categories, i.e., autogenic life cycles that involve the parasites infecting hosts that are restricted to a single aquatic habitat and allogenic cycles in which the helminths infect at least 1 host that is capable of moving between bodies of water (Esch et al., 1988; Criscione and Blouin, 2004; Fellis and Esch, 1995). These differences in life cycles can play important roles in the transmission dynamics between hosts, particularly the definitive and first intermediate hosts. Additionally, the biology and ecology of parasite hosts, both intermediate and
2 definitive, are important in the transmission dynamics of the trematodes (Smith, 2001;
Huspeni and Lafferty, 2004; Hechinger and Lafferty, 2005; Fredensborg et al., 2006).
Furthermore, the abundance of not only a focal host species, i.e., a natural host for the parasite, but decoy or alternative host species can play important roles in the infection patterns of trematodes (Ostfeld and Keesing, 2000a, 2000b; LoGuidice et al., 2003;
Keesing et al., 2006).
One important group of digenetic trematodes, at least in terms of wildlife disease, includes echinostomes. These species are responsible for zoonotic disease, particularly in the Far East (Graczyk and Fried, 1998), and are of growing concern in North America due to their high abundance, ubiquity, and low host specificity (Johnson and McKenzie,
2009). The family Echinostomatidae i ncludes 91 genera, with 85 species parasitic in vertebrates. A majority of these parasites infect wild animals, with Echinostoma being the most widely studied genus in the family. These parasites are distinguished by having a row of collar spines around t he oral sucker (Figs. 1, 2). Within this genus, the
‘revolutum ’ group is characterized by having 37 collar spines; they are abundant in North
America, with a cosmopolitan distribution across the continent (Johnson and McKenzie,
2009).
3 THE BIOLOGY OF ECHINOSTOMA SPP.
Life cycle
Echinostoma spp. exhibit a complex life cycle utilizing 3 hosts. Eggs are released from the adult worms and are deposited into a freshwater ecosystem, where they hatch.
A ciliated miracidium emerges from the egg and swims until it encounters its first intermediate host, an aquatic snail. The miracidium is a non-feeding stage of the parasite and can survive for only 12-24 hr (Anderson et al., 1982; Muñoz-Antoli et al., 2000;
Muñoz-Antoli et al., 2003). Due to the short window of infectivity, these larval stages utilize chemical cues to aid in host location (Haberl et al., 2000; Kalbe et al., 2000).
There is a high degree of specificity for the first intermediate hosts by Echinostoma spp. miracidia, with species of Helisoma , Lymnaea , and Physa , being the most commonly employed, although only the former 2 species serve as first intermediate hosts in North
America (Johnson and McKenzie, 2009). A variety of glycoproteins (MAGs) are used by these species and serve as cues for attracting miracidia (Haberl et al., 2000; Kalbe et al.,
2000; Kostadinova and Gibson, 2000). After penetrating the snail host, the miracidium sheds its sternal plates, develops into a sporocyst, and migrates to a species-specific site, usually the ventricle or aorta arteries (Esteban and Muñoz-Antoli, 2009).
After a period of 5-8 days, the sporocysts reproduce asexually and give rise to mother rediae, which migrate to the ovotestis and produce daughter rediae (Esteban and
Munoz-Antoli, 2009). Rediae have a mouth and rudimentary gut, which they use to actively consume host tissue and hemolymph (Esteban and Munoz-Antoli, 2009).
Additionally, rediae are predatory on the larval stages of other trematode species and other parasites that inhabit the snail (Kuris, 1990; Fernandez and Esch, 1991; Suhardono
4 et al., 2006; Zimmermann et al., 2011a). The daughter rediae then produce asexual clones, called cercariae, which is another free-swimming larval stage.
Echinostoma spp. cercariae are shed from the snail host and search for a second intermediate host, typically another gastropod. Similar to miracidia, cercariae are non- feeding larval stages and can only survive for 24-48 hr before depleting their energy stores (McCarthy, 1999; Toledo et al., 1999; Muñoz-Antoli et al. 2003). Similar to miracidia, echinostome cercariae also use chemical cues to aid in host location, but due to decreased host specificity, these cues are much more generalized than those from miracidia (Haas, et al., 1995; Haberl et al., 2000; Haas, 2003). Echinostome cercariae respond to amino acids, urea, ammonia, and other peptides produced by nearly all aquatic molluscs (Haberl et al., 2000; Haas, 2003). In addition to gastropods, amphibians are also common second intermediate hosts, where the parasite may cause considerable pathology and even death (Johnson and McKenzie, 2009). In amphibian tadpoles, cercariae frequently encyst as metacercariae in the renal region of both snail and amphibian hosts (Keeler and Huffman, 2009) (Fig. 3). Upon consumption by a definitive host, the metacercariae excyst in the intestine and become sexually mature.
Echinostoma spp. differs from most digenetic trematode parasites in that there is very little specificity for the definitive host. For example, E. revolutum has been documented in over 40 definitive hosts, and is considered to be one of the most ubiquitous of all trematode species in its distribution (Johnson and McKenzie, 2009).
Pathology
Pathology in the first intermediate host emanates from 2 sources, i.e., both rediae and cercariae. Rediae are predatory on other organisms that inhabit the snail and also
5 consume host tissue, typically the ovotestis. This ultimately results in castration of the snail, which may be highly detrimental to the host’s population biology. C ercariae induce a different kind of pathology, especially among juvenile snails. Cercariae must leave the first intermediate host in search of a new one and heavy infections can induce significant pathology and even mortality due to the migration of cercariae through host tissue (Kuris, 1980; Sorenson and Minchella, 1998).
Pathology in second intermediate hosts tends to be far more severe than in the molluscan first intermediate host, particularly among amphibians. The metacercariae aggregate in the kidney, where they stimulate localized immediate hypersensitivity reactions and edema, resulting in the death of young tadpoles that are still developing renal function. Heavy infections in the kidney of larval amphibians severely curtail the development to the adult frogs (Johnson and McKenzie, 2009). It has been asserted that echinostome infections are one of the major causes of amphibian decline in North
America (Schotthoefer et al., 2003; Skelly et al., 2006; Holland et al., 2007), and have been reported in a third of all amphibians sampled across the U.S. (Johnson and
McKenzie, 2009).
Although pathology caused by echinostomes in the definitive host is typically low, some detrimental effects have been reported (Chai, 2009). For instance, migratory birds harboring heavy infections with Echinostoma spp. may become debilitated during the migration (Maldonato and Lanfredi, 2009). Heavy infections by these parasites are known to produce enteritis followed by hemorrhagic diarrhea in humans and other definitive hosts. Most zoonotic echinostome infections occur in southeast Asia, due to cultural mores of consuming raw or undercooked animal tissue (Chai, 2009). In
6 immunocompromised hosts, typically the result of malnutrition and anemia, infection may be fatal; healthy immune systems, however, tend to subdue infections in a matter of weeks (Chai, 2009).
Genomics
Although there are no full genome projects currently underway, there has been a fair amount of sequencing performed on Echinostoma spp. (Morgan and Blair, 1995;
Morgan and Blair, 1998; Kostadinova et al. 2003; Detwiler et al., 2010; Detwiler et al.,
2012; Georgieva et al., 2013). Most sequencing has involved genomic DNA of
Echinostoma spp., with emphasis on the internal transcribed spacers (ITS-1 and ITS-2).
Additional sequences of the complete 18S and 5.8S genes, as well as partial sequences of the 28S genes, have also been catalogued. Mitochondrial markers such as NADH dehydrogenase subunit 1 (ND1) and cytochrome oxidase 1 (CO1) have also been sequenced and, in combination with the genomic markers, are used for taxonomy and phylogeny. Echinostoma spp. exhibit high taxonomic diversity, which can be attributed to the wide range of hosts and large geographical distribution these parasites employ
(Detwiler et al., 2010; Detwiler et al., 2012; Georgieva et al., 2013). Among the genomic sequences, there is very little divergence among the ‘ revolutum’ group (Marcilla, 2009).
This group includes 5 species of 37 collar spine echinostomes, i.e., E. revolutum , E. trivolvis , E. caproni , E. echinatum , and E. jurini (Fried and Graczyk, 2004). Within this group, the ND1 gene is a suitable marker for species or strain detection (Morgan and
Blair, 1998), whereas intraspecific differences of Echinostoma spp. can be detected using the ITS-2 marker (Marcilla, 2009). A comparison of both the ND1 and ITS markers allows clear classification of these echinostome species (Kostadinova et al., 2003).
7 Additionally, there is an elevated degree of variation in the ‘ revolutum’ group, suggesting high genetic diversity (Kostadinova et al., 2003), making it an intriguing group in which to study population genetics.
Population biology
Echinostoma spp., particularly E. revolutum , use a wide array of vertebrate animals as definitive hosts. More than 40 definitive hosts have been reported, with a majority being aquatic waterfowl (Johnson and McKenzie, 2009). The distribution of digenetic trematode parasites is often intimately tied to the behavior of their definitive hosts (Smith, 2001; Huspeni and Lafferty, 2004; Hechinger and Lafferty, 2005;
Fredensborg et al., 2006). In North America, waterfowl migrate between the breeding and wintering grounds on 4 distinct flyways (Fig. 4). The Atlantic flyway runs up and down the eastern seaboard of the U.S and encompasses all of the states that border the
Atlantic Ocean. The Mississippi flyway extends vertically from the eastern side of the
Mississippi River to edge of Atlantic Flyway. The central flyway extends from the western side of the Mississippi River to the eastern edge of the Rocky Mountains and the
Pacific flyway runs along the western seaboard of the United States extending to the western edge of the Rocky Mountains (Fig. 4).
8 STUDY SITES
Mallard Lake
Mallard Lake is a 4.9 ha pond located in the Piedmont region of North Carolina
(36 o00’03” N, 80 o24’07” W). Nestled within Tanglewood Park, Mallard Lake is frequented by the public for fishing, non-motorized boating, and other general uses, particularly in the summer mo. The majority of the eastern bank is part of a public access recreation park, while the northeastern portion is adjacent to a public golf course owned by Tanglewood. The western bank consists primarily of dense wooded vegetation and has limited access to the public, except for the occasional fisherman (Fig. 5).
There are 4 snail species present in Mallard Lake, including Helisoma anceps ,
Physa acuta , Lymnaea columella , and Menetus dilatus . Helisoma anceps was by far the most abundant snail species in the lake based on snail density surveys performed throughout the study. Although there is a limited littoral zone, the snails were ubiquitously distributed across the pond. Measurements of snail density were similar throughout Mallard Lake with the exception of highly aggregated populations within 2 man-made fishing stands, 1 on the southwest end and another on the northwest portion of the pond (Fig. 4; transects 48, 49, and 96). These areas were flat concrete barricades submerged in 10 cm of water and extended 1.5 m from the shoreline. These former fishing stands were partially covered in silt substratum and leaf debris, but offered an extended littoral zone for the snails to inhabit.
Species diversity of vertebrates in Mallard Lake was high, with 9 species of fish,
3 species of frogs, 4 species of turtles, and water snakes all being found within the pond
(Table I). The most abundant fish species in Mallard was bluegill sunfish ( Lepomis
9 macrochirus ), followed by redear sunfish ( Lepomis microlophus ). The largemouth bass
(Micropterus salmoides ) population was small and because of its importance as a sport fish and in an attempt to increase fishing activity within the pond, 250 largemouth bass, and 500 bluegill sunfish were added to Mallard Lake in the spring of 2008. An additional
10,000 fingerling bluegill and redear sunfish were added to the pond as forage for the bass (C. Weavil, pers. comm.). Frog sightings were sparse, but primarily occurred on the southern end of the pond near the island where there was a more extensive littoral zone as well as vegetative cover on the island itself. Yellow-bellied sliders ( Trichemys scripta scripta ) were the most commonly observed reptile in the pond, primarily inhabiting the concrete fishing stand and adjacent fallen timber in the southwest portion of Mallard
Lake.
There were both permanent and visiting waterfowl residents of Mallard Lake due to visitors consistently providing food for the birds (Table II). Canada geese ( Branta canadensis ) and mallard ducks ( Anas platyrhyncos ) were the most abundant species visiting the pond, with highest numbers in June and July (see Chapter III). The other migratory waterfowl were only observed over a short period of time and presumably just visited Mallard Lake as part of their migrations to their breeding or wintering grounds.
Belted kingfishers ( Megaceryle alcyon ) were most frequently observed in the summer mo, while 2 heron species were most abundant in the fall.
Prior to snail collections in the fall of 2008, the pond was divided into 148, 10-m transects (Fig. 6). Each transect was then surveyed to determine the dominant substratum type within each segment and classified into 1 of 3 categories. Leaf substratum was characterized as having at least two-thirds of the benthos being covered by leaves and
10 other decaying matter, clay/silt substratum was classified as having two-thirds of the bottom of the pond exposed with no decaying material, and mixed substratum had intermediate levels of benthic cover. A majority of the transects were classified as having leaf substratum (71%), while clay/silt (15%) and mixed (14%) substrata were similar in their abundance. A total of 4,219 H. anceps snails was collected between
September 2008 and June 2010.
Eight parasite species were recovered from H. anceps in Mallard Lake, as well as a commensalistic annelid, Chaetogaster limnaei limnaei (Table III). Although digenetic trematodes were the most speciose group represented, only Echinostoma sp. occurred consistently in prevalences > 3.0%. The nematode parasite Daubaylia potomaca and C. l. limnaei were also frequently found in the snail population (Zimmermann et al., 2011a,
2011b).
In the summer of 2011, Mallard Lake was overrun with an unidentified aquatic plant that covered ~70% of the lake’s surface. Although none of animal fauna appeared to be adversely affected, the pond was chemically treated to reduce the abundance of this vegetation. This decimated the snail population and concomitantly eradicated a parasitic nematode, Daubaylia potomaca , from the lake. In the summer of 2012, the water levels in Mallard Lake dropped 5-7 m to allow for the construction of a new drainage system from the adjacent golf course into the pond. This drastic decline in the water level further reduced what was left of the snail populations and the water levels have only recently been restored
11 Coon Lake
Coon Lake is a 1.5 ha pond also located within Tanglewood Park in Forsyth
County, North Carolina (35 o59’50” N, 80 o24’31” W). Although it is located within the public park, there is very little visitation to this pond due to its proximity to a golf course and lack of trails leading to it. The western edge of Coon Lake is adjacent to a public golf course and has been cleared of all vegetation due to the construction of a cart path along the bank. The remainder of the pond is surrounded by dense vegetation that provides cover and forage for the animals found in, and around, the lake (Fig. 7).
There are 3 species of pulmonate snails in Coon Lake, including Physa gyrina ,
Lymnaea columella , and Menetus dilatus that comprise 75.0%, 24.3%, and 0.7% of the snail fauna, respectively. A majority of the snails were found in the northwestern and southwestern portions of the lake due to the extensive littoral zone available for the gastropods. The western bank adjacent to the golf course had almost no littoral zone due to the man-made construction of the bank; in this location, there was a steep drop off; the southern end received very little sunlight and presumably had little macrophyte production due to limited littoral zone. Snail distribution was ubiquitous in the northwest and southwest portions of the lake.
Vertebrate diversity in and around the pond was much less than that of Mallard
Lake, but abundance of these animals was high (Table IV). There were healthy and abundant populations of all 3 fish species and much larger populations of amphibians in
Coon Lake, due to the abundance of suitable habitat surrounding the pond. However, reptile and bird diversity were significantly less in Coon Lake than in Mallard Lake. Due
12 to the healthy fish populations, no stocking efforts took place during the course of the work at the pond.
Prior to collections in 2013, the western half of the pond was divided into 40, 5-m transects (Fig. 8). A point in the center of the western bank, equidistant from the tree lines on either side, was considered to be the ‘point of maximum disturbance’ for the study site and the remainder of the transects were put into 1 of 3 categories. Open habitat was devoid of all trees and vegetation and was located exclusively on the western bank of the pond, wooded habitat were transects that were surrounded by dense vegetation and trees, and edge habitat were transects within 5 m of the edge of the tree line. A total of
2,798 pulmonate snails was collected between May and July in the summer of 2013.
Despite fewer vertebrate species inhabiting or visiting Coon Lake, increased vertebrate and gastropod abundance led to increased species diversity among trematode parasites
(Table V; Figs. AI-1 – AI-6).
In the summer of 2012, the water levels in Coon Lake were dropped 5-7 m to allow for the construction of a new drainage system on the western edge of the pond.
This drastic drop in water level did not affect the fish or amphibian populations, but did lead to the local extinction of a one-time abundant snail species in the pond, Helisoma anceps . The water was restored to normal levels after 2 mo, consequently, vertebrate and invertebrate populations recovered. During the warmest summer mo, i.e., July and
August, the pond is nearly covered in dense mats of Eurasian watermilfoil ( Myriophyllum spicatum ). This caused a majority of the snails to abandon the benthic habitat adjacent to the bank and occupy the dense plant mats that were rich in macrophytes and aufwuchs in the deeper water.
13 Other U.S. sample sites
A total of 11,227 aquatic snails was collected from 139 bodies of water from across the continental U.S., east of the Rocky Mountains, in the summers of 2012 and
2013 (Table AI-1). There were 15 species of snails collected from 3 different orders, but the majority were pulmonate snails belonging to the Basommatophora (Table VI). Snails were collected from 26 states, ranging from Maryland to Wyoming in latitude and North
Dakota to Louisiana in longitude (Table AI-1; Fig. 9). A variety of trematode parasites was recovered from these snails, but by far the most common were Echinostoma spp. metacercariae. These metacercariae were recovered from 58 (41.7%) of the sample sites and, although they are known to have a cosmopolitan distribution, they were rarely seen in the southern U.S. below the 35 th parallel north (Moran’s I, P = 0.0219; Fig. 9). Within the bodies of water in which Echinostoma spp. metacercariae were recovered, 19.8% of the snails were infected by the parasite.
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21 Table I. Fish, amphibian, and reptile species observed in Mallard Lake.
Vertebrate Species
Fish species Order Cypriniformes Family Cyprinidae American Gizzard Shad ( Dorosoma cepedianum ) Common Carp ( Cyprinus carpio ) Order Cyprinodontiformes Family Poeciliidae Mosquitofish ( Gambusia affinis ) Order Perciformes Family Centrarchidae Bluegill Sunfish ( Lepomis macrochirus ) Green Sunfish ( Lepomis cyanellus ) Largemouth Bass ( Micropterus salmoides ) Redear Sunfish ( Lepomis microlophus ) Warmouth ( Lepomis gulosus ) Order Siluriformes Family Ictaluridae Channel Catfish ( Ictalurus punctatus )
Amphibian species Order Anura Family Ranidae Bullfrog ( Rana catesbiana ) Green Frog ( Rana clamitans ) Pickeral Frog ( Rana palustris ) Reptile species Order Squamata Family Colubridae Northern Watersnake ( Nerodia sipedon ) Order Testudines Family Kinosternidae Common Musk Turtle ( Sternotherus odoratus ) Family Emydidae Painted Turtle ( Chrysemys picta ) Red Eared Slider ( Trachemys scripta elegans ) Yellow Bellied Slider ( Trachemys scripta scripta )
22 Table II. Bird species observed visiting Mallard Lake and the month when they most commonly visited the pond.
Month Most Bird Species Frequently Observed Order Anseriformes Family Anatidae American Black Duck ( Anas rubripes ) March Blue-Winged Teal ( Anas discors ) September Canada Goose ( Branta candiensis ) June, July Mallard ( Anas platyrhyncos ) July Peking Duck ( Anas platyrhyncos domesticus ) Year round
Order Coraciformes Family Cerylidae Belted Kingfisher ( Megaceryle alcyon ) May, June
Order Pelicaniformes Family Ardeidae Great Blue Heron ( Ardea herodias ) August Green Heron ( Buteroides virescens ) August
23 Table III. Parasite species, the hosts from which they were recovered, and the parasitic stage recovered from Mallard Lake.
Parasite Species Parasite Phylum (Class) Host Parasitic Stage
Chaetogaster limnaei limnaei Annelida (Oligochaeta) H. anceps Adult (Commensal)
Chaetogaster limnaei vaghini Annelida (Oligochaeta) H. anceps Adult
Sporocysts/Rediae Clinostomum marginatum Platyhelminthes (Trematoda) H. anceps Cercariae
Sporocysts Cotylurus sp. Platyhelminthes (Trematoda) H. anceps Cercariae
Daubaylia potomaca Nematoda H. anceps Egg - Adult
Sporocysts/Rediae Echinostoma sp. Platyhelminthes (Trematoda) H. anceps Cercariae Metacercariae
Sporocysts/Rediae Megalodisucs temperatus Platyhelminthes (Trematoda) H. anceps Cercariae
Unknown Mermithid Nematoda H. anceps Unknown
Sporocysts Uvulifer ambloplitis Platyhelminthes (Trematoda) H. anceps Cercariae
24 Table IV. Vertebrate species observed in Coon Lake in the summer of 2013.
Vertebrate Species
Fish species Order Cyprinodontiformes Family Poeciliidae Mosquitofish ( Gambusia affinis ) Order Perciformes Family Centrarchidae Bluegill Sunfish ( Lepomis macrochirus ) Largemouth Bass ( Micropterus salmoides )
Amphibian species Order Anura Family Ranidae Bullfrog ( Rana catesbiana ) Green Frog ( Rana clamitans ) Pickeral Frog ( Rana palustris )
Reptile species Order Testudines Family Emydidae Painted Turtle ( Chyrsemys picta )
Bird species Order Anseriformes Family Anatidae Canada Goose ( Branta candiensis ) Mallard ( Anas platyrhyncos ) Order Podicepiformes Family Podicepidae Pied-Billed Grebe ( Podilymbus podiceps )
25 Table V. Parasite species, the hosts from which they were recovered, and the parasitic stage recovered from Coon Lake.
Parasite Species Parasite Phylum (Class) Host Parasitic Stage
Sporocysts Australapemonus burtii Platyhelminthes (Trematoda) L. columella Cercaraie
Sporocysts Australapemonus canadiensis Platyhelminthes (Trematoda) L. columella Cercaraie
P. gyrina Chaetogaster limnaei limnaei Annelida (Oligochaeta) Adult (Commensal) L. columella
Sporocysts/Rediae Echinoparyphium recurvatum Platyhelminthes (Trematoda) L. columella Cercaraie Metacercariae
Sporocysts Glypthelmins quieta Platyhelminthes (Trematoda) P. gyrina Cercaraie
Sporocysts Haematoloechus complexus Platyhelminthes (Trematoda) P. gyrina Cercaraie
Sporocysts/Rediae Megalodiscus temperatus Platyhelminthes (Trematoda) P. gyrina Cercaraie
Sporocysts/Rediae Notocotylus attenuatus Platyhelminthes (Trematoda) P. gyrina Cercaraie Metacercariae
Sporocysts Plagiorchis sp. Platyhelminthes (Trematoda) P. gyrina Cercariae
L. macrochirus Pisciamphistoma stunkardi † Platyhelminthes (Trematoda) Adult M. salmoides
L. macrochirus Posthodiplostomum minimum † Platyhelminthes (Trematoda) Metacercariae M. salmoides
Sporocysts Tylodelphis sp. Platyhelminthes (Trematoda) L. columella Cercariae
Sporocysts Unknown strigeid Platyhelminthes (Trematoda) M. dilatus Cercariae † Luth, unpublished data
26 Table VI. The species of snail and number collected from 139 collection sites across the continental U. S.
Snail Species Number Collected
Order Architaenioglossa Family Ampullariidae Pomacea paludosa 50 Family Viviparidae Lioplax subcarinata 38
Order Basommatophora Family Ancylidae Ferrissia froquilus 3 Family Lymnaeidae Lymnaea columella 627 Lymnaea elodes 154 Family Physidae Physa acuta 4,500 Physa gyrina 2,792 Family Planorbidae Helisoma anceps 1,065 Helisoma trivolvis 1,360 Menetus dilatus 82
Order Heterstropha Family Valvatidae Valvata tricarinata 54
Order Neotaenioglossa Family Hydrobiidae Amnicola limosa 181 Somatogyrus parvulus 1 Family Pleurocercidae Pleurocerca canaliculata 295 Pleurocerca catenaria dislocata 25
27 FIGURE 1. Echinostome cercariae recovered from L. columella in Coon Lake.
28 29 FIGURE 2. Adult echinostome parasite isolated from an American herring gull ( Larus smithsonianus )
30 31 FIGURE 3. Helisoma anceps with the shell removed. The arrow indicates an infection by Echinostoma sp. metacercariae encysted in the kidney region of the snail.
32 33 FIGURE 4. Map of the waterfowl flyways in the United States.
34 35 FIGURE 5. Satellite image of Mallard Lake. Credit: U.S. Fish and Wildlife Services.
36 37 FIGURE 6. Map of Mallard Lake divided into 148, 10 m transects with the substratum type of each transect.
38 39 FIGURE 7. Satellite image of Coon Lake. Credit: U.S. Fish and Wildlife Services.
40 41 FIGURE 8. Map of Coon Lake with the western half of the pond divided into 40, 10 m transects with the point of maximum disturbance and the start of the tree lines indicated.
42 43 FIGURE 9. Map of the ponds and lakes sampled across the U.S. with the 4 waterfowl flyways. Dark circles denote where Echinostoma spp. metacercariae were present and light circles denote where the parasites were absent.
44 45 CHAPTER II
DIGENETIC TREMATODE LIFE CYCLES SHAPE THEIR INFECTION PATTERNS
IN THEIR PULMONATE SNAIL FIRST INTERMEDIATE HOSTS
46 ABSTRACT
Digenetic trematodes are parasites characterized by having complex life cycles that can be divided into 2 categories; allogenic life cycles, which have at least 1 host capable of moving between bodies of water and autogenic species in which all hosts are restricted to a single aquatic habitat. The transmission characteristics of trematode parasites having these 2 life cycle patterns were observed in pulmonate snail first intermediate hosts and compared with the abundance and behavior of their definitive hosts. The transmission blueprints of the trematode parasites were intimately tied to the abundance and seasonal dynamics of their definitive hosts. Allogenic parasites, particularly those cycling through aquatic waterfowl, exhibited distinct seasonal recruitment patterns closely associated with the ephemeral visitation of their definitive hosts, while autogenic parasites displayed continuous recruitment patterns due to the constant presence of their definitive hosts. Moreover, the seasonal dynamics of trematode parasites, regardless of life cycle type, were strongly correlated with the abundance of their definitive hosts and the size of the gastropods, while the infection patterns in the snails could possibly be used as a proxy for the abundance and species of definitive hosts present in an aquatic habitat.
47 INTRODUCTION
Digenetic trematodes are a group of parasitic flatworms characterized by having complex life cycles. Although there are many variations, most of these parasites include
3 hosts for completion of their life cycles. There is high specificity for the first intermediate host, commonly aquatic gastropods, which decreases in second intermediate and definitive hosts. Trematode life cycles can be divided into 2 categories. Allogenic life cycles include at least 1 host that is capable of moving between bodies of water, e.g., avian and mammalian hosts, while autogenic species are restricted to a single aquatic habitat, e.g., in fish, amphibian, and reptile hosts (Esch et al., 1988; Criscione and Blouin,
2004; Fellis and Esch, 2005). It is thus clear that the migratory, and non-migratory behavior of the definitive hosts plays an important role in shaping the seasonal periodicity of parasite infections in the intermediate hosts within an aquatic system.
Parasites with allogenic life cycles have greater colonization potential and dispersal ability due to both the aquatic and terrestrial nature of their definitive hosts, while autogenic species are limited in these aspects due to the completely aquatic dependence of their definitive hosts (Esch et al., 1988; Criscione and Blouin, 2004; Fellis and Esch,
2005).
The typical digenetic trematode life cycle involves eggs being shed into an aquatic ecosystem by a definitive host. Once the eggs are in water, they are either consumed directly by the first intermediate host or hatch into a non-feeding, free- swimming stage called a miracidia, which penetrates the first intermediate host, most commonly a gastropod. Once inside the snail, miracidia develop into asexually reproducing larval stages called sporocysts and rediae that inhabit the gonad or
48 hepatopancreas of their first intermediate hosts. After a period of 3-8 wk, a second free- swimming larval stage, asexually produced clones called cercariae, are derived from sporocysts/rediae and exit the first intermediate host into the water column to search for a second intermediate host (Meuleman, 1971; Lo and Cross, 1975; Zelmer and Esch, 2000;
Pinheiro et al., 2009). A notable exception is Halipegus spp., which produces immobile cystopherous cercariae that have increased longevity, but must be consumed directly by a second intermediate host (Krull, 1935; Thomas, 1939; Shostak and Esch, 1990; Goater et al., 1990; Bolek et al., 2010). Cercariae locate and infect a wide array of second intermediate hosts, ranging from macroinvertebrates to vertebrates, depending on the parasite, and penetrate and encyst in host tissue as metacercariae. Metacercariae remain encysted and viable for many weeks until the second intermediate host is consumed by the definitive host, where the parasite excysts, migrates to its final infection site, and develops into an adult.
These complex life cycles make the interactions between hosts integral for the transmission and understanding of the infection dynamics of trematode parasites. Many studies have shown the importance of second intermediate hosts for the transmission of trematode parasites to their definitive hosts. Often, natural trophic dynamics are utilized by these parasites to increase the chances of transmission (Lafferty et al., 2006; Lafferty,
2006; Kuris et al., 2008) and, on occasion, trematodes alter the behavior of their hosts to increase the probability of life cycle completion (Dobson, 1984; Poulin, 2010). Various aspects of host ecology are known to be linked to the transmission and accumulation of parasites in their hosts (Ezenwa, 2004; Loys et al., 2010; Chapter VII). However, far less
49 research has been performed in coupling definitive host behavior and the infection patterns of their gastropod first intermediate hosts.
The presence, abundance, and frequency of visitation by definitive hosts have been shown to impact parasite infection in first intermediate hosts, with a majority of these studies focusing on waterfowl definitive hosts and parasite transmission to gastropods (Smith, 2001; Huspeni and Lafferty, 2004; Hechinger and Lafferty, 2005;
Fredensborg et al., 2006). Although the life histories of these allogenic parasites have been frequently connected with definitive host behavior, fewer studies have addressed the infection patterns of autogenic parasites in their snail hosts. Definitive hosts for allogenic parasites, particularly migratory waterfowl, are usually ephemeral in their visitation, while autogenic parasite definitive hosts are consistently present in a particular aquatic system. It was predicted that the differences in the nature of the definitive hosts in these life cycle types play an important role in the infection dynamics and transmission patterns of the larval stages in snail first intermediate hosts, i.e., seasonal patterns in the presence and abundance of definitive hosts in an aquatic system will result in changes in the periodicity of parasite infection in their gastropod first intermediate hosts. This idea was tested by observing the abundance and frequency of definitive hosts visiting an aquatic system and then comparing these parameters with the infection patterns of the trematode parasites infecting snail first intermediate hosts. A comparison was also made with a nearby study site that has been researched extensively over the past 30 yr to help elucidate any trends in the patterns of infection associated with the life cycle variations.
50 MATERIALS AND METHODS
Study sites
Mallard Lake: Mallard Lake is a 4.9 ha pond located in Forsyth County, North
Carolina (36 o00’03”, 80 o24’07” W). This impoundment is adjacent to a public go lf course and park that are frequented by visitors and fisherman, particularly in the summer mo. The lake possesses 4 snail species, including Helisoma anceps , Physa acuta ,
Lymnaea columella , and Menetus dilatus , with H. anceps by far the most abundant. It also harbors several centrarchid fish species, as well as Gambusia affinis . Rana clamitans, R. catesbeiana, and R. palustris all inhabit the pond, although the abundance of these species is low. Additionally, due to high human traffic and feeding of the waterfowl, both permanent and visiting populations of Canada geese ( Branta canadensis ) and mallard ducks ( Anas platyrhynchos ) are present in the lake year round. Visiting piscivorous birds and other waterfowl species are also present.
Charlie’s Pond: Charlie’s Pond is a 1.7 ha impoundment located in Stokes
County, North Carolina (36 o17’17.7” N, 80 o03’44.7” W). The pond is spring fed and maintains fairly consistent water levels even with seasonal changes in precipitation and groundwater. The pond is situated on property owned by Duke Energy and public access is restricted (Esch et al., 1997). Charlie’s Pond has an extensive littoral zone and harbors abundant populations of H. anceps and P. gyrina snails, as well as smaller populations of
Gyraulus sp. and L. columella . Both R. clamitans and R. catesbeiana have been permanent residents of the pond since research began in 1984 (Crews and Esch, 1986).
Additionally, healthy populations of G. affinis and a variety of centrarchid fish species also inhabit the system. Aquatic waterfowl occasionally visit the pond, particularly in the
51 spring and autumn migrations, but there are no permanent residents (Esch et al., 1997).
Extensive research on the trematode communities infecting both H. anceps and P. gyrina has been performed in Charlie’s Pond over the last 30 yr, making it an ideal site to compare the infection patterns of various trematode species in their snail first intermediate hosts.
Data collection
Mallard Lake: A total of 3,384 H. anceps snails was collected from Mallard Lake between March and October of 2009. The pond was divided into 148, 10-m transects and snails were collected twice per wk. Prior to each collection, snail density and a census of the aquatic waterfowl was conducted, identifying both the species and numbers of birds present in, and around, the pond . Each snail was returned to the lab and isolated in a 55- mm fingerbowl for 48 hr before necropsy to check for patent trematode infections. The snails were then measured (to the nearest 0.05 mm) and necropsied to identify pre-patent infections of digenetic trematode species. All trematodes were identified to at least the genus level. Although snails were collected twice per wk, data were compiled monthly to maintain consistency with other studies.
Charlie’s Pond: Extensive seasonal data of digenetic trematodes infecting their gastropod first intermediate hosts has been collected in Charlie’s pond over the past 30 yr.
Helisoma anceps were collected and screened for trematode infections between March and November in 8 different collection seasons (Crews and Esch, 1986; Williams and
Esch, 1991; Fernandez and Esch, 1991b; Sapp and Esch, 1994; Schotthoeffer, 1998;
Negovetich and Esch, 2007); 18 different trematode species were isolated from these snails throughout the duration of this long-term study. For a complete list of these
52 trematode species see Negovetich and Esch (2007). Additionally, P. gyrina were sampled and checked for trematodes during the same seasonal periods in 3 yr of collection (Snyder and Esch, 1993; Sapp and Esch, 1994; Schotthoeffer, 1998) with at least 7 different species found infecting these snails. No new data were collected from
Charlie’s Pond for this study, but the previous collections were analyzed to determine if infection patterns relating to definitive host biology or life cycle type could be ascertained from this extensively studied system. Only trematode species with an overall prevalence of at least 1.0%, within a given year, were used in the analyses.
Statistical analysis
Trematode infections in snail first intermediate hosts were described in terms of prevalence according to the terminology of Bush et al. (1997). Linear and quadratic polynomial regression models were each fit to these data and the Akaike information criterion (AIC) was used to determine which model better explained the trends in prevalence, with a lower AIC score indicative of a better model fit. The better fit model was then compared to a null model, i.e., fluctuation around the mean, using one-way
ANOVA. A resulting p-value greater than 0.05 meant a failure to reject the null model and that neither the linear or quadratic models adequately explained the observed trends.
Additionally, a mixed effects regression model was performed on the prevalence of
Echinostoma spp. and Cotylurus spp. from H. anceps in Mallard Lake. Snail density, snail shell length, the abundance of waterfowl, and water temperature were used as fixed effects and the transect from which the snails were collected was used as a random effect.
All models and AIC analyses were performed using the R software (R Development Core
Team, Vienna, Austria).
53 RESULTS
Mallard Lake
Bird surveys
Canada geese and mallards were the most commonly observed waterfowl visiting
Mallard Lake and were most abundant in June and July (Fig. 1). Belted kingfishers
(Megaceryle alcyon ) were frequent visitors of the pond year round, often observed feeding in the early morning. Great blue herons ( Ardea herodias ) were largely absent between March and July, but were abundant in August, often with multiple herons visiting the pond in the remainder of the fall mo and into the winter.
Allogenic parasites
There were 4 species of trematode parasites found in Mallard Lake that had allogenic life cycles (Table I). Echinostoma sp., Diplostomum schueringi , and Uvulifer ambloplitis all showed similar trends in their seasonal parasite recruitment patterns in H. anceps (Fig. 2). Initial prevalence was very low in March, steadily increased until it peaked in the summer mo, and then declined in all 3 species as the winter mo approached.
The prevalence of Echinostoma sp. ( P = 0.048, R 2 = 0.49) and U. ambloplitis (P = 0.0160,
R2 = 0.67) were significantly explained by the polynomial model. Cotylurus sp., on the other hand, was not significantly explained by either the polynomial ( P = 0.0739) or the linear model ( P = 0.8182). However, the prevalence of this parasite species was best explained by the polynomial model (Table AII-1). The infection patterns of
Clinostomum marginatum differed significantly from those of the other allogenic parasites. The prevalence of infection of this parasite increased significantly as the
54 collection season progressed. These data were best explained by a linear model, which was statistically significant ( P = 0.0376, R 2 = 0.48) (Table AII-1, Fig. 2D).
There were no significant correlations between the prevalence of Echinostoma spp. infection in the snail hosts and snail density (Mixed Effects Regression , P = 0.2974) or water temperature (Mixed Effects Regression , P = 0.2181). However, significant correlations were observed between prevalence of Echinostoma sp. and the abundance of waterfowl (Mixed Effects Regression , P = 0.0011) and snail shell length (Mixed Effects
Regression, P = 0.0357). Similar trends were observed in Cotylurus sp. in which waterfowl abundance (Mixed Effects Regression , P = 0.0083) and shell length (Mixed
Effects Regression , P = 0.0394) showed a significant correlations with the prevalence of infection in the gastropod hosts
Autogenic parasites
Only 1 trematode species, Megalodiscus temperatus , with an autogenic life cycle was present in Mallard Lake (Table I). The prevalence of M. temperatus was low throughout the duration of the study period and neither the linear ( P = 0.4038) or polynomial model ( P = 0.5291) were statistically significant in explaining the observed trends. A failure to reject the null model indicated that the prevalence of M. temperatus fluctuated around a mean value (Table AII-1; Fig. 3).
Charlie’s Pond
Allogenic species
There were 5 species of allogenic trematodes analyzed from Charlie’s Pond
(Tables II, III). Echinostoma trivolvis, Echinoparyphium parvum, D. scheuringi , an unidentified strigeid, and Posthodiplostomum minimum all showed similar trends in the
55 prevalence of infection in their snail first intermediate hosts. Low levels of infection in
March were followed by rapid increases in prevalence as the collection season progressed.
After a peak in the summer mo, a rapid decline was observed (Fig. 4, AII-1, AII-2). In all cases, the polynomial model provided a better fit for the prevalence patterns than the linear model (Tables AII-2, AII-3). In H. anceps, the prevalence of both E. trivolvis (P =
0.0090, R 2 = 0.77) and D. scheuringi ( P = 0.0301, R 2 = 0.65) in 1992 were significantly explained by the polynomial model, while E. trivolvis (P = 0.0132, R 2 = 0.01), E. parvum (P = 0.0381, R 2 = 0.04), the unknown strigeid ( P = 0.0451, R 2 = 0.74), and P. minimum (P = 0.0476, R 2 = 0.67) were all significantly explained by the polynomial model in the only year they were found.
Autogenic parasites
Five species of trematodes with autogenic life cycles were analyzed from various yr of collection in Charlie’s Pond (Tables II, III). Both Halipegus occidualis and
Halipegus eccentricus showed similar trends in the prevalence of infection in H. anceps and P. gyrina snails, respectively. Prevalence of infection by these parasites increased until mid-summer when a rapid decline in prevalence was observed due to turnover of the snail population, a period when the older snail cohort dies and is replaced by the younger group of snails. The parasite prevalence began to rise again until temperature declines led to fluctuations in the frequency of Halipegus spp. in the snails (Fig. 4A, B). With the exception of H. occidualis in 1992, when a negative polynomial model showed a significant fit ( P = 0.0034, R 2 = 0.72; Fig. AII-7), neither the linear or polynomial models explained the prevalence of either H. occidualis or H. eccentricus and the null model best explained the observed trends (Table AII-2, AII-3).
56 Haematoloechus complexus exhibited relatively consistent trends in the prevalence of infection and were best described by the null model (Table AIII; Fig. 4C).
Megalodiscus temperatus and G. quieta showed differing trends in the prevalence of infection from the other autogenic parasites. In M. temperatus , low prevalence of infection early in the collection season gradually increased until it peaked in mid-summer, typically in July or August, before declining as winter approached (Fig. 5D, AII-7, AII-8).
The seasonal prevalence trends in this parasite were best explained by the polynomial model when infecting H. anceps , with a significant fit in both 1988 ( P = 0.0433, R 2 =
0.61) and 1992 ( P = 0.0137, R 2 = 0.75; Table AII-2). The null model explained the prevalence of M. temperatus in P. gyrina in each yr of collection (Table II-3). The prevalence of G. quieta infection showed mixed results. It was best described by the null model in 1991, the linear model in 1992 ( P = 0.0315; R 2 = 0.56), and the polynomial model in 1996 ( P = 0.0375; R 2 = 0.71; Table AII-3).
57 DISCUSSION
There were only 5 species of digenetic trematodes observed in H. anceps from
Mallard Lake (4 allogenic and 1 autogenic) compared to 18 trematode species that were identified from the same snail species in Charlie’s Pond (10 allogenic and 8 autogenic; ranging from 6-11 species in any given year) (Negovetich and Esch, 2007). A few factors could have led to the differences in trematode diversity between the 2 impoundments. Mallard Lake is a public park frequented by recreational visitors, particularly in the summer mo, while Ch arlie’s Pond is private and was accessed only by researchers. Probably as a result, the diversity of wildlife, particularly visiting birds, differed significantly between the 2 sample sites. Mallard Lake was dominated by the presence and abundance of 2 waterfowl species, i.e., Canada geese and mallards, while
Charlie’s Pond was visited by a number of duck, goose, and grebe species passing through on their migration routes (Esch et al., 1997). Furthe rmore, Charlie’s Pond is almost completely surrounded by dense wooded vegetation, as well as emergent vegetation within the pond for a majority of the 30-yr study period, while Mallard Lake had only a small part of the shoreline adjacent to woods and lacks emergent vegetation.
Additionally, Charlie’s Pond has a much more extensive littoral zone than Mallard Lake, resulting in a larger frog population (Wells, 1977; Wetzel and Esch, 1996; Zelmer et al.,
1999). This represents a greater potential for the presence of autogenic parasites that use frogs as definitive hosts to establish infection, as indicated by 8 autogenic species in
Charlie’s pond and only M. temperatus , in Mallard Lake (Negovetich and Esch, 2007).
Additionally, the trematode species composition is completely different in the 2 ponds and may have contributed to the discrepancies in species diversity. Trematodes are
58 known to form distinct guilds that often have set hierarchies resulting in predation, competition, and limitations to infection establishment of trematode larval stages in certain molluscan hosts (Kuris, 1990; Fernandez and Esch, 1991a; Sousa, 1993; Esch and
Fernandez, 1994; Lafferty et al., 1994). The dominant species in Mallard Lake was
Echinostoma sp. with peak prevalence of 24.9% in July. Echinostoma spp. are the top predators within snail hosts due to the large size and active nature of their rediae, which are able to both consume, therefore, out-compete the larval stages of other trematode species. Accordingly, echinostomes are often the dominant species in a molluscan trematode hierarchies (Kuris, 1990; Fernandez and Esch, 1991b; Esch and Fernandez,
1994; Suhardono et al., 2006). Their high prevalence in Mallard Lake and the fact that they were by far the most abundant trematode species infecting H. anceps , may limit the colonization success of other parasites in the system. This was evidenced by the prevalence of the other parasite species never exceeding 2.7% in Mallard Lake in any mo.
Furthermore, of the 560 snails infected by trematode parasites, only 1 snail (0.2%) harbored a double infection of trematode larval stages.
Charlie’s Pond on the other hand had dominant populations of H. occidualis in H. anceps and H. eccentricus in P. gyrina , with prevalence of these species exceeding 50% in some mo and yr (Crews and Esch, 1986; Williams and Esch, 1989; Fernandez and
Esch, 1991; Snyder and Esch, 1993; Sapp and Esch, 1994). Although these trematodes possess predatory rediae, co-infections with H. occidualis and H. eccentricus are much more common and have less antagonistic effects, especially the latter, than co-infections with Echinostoma sp. (Fernandez and Esch, 1991a; Snyder and Esch, 1993). These
59 reduced antagonistic effects could allow for greater diversity of trematode parasites within Charlie’s Pond.
A number of factors, both abiotic and biotic, have been associated with the infection patterns of trematodes in gastropod first intermediate hosts, including microhabitat differences (Williams and Esch, 1991; Sapp and Esch, 1994; Chapter IV), host size (Fernandez and Esch, 1991b; Sorensen and Minchella, 1998; Byers et al., 2008;
Detwiler et al., 2009; Chapter VII), host density (Ewers, 1964; McCarthy and Kanev,
1990), seasonal components (Crews and Esch, 1986; Williams and Esch, 1991; Sapp and
Esch, 1994), and definitive host abundance (Smith, 2001; Huspeni and Lafferty, 2004;
Hechinger and Lafferty, 2005; Fredensborg et al., 2006). The infection patterns of the trematode species in H. anceps from Mallard Lake in the current study were significantly correlated with the size of the snail hosts and the abundance of their definitive hosts.
Larger snails are oftentimes more frequently infected than smaller individuals due to increased exposure time. Also, larger snails tend to be older and more easily located by trematode larval stages (Fernandez and Esch, 1991b; Sorensen and Minchella, 1998;
Byers et al., 2008; Detwiler et al., 2009). High prevalence of Echinostoma sp. first intermediate larval stages occurred just prior to the turnover of the snail population in
Mallard Lake in late May. This correlated with the oldest, and consequently largest, snails being frequently infected with the trematode miracidia. Older snails have increased exposure time to the trematode larval stages and are easier to locate due to the production of more distinct chemical cues (Haas, 2003; Byers et al., 2008; Detwiler et al.,
2009; Chapter VI).
60 Additionally, both Echinostoma sp. and Cotylurus sp. use aquatic waterfowl as definitive hosts (Crichton and Welch, 1972; Campbell, 1973; Dronen et al., 1994;
Johnson and McKenzie, 2009). In Mallard Lake, both Canada geese and mallard ducks are presumably definitive hosts for both of these parasites and the highest prevalence of infection in H. anceps mirrors the periods of highest abundance in these waterfowl species (Fig. 6). However, when the waterfowl abundance declined rapidly in August and September, so did the prevalence of these allogenic parasites. The mixed effect regression model revealed that waterfowl abundance correlated significantly with the prevalence of the trematodes and shell length, density, and water temperature failed to yield significant trends. The importance of waterfowl abundance was further illustrated following snail turnover in late May when the older snail cohort died and was replaced by the younger cohort. Turnover resulted in a drastic decrease in the prevalence of
Echinostoma sp. infection. Waterfowl abundance continued to increase in June and July, which resulted in increased prevalence of infection in the younger cohort of snails.
Similar fluctuations were also found linking the infection patterns of both U. ambloplitis and C. marginatum to the presence and abundance of their piscivorous avian hosts. Uvulifer ambloplitis uses belted kingfishers as definitive hosts (Lemly and Esch,
1984a,b), birds that inhabited the wooded areas adjacent to Mallard Lake year round, but were more frequently observed feeding in the summer mo. As a result, the prevalence of
U. ambloplitis followed a parabolic distribution that peaked in the summer mo, coinciding with the greatest abundance and activity of the definitive host. Additionally,
C. marginatum exhibited a significant upward trend in prevalence as the collection season progressed. This significant increase corresponded with amplified frequency and
61 abundance of great blue herons visiting Mallard Lake, the definitive host for this parasite
(Dzikowski et al., 2004), indicating that host presence and abundance are important in explaining these parameters in trematode infections in the snail first intermediate hosts
(Smith, 2001; Huspeni and Lafferty, 2004; Hechinger and Lafferty, 2005; Fredensborg et al., 2006).
The same patterns did not emerge from the lone autogenic parasite, M. temperatus , in Mallard Lake. The null model best described the prevalence of this parasite in H. anceps , suggesting that the prevalence of infected snails may undergo fluctuations associated with continuous parasite recruitment. Unlike the allogenic parasites described, the definitive host of this autogenic parasite is not ephemeral, and can potentially introduce parasite eggs into the aquatic system yr round. As a result, snails can acquire
M. temperatus infections throughout the collection period as opposed to seasonal recruitment events like the allogenic parasites utilizing aquatic waterfowl as definitive hosts.
Data from Charlie’s Pond confirmed both the importance of definitive host behavior and the general trends of allogenic and autogenic parasite infection. Each of the allogenic parasites from Charlie’s Pond analyzed in this study use aquatic waterfowl as the definitive hosts (Tables II, III). These hosts are ephemeral in Charlie’s Pond and only visit as part of their migration route to their breeding grounds (Esch et al., 1997). As a result, they are capable of depositing eggs of parasites with which they are infected into the pond before continuing their migration, resulting in variations in the presence and abundance of allogenic parasite species from yr to yr (Esch et al., 1988; Fellis and Esch,
2005; Negovetich and Esch, 2007). Most of these migratory birds cross through the
62 Piedmont region of North Carolina in the spring, a pattern that was observed in the trematode infections of the snail first intermediate hosts (Malecki et al., 2006).
The life cycles of these trematode parasites may play an important role on the infection patterns in their snail hosts. Even though the waterfowl definitive hosts are ephemeral and leave their parasites behind during their short visits, there can be lasting residual effects of infection by these parasites. Trematode infections can persist in snails for several mo, increasing the longevity of a single visitation event. Based on bird surveys in Mallard Lake, waterfowl are most abundant in the Piedmont region of North
Carolina in the summer mo, which coincides with prevalence peaks of the parasites they carry in snail intermediate hosts. Although waterfowl abundance was never recorded in
Charlie’s Pond, due to its similar qualities and proximity to Mallard Lake, the highest waterfowl traffic was probably in the summer mo, a trend that is also indicated by high prevalences of trematodes that use these birds as definitive hosts. Based on these trends, it may be possible to use trematode infections in snails as an indirect approximation of the abundance and species of avian visitors to an aquatic habitat (Huspeni and Lafferty,
2004; Hechinger and Lafferty, 2005; Fredensborg et al., 2006)
In most cases, the recruitment patterns of autogenic parasites were not adequately described by either the linear or polynomial models, suggesting fluctuation around a mean value. This is indicative of continuous recruitment for these autogenic parasites.
Halipegus occidualis , H. eccentricus , and H. longiplexus all showed relatively constant levels of infection across mo of collection in Charlie’s Pond, in dicating that H. anceps was recruiting these parasites throughout the collection season. In either June or July, depending on the yr of collection, dramatic decreases in the prevalence of these
63 trematode species were observed due to snail turnover, a period when the older snail cohort dies and are replaced by the younger generation of snails (Crews and Esch, 1986).
The decreases in prevalence associated with snail turnover are followed by increases in infection of these parasites in the new snail cohort, demonstrating that as the younger generation of snails becomes larger and has more exposure time to the infective stages of the parasites, they acquire more infections. This reinforces the notion of continuous recruitment by these autogenic parasite species resulting from a consistent presence of their definitive hosts.
Halipegus occidualis has a 4-hosts life cycle, with H. ancesps as first intermediate hosts, ostracods as second intermediate hosts, dragonflies as paratenic hosts to bridge a trophic gap, and R. clamitans as definitive hosts (Krull, 1935). Upon consumption of infected dragonflies, the metacercariae excyst and migrate out of the intestine and settle under the tongue of the frog host. Occasionally, heavy infections are lost when the skin in the mouth is sloughed off with the parasites still attached (Goater et al., 1989; Wetzel and Esch, 1996; Zelmer et al., 1999).
There were 3 yr of collection (1988, 1992, and 1996) when both H. anceps and R. clamitans were surveyed for H. occidualis infections. The patterns of adult worm abundance in R. clamitans were similar to the prevalence of the snails in each year they were studied; however, a 2-mo lag was observed in 1988 and 1992 (Fig. 7). This lag most likely corresponded to a 6-8 wk pre-patent period of H. occidualis when sporocysts and rediae had been produced, but cercariae had yet to be shed from the snail host
(Zelmer and Esch, 2000). Once the snails began shedding cercariae, the frogs began to accumulate the infections as the parasites pass through the intermediate hosts. Snail
64 turnover in June and July corresponded with significant decreases in the abundance of H. occidualis adults in August and September because the older snail cohort that had a high prevalence of infection died and the younger uninfected cohort begans to flourish (Crews and Esch, 1986; Fernandez and Esch, 1991b). An additional reason the abundance of adult worms matches the prevalence of snail infection by the larval stages is because large infrapopulations of adult parasites are occasionally shed from the frogs (Goater et al., 1989; Wetzel and Esch, 1996; Zelmer et al., 1999).
There was 1 notable exception to these continuous recruitment patterns by autogenic parasites, specifically with M. temperatus . The life cycle of this parasite involves cercariae leaving the snail host, penetrating the skin of a Rana spp. tadpole, and encysting as a metacercariae. These metacercariae remain until the skin is shed and consumed. Upon consumption of the skin and concomitantly the parasite metacercariae, the frog, or in some cases the tadpole, becomes infected with the adults of these parasites
(Leigh, 1946; Bolek and Janovy, 2008). This life cycle plays an important role in the transmission dynamics of M. temperatus in their snail hosts.
Trematode infections in snail intermediate hosts may be lost in the winter mo, coupled with higher mortality of infected snails (Goater et al., 1989; Zimmermann et al.,
2011 ). Additionally, snails begin to emerge from torpor in both Charlie’s Po nd and
Mallard Lake in March and frogs emerge from their winter hibernations during the same time period, suggesting little interaction between the definitive and first intermediate hosts at the beginning of the collection period (Crews and Esch, 1986; Regosin et al.,
2003; Zimmermann et al., 2011). These factors account for low prevalence of infection by M. temperatus in the pulmonate snails at the beginning of the collection season.
65 Furthermore, tadpoles that recruit metacercariae infections in the previous yr and survived winter have yet to metamorphose and are not yet contributing parasite eggs to the aquatic system. Tadpoles begin to metamorphose into adults as temperatures increase and would subsequently contribute more parasites into the aquatic system (Alvarez and
Nicieza, 2002; Loman, 2006). This could potentially lead to significant increases in parasite prevalence of M. temperatus in the summer mo in Charlie’s Pond, corresponding to the greatest abundance of frogs in the population (Wetzel and Esch, 1996; Zelmer et al.,
1999); further testing of this hypothesis is needed. The abundance of frog definitive hosts in Charlie’s Pond was not determined on a monthly basis throughout the collection season in these studies. Linking the role of host abundance to the infection patterns of trematodes with autogenic life cycles is an avenue for further study.
Another explanation for variations in the prevalence trends of M. temperatus compared the other autogenic parasite species may be antagonistic interactions with H. occidualis . Both parasites use R. clamitans as definitive hosts, one in the gut ( M. temperatus ) and the other in the oral cavity ( H. occidualis ), but eggs are probably released in feces simultaneously, leading to a potential overlap in the infections of their snail hosts (Esch et al., 1997). However, co-infections of these species were never found in Charlie’s Pond (Crews and Esch, 1986; Williams and Esch, 1991; Fernand ez and Esch,
1991a; Esch et al., 1997). Fernandez and Esch (1991a) demonstrated that a distinct guild structure of trematode infection in Charlie’s Pond was driven by the presence and nature of rediae infection. Echinostomes were at the top of the hierarchy, H. occidualis were dominant over trematode species having only sporocysts, and challenge infections with M. temperatus were inconclusive (Fernandez and Esch, 1991b). Since H. occidualis and M.
66 temperatus both have rediae stages, antagonistic effects could be occurring. Data regarding the prevalence of these parasites may suggest the former to be the dominant species in the trematode guild. The prevalence of M. temperatus consistently increased when the prevalence of H. occidualis declined, suggesting that antagonistic interactions of the latter parasite could be involved (Fig. 8).
These antagonistic trends were not evident in M. temperatus infections in P. gyrina in Charlie’s Pond. Halipegus occidualis does not infect P. gyrina and the null model best described the prevalence patterns of M. temperatus in physid snails, indicating that the prevalence fluctuated around a mean value. Although H. eccentricus was found to infect P. gyrina in Charlie’s Pond, it did not have the same antagonistic interacti ons as
H. occidualis did in H. anceps . Co-infections in the physid snails were much more common than in H. anceps in Charlie’s Pond (Snyder and Esch, 1993; Esch et al., 1997).
This indicates that there were far fewer antagonistic interactions taking place in these snails, particularly with H. eccentricus that was involved in 83.7% of double infections in
P. gyrina (Snyder and Esch, 1993; Esch et al., 1997). The idea of reduced antagonistic effects of H. eccentricus compared to its congener in H. anceps was reinforced by the interactions between the former parasite and G. quieta . These species were commonly found infecting the same snail, presumably due to utilization of the same definitive and first intermediate hosts, and showed similar patterns in the prevalence of infection, suggesting little antagonism despite H. eccentricus having rediae larval stages and G. quieta having only a sporocyst larval stage (Snyder and Esch, 1993; Esch et al., 1997)
(Fig. 9). This suggests that antagonism against M. temperatus by H. occidualis may have
67 led to the discrepancies in the prevalence trends of the former species from the other autogenic parasites.
A few important patterns emerged from the prevalence of autogenic and allogenic trematode parasites from these 2 study sites. First, the life cycle of the trematodes and the behavior, ecology, and seasonal dynamics of the definitive hosts, as well as snail size, are important factors in shaping the transmission patterns of these parasites in their snail first intermediate hosts. The infection patterns of trematode parasites in the gastropods can potentially be used as a proxy for the presence and abundance of definitive hosts in an aquatic system. Additionally, differences in the patterns of infection in the different life cycle types were observed. Allogenic parasites exhibit strong seasonal recruitment trends in their snail hosts while autogenic parasites display continuous recruitment patterns in gastropods. These factors make the study of snail trematode interactions important for understanding the interplay between parasites, their life cycles, and their hosts in aquatic ecosystems.
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76 Table I. Infective stage, molluscan stage, location, hosts and life cycle classification of digenetic trematode species infecting H. anceps in Mallard Lake.
Trematode Infective Molluscan Location 2nd Intermediate Definitive Life Cycle Species Stage Stage in Snail Host Host Type Sporocyst Clinostomum marginatum Miracidium Gonad Fish Ardea herodias Allogenic Rediae Diplostomum scheuringi Miracidium Sporocyst Gonad Gambusia affinis Podilymbus podiceps Allogenic Sporocyst Hepatopancreas Waterfowl Echinostoma sp. Miracidium Snail Allogenic Redia Gonad Mammals Sporocyst Rana clamitans Megalodiscus temperatus Miracidium Gonad Tadpole Autogenic Redia Rana catesbiana Uvulifer ambloplitis Miracidium Sporocyst Gonad Fish Megaceryle alcyon Allogenic
77 Table II. Infective stage, molluscan stage, location, hosts and life cycle classification of digenetic trematode species infecting Helisoma anceps in Charlie's Pond that were analyzed in this study.
Trematode Infective Molluscan Location 2nd Intermediate Definitive Life Cycle Species Stage Stage in Snail Host Host Type Sporocyst Diplostomum scheuringi ‡§‖ Miracidium Gonad Gambusia affinis Podilymbus podiceps Allogenic
Sporocyst Hepatopancreas Waterfowl Echinostoma trivolvis ‡§‖ Miracidium Snail Allogenic Redia Gonad Mammals Sporocyst Hepatopancreas Ostracod Halipegus occidualis * †‡§‖ # Egg Rana clamitans Autogenic Redia Gonad Dragonfly Sporocyst Rana clamitans Megalodiscus temperatus ‡§‖ Miracidium Gonad Tadpole Autogenic Redia Rana catesbiana * Data from Crews and Esch (1986) † Data from Williams and Esch (1991) ‡Data from Fernandez and Esch (1991b) § Data from Sapp and Esch (1994) ‖ Data from Schotthoeffer (1996) # Data from Negovetich and Esch (2007)
78 Table III. Infective stage, molluscan stage, location, hosts and life cycle classification of digenetic trematode species infecting Physa gyrina in Charlie's Pond that were analyzed in this study.
Trematode Infective Molluscan Location 2nd Intermediate Definitive Life Cycle Species Stage Stage in Snail Host Host Type Sporocyst Hepatopancreas Echinoparyphium parvum ‖ Miracidium Snail Waterfowl Allogenic Redia Gonad Sporocyst Hepatopancreas Waterfowl Echinostoma trivolvis § Miracidium Snail Allogenic Redia Gonad Mammals Rana clamitans Glypthelmins quieta ¶§‖ Egg Sporocyst Hepatopancreas Tadpole Autogenic Rana catesbiana Hepatopancreas Rana clamitans Haematoloechus complexus ¶§ Egg Sporocyst Dragonfly Autogenic Gonad Rana catesbiana Sporocyst Hepatopancreas Copepod Halipegus eccentricus ¶§ Egg Rana clamitans Autogenic Redia Gonad Damselfly/Dragonfly Sporocyst Rana clamitans Megalodiscus temperatus §‖ Miracidium Gonad Tadpole Autogenic Redia Rana catesbiana Hepatopancreas Posthodiplostomum minimum ‖ Miracidium Sporocyst Fish Ardea herodias Allogenic Gonad Hepatopancreas Strigeid§ Miracidium Sporocyst Unknown Waterfowl Allogenic Gonad ¶ Data from Snyder and Esch (1993) § Data from Sapp and Esch (1994) ‖ Data from Schotthoefer (1996)
79 FIGURE 1. Mean (+/- standard error) monthly abundance of the 2 most commonly observed waterfowl species from Mallard Lake, Canada Geese ( Branta canadiensis ) and
Mallards ( Anas platyrhynchos ).
80 81 FIGURE 2. The monthly prevalence of allogenic parasite species infection in H. anceps from Mallard Lake. The infection prevalence is fit with the best fit linear or polynomial model (dashed line). (A) Echinostoma sp., (B) Cotylurus sp., (C) Uvulifer ambloplitis , and (D) Clinostomum marginatum
82 83 FIGURE 3. The monthly prevalence of an autogenic parasite, Megalodiscus temperatus, infection in H. anceps from Mallard Lake. The infection prevalence is fit with the average prevalence of infection (dashed line) since neither the linear or polynomial models fit these data significantly.
84 85 FIGURE 4. Prevalence of infection of allogenic parasites from pulmonate snails in
Charlie’s Pond. The dashed lines represent the best fit polynomial curve of the prevalence. (A) Echinostoma trivolvis (Sapp and Esch, 1994), (B) Diplostomum scheuringi (Sapp and Esch, 1994), (C) Echinoparyphium parvum (Schotthoeffer, 1996),
(D) unknown strigeid (Sapp and Esch, 1994), (E) Posthodiplostomum minimum
(Schotthoeffer, 1996).
86 87 FIGURE 5. Prevalence of infection of autogenic parasites from pulmonate snails in
Charlie’s Pond. The dashed lines represent th e best fit lines, polynomial curve, or the average prevalence if neither model showed a statistically significant fit. (A) Halipegus occidualis (Crews and Esch, 1986), (B) Halipegus eccentricus (Sapp and Esch, 1994), (C)
Haematoloechus longiplexus (Sapp and Esch, 1994), (D) Megalodiscus temperatus (Sapp and Esch, 1994), (E) Glypthelmins quieta (Sapp and Esch, 1994), and (F) Glypthelmins quieta (Schotthoeffer, 1996).
88 89 FIGURE 6. Monthly prevalence of Echinostoma sp. and Cotylurus sp. infection in H. anceps and the mean monthly abundance (± standard error) of waterfowl present in
Mallard Lake.
90 91 FIGURE 7. The prevalence of H. occidualis larval stages in H. anceps and the abundance of adult H. occidualis in R. clamitans from Charlie’s pond in 3 different collection seasons: (A) 1988 (Goater, 1989; Williams and Esch, 1991), (B) 1992 (Sapp and Esch, 1992; Wetzel and Esch, 1996; Zelmer and Esch, 1999), (C) 1996 (Schotthoefer,
1996; Zelmer and Esch, 1999).
92 93 FIGURE 8. The prevalence of H. occidualis and M. temperatus infection in H. anceps from Charlie’s Pond in 3 different collection seasons: ( A) 1988 (Williams and Esch,
1991), (B) 1989 (Fernandez and Esch, 1991b), (C) 1992 (Sapp and Esch, 1994).
94 95 FIGURE 9. The prevalence of H. eccentricus and G. quieta infection in P. gyrina from
Charlie’s Pond in 2 different collection seasons: ( A) 1991 (Snyder and Esch, 1993) and
(B) 1992 (Sapp and Esch, 1994).
96 97 CHAPTER III
INFECTION PATTERN DIFFERENCES OF DIGENETIC TREMATODE
PARASITES ACROSS THE NORTH AMERICAN WATERFOWL FLYWAYS
98 ABSTRACT
Changes in the environmental landscape associated with both natural and anthropogenic disturbance can have a major impact on the abundance and distribution of wildlife. The migration patterns and declines of North American waterfowl populations since the 1950’s associated with changes in habitat and climate have been areas of major focus of research due to their role in zoonotic disease and their economic importance.
Continuing efforts to track the movement and population size of these bird populations is also employed to aid in conservation and the restoration of wetlands and the waterfowl populations. The present study examined the prevalence and abundance of a ubiquitous group of waterfowl parasites infecting gastropod intermediate hosts across 3 North
American waterfowl flyways. A significant spatial correlation of Echinostoma spp. distribution showed a lack of worms in the southeastern U.S. below 35 o N latitude, possibly attributed to declines in migratory populations associated with changes in climate and the environmental landscape. Significantly increased infection of
Echinostoma spp. was observed in the Atlantic flyway, particularly in the mid-Atlantic states, probably as a result of increased permanent residency of the waterfowl, due to increased in agriculture in the region creating an abundant supply of food for the winter mo, and increased precipitation benefiting molluscan intermediate hosts. The prevalence and abundance of Echinostoma spp. metacercariae in their gastropod intermediate hosts can potentially be used as an indirect indicator of waterfowl movement and visitation because of the intimate associations between bird and snail hosts in trematode life cycles.
99 INTRODUCTION
Changes in the environmental landscape, both natural and anthropogenic, and their impact on wildlife, have been a topic of growing concern (Daszak et al., 2001; Frid and Dill, 2002; Sawyer et al., 2011; Chan and Blumstein, 2011; Bennett and Zurcher,
2013). Deforestation, increased agriculture, and other forms of habitat disturbance can have massive impacts on the distribution and ecology of wildlife across North America
(Freemark and Boutin, 1995; Rempel et al., 1997; Alin et al., 1999; Laurence et al., 2001;
Vickery et al., 2001). These effects become particularly important when the wildlife affected are of economic and recreational importance, such as waterfowl, which are frequently hunted for both sustenance and sport. These avian species are affected by environmental alterations associated with disturbance to both aquatic and terrestrial habitats, particularly in the breeding grounds, leading to heavy conservation efforts to maintain their populations (Williams et al., 1999; Williams, 2003; Perez-Arteaga et al.,
2006; Brasher et al., 2007). However, despite these efforts, the environmental landscape, abundance, and distribution of these birds are under a constant state of flux, but with significant population dec reases since the 1950’s (Bethke and Nudds, 1995; Link et al.,
2006; Zimpfer et al., 2013).
In North America, waterfowl typically migrate along 4 designated routes referred to as flyways. The Atlantic flyway along the eastern seaboard, the Mississippi flyway adjacent to the eastern bank of the Mississippi River, the Central flyway covering the
Great Plains to the eastern edge of the Rocky Mountains, and the Pacific flyway along the western seaboard of the United States, each harbor distinct bird populations. These flyways result in isolated courses for migration between breeding and wintering grounds;
100 tracking the birds along these routes has been an area of recent attention due to concerns regarding zoonotic pathogens these birds carry (Krauss et al., 2004; Senne et al., 2006;
Kim et al., 2007). Aerial surveys, banding efforts, and radio tracking devices have been used to aid in following the migration patterns of waterfowl in North America.
Disease associated with aquatic waterfowl, particularly avian influenza, is of great interest due to its pathogenicity and recent outbreaks in Europe and Asia (Olsen et al.,
2006; Latorre-Margalef et al., 2007). Growing concern over this disease has taken focus away from other pathogens negatively affecting these bird hosts, including digenetic trematode parasites. Waterfowl are commonly infected by these parasites, often in high numbers, and frequently have distinct suites of parasites based on host dietary preferences (Bush and Holmes, 1986a, 1986b; Feynich et al., 1996). Many digenetic trematodes have high specificity for a genus or species of definitive host, making generalized assumptions of parasite infection across many waterfowl species difficult.
However, Echinostoma spp. have very low specificity for definitive hosts, having been reported in over 60 different vertebrate species (a rarity among trematode parasites), including a number of waterfowl hosts (Johnson and McKenzie, 2009).
Echinostoma spp. are allogenic trematode parasites, i.e., a species that involves at least 1 host capable of moving between bodies of water (Esch et al., 1988; Criscione and
Blouin, 2004; Fellis and Esch, 2005), with complex life cycles incorporating 3 hosts.
Eggs are passed into water where they hatch into a non-feeding, free-swimming stage called a miracidium. Miracidia locate pulmonate snail first intermediate hosts, Lymnaea spp. or Helisoma spp. in North America, with the aid of species-specific chemical cues
(Haas and Haberl, 2000; Haas, 2003). Once this larval stage infects a gastropod first
101 intermediate host, it develops into asexually reproducing sporocysts, mother rediae, and daughter rediae. These stages ultimately give rise to free-swimming clones, called cercariae, which leave the gastropod in search of a second intermediate host. Specificity in this larval stage is far less than miracidia; second intermediate hosts include a variety of gastropod, amphibian, and fish species, although the former is most frequently used in nature (Haberl et al., 2000; Johnson and McKenzie, 2009; Haas, 2003). Once cercariae infect the second intermediate host, they encyst, become metacercariae, and remain in this state until they are consumed by a definitive host, most commonly aquatic waterfowl.
These echinostome parasites are useful ecological models because they utilize aquatic gastropods not only as first intermediate hosts, but as second intermediate hosts as well. The transmission of Echinostoma spp. larval stages is often intimately tied to the behavior and seasonal dynamics of their hosts (Smith, 2001; Huspeni and Lafferty, 2004;
Hechinger and Lafferty, 2005; Fredensborg et al., 2006; Chapter II). This makes these parasites potentially useful indicators of waterfowl presence and abundance in aquatic habitats. It was hypothesized that the abundance and distribution of Echinostoma spp. parasites were related to the presence, abundance, and behavior of their waterfowl hosts.
To test this hypothesis, the infection patterns of Echinostoma spp. metacercariae in gastropod second intermediate hosts across the North American waterfowl flyways were investigated to determine if the trends in parasite frequency and abundance were related to the ecology and abundance of their waterfowl hosts. The study involved the collection and survey of aquatic snails from 3 of the 4 North American waterfowl flyways in a broad latitudinal range.
102 MATERIALS AND METHODS
A total of 11,227 aquatic snails was collected from 139 bodies of water across 3
North American waterfowl flyways between June and August in 2012 and June and July in 2013 (Table AI-1; Table I-6). After collection, snails were preserved in alcohol until necropsy. Prior to dissection, each snail was identified to the species level using the taxonomic key developed by Burch (1982); each snail was measured to the nearest 0.05 mm. The snails were divided according to the waterfowl flyways (Fig. I-4) and the number of Echinostoma spp. metacercariae present within each host was recorded. Only individuals in the ‘revolutum’ group, i.e., those possessing 37 collar spines, were analyzed in this study.
The parasite population was described in terms of prevalence, i.e., the number of infected hosts divided by the total number of hosts, and mean abundance, i.e., the total number of parasites recovered divided by the total number of hosts sampled (according to the terminology of Bush et al., 1997). Moran’s I spatial autocorrelation was performed to determine if there were any significant patterns to the distribution of Echinostoma spp. across the large sample range. Chi square analysis was used to compare the prevalence of Echinostoma spp. metacercariae and other allogenic trematode species in the snail populations between the year of collection and waterfowl flyways. Kruskal-Wallace tests were used to compare the mean abundance of Echinostoma spp. metacercariae between yr of sample and waterfowl flyways, since the parasites were overdispersed and failed the
Fmax test. Steel-Dwass post-hoc tests were employed to elucidate differences in metacercariae abundance between waterfowl flyways. All statistical analyses were performed using the R software package (R Core Team, Vienna, Austria).
103 RESULTS
There was a statistically significant spatial correlation in the distribution of
Echinostoma spp. observed in this study, due to the lack of the parasite below the 35 o N latitude and increased aggregation of the parasite in the Mid- Atlantic states (Moran’s I, P
= 0.0021; Fig. 1). The prevalence of Echinostoma spp. was greater in the summer of
2013 compared to the previous yr ( χ2 = 40.7, P < 0.0001; Fig. 2A). Additionally, the mean abundance echinostome metacercariae infecting snail hosts was greater in the 2013 collection season than in 2012 (Kruskal -Wallace, P < 0.0001; Fig. 2B). These discrepancies between yr of collection were driven by significantly smaller prevalence of metacercariae in the Mississippi flyway (Fig. 3A) and significantly greater the mean abundances of these helmin ths in the Atlantic flyway in 2013 compared to the previous year (Fig. 3B).
Both the prevalence (Chi square, P < 0.0010) and mean abundance (Kruskal-
Wallace, P < 0.0001) of Echinostoma spp. metacercariae were significantly greater in the
Atlantic flyway than in either of the other 2 flyways (Steel-Dwass, P < 0.05), regardless of the year of collection (Fig. 3). There were 5 ponds in the study that had permanent, year round waterfowl residents (Table I). The prevalence ( χ2 = 56.7, P < 0.0001) and intensit y (Kruskal -Wallace, P < 0.0001) of Echinostoma spp. metacercariae was significantly greater in ponds with permanent waterfowl residents compared to those with only ephemeral avian visitations (Fig. 4).
104 DISCUSSION
Collections in consecutive summers revealed significant differences in both the prevalence and intensity of Echinostoma spp. in their snail hosts, with increased parasitism in 2013 compared to the previous yr. One possible explanation for the differences between yr of collection is precipitation in the study areas. All snails collected in this study are dependent on aquatic environments for habitat, food, and reproduction (Dillon, 2000; Johnson and McKenzie, 2009). As a result, their populations can be significantly affected by precipitation. Increased rainfall can potentially lead to habitat expansion and increase the abundance of snail populations, which can also impact the infection dynamics of trematode parasites (Marin et al., 1998;
Remais et al., 2007; Remais et al., 2008), while also resulting in more potential hosts for
Echinostoma spp. larval stages. The spring and summer of 2013 saw record levels of precipitation in many of the states in the Atlantic flyway, particularly in the mid-Atlantic region, which were comparatively greater than the previous year (NOAA 2012, 2013)
(Fig. 5). These differences in rainfall may have contributed to, at least in part, the discrepancies in Echinostoma spp. infection between years.
Another possible explanation leading to differences in parasite spatial patterns was the distribution and behavior of definitive hosts for Echinostoma spp., namely waterfowl. A spatial autocorrelation revealed that Echinostoma spp. were not found below 35 o N latitude, with the exception of a single infected snail in Alabama, which was clearly an outlier. Additionally, aggregation in the prevalence of infection was observed in the mid-Atlantic states. Waterfowl populations in North America have been on the decline since the 1950’s du e to increases in agricultural practices and other anthropogenic
105 disturbances, but some areas have been affected more than others (Bethke and Nudds,
1995; Krapu et al., 1997; Murray et al., 2010).
Population, migration, and hunting reports indicate that although waterfowl are still present in the southeastern U.S., their abundance has declined significantly since the late 1990’s (Link et al., 2006; U.S. Fish and Wildlife Service, 2012, 2013a, 2013b;
Raftovich and Wilkins, 2013; Zimpfer et al., 2013) due to hydrologic and geomorphic changes, increased agriculture, and urbanization in the southern floodplains (King et al.,
2009). For example, the coastal marshes of Louisiana, which cover over 1.6 million ha, provide wintering and migration habitat for nearly two-thirds of the waterfowl inhabiting the Mississippi flyway. In recent decades, substantial declines in both quantity and quality of these marshes, through degradation and conversion to saline environments, have occurred in these coastal regions of the southeastern U.S. (Louisiana, 2014). Aerial surveys of waterfowl abundance between November and January have been conducted annually since 1977 and September surveys since 2004. These aerial surveys have shown significant reductions in waterfowl abundance over the last 25 yr, most likely associated with the changes in the environmental landscape of the Louisiana coastal wetlands
(Louisiana, 2014; Fig. 6). Additionally, predictive modeling based on changes in climate and the environmental landscape have projected a northward shift in the wintering grounds and decreased breeding success in a number of waterfowl species (Matthews et al., 2004; Forcey et al., 2007), thus exacerbating the problem. It is suggested that these declines in waterfowl abundance in the southeastern U.S. may be responsible for the decreases in parasite prevalence and abundance in this region.
106 In both yr of collection, there was a significantly higher prevalence and mean abundance of Echinostoma spp. metacercariae in the Atlantic flyway in comparison to the
Central and Mississippi flyways, despite the latter 2 flyways having significantly more waterfowl in their ranges (U.S. Fish and Wildlife Service, 2013a). One potential explanation for this phenomenon was a lack of trematode parasites in the southeastern
U.S. Decreased parasitism by Echinostoma spp. in this region resulted in a number of samples being collected in areas where there were very few parasites, potentially skewing the prevalence and abundance of these trematodes. A great deal of wetland degradation and disturbance has occurred in the southeastern U.S. in the past 2 decades through both natural disasters, e.g., hurricanes and tornadoes, and anthropogenic causes, e.g., agricultural practices, forestry, and pollution (King et al., 2009). Additionally, an affinity for, and reliance on, shallow water habitats with a high degree of connectivity are important for sustaining waterfowl populations (Perry and Deller, 1996). It has been shown that degradation of shallow water habitats as a result of increased human use for fishing, boating, and other recreation activities can lead to substantial declines in waterfowl populations, similar to what is currently happening in the southeastern U.S.
(Perry and Deller, 1996; King et al., 2009). These factors may account for the low prevalence and abundance of parasites in the Mississippi flyway
Additonally, differences in precipitation, not only between yr of collection, but between flyways were observed (Fig. 5). Significantly more rainfall was observed in the
Atlantic flyways in each yr of collection, particularly in 2013 when record rainfalls occurred in many states in the Atlantic flyway (NOAA, 2012; 2013). This additional rainfall increased suitable habitat for the gastropod hosts, potentially increasing their
107 population sizes and aggregation (Marin et al., 1998; Remais et al., 2007). These factors can have significant effects on the infection patterns and success of Echinostoma spp., particularly snail density (McCarthy, 1990; Chapter VI).
Another factor that could have contributed to increased parasitism by
Echinostoma spp. in the Atlantic flyway is change in the migration patterns by ducks and geese in this region. Waterfowl in this flyway, particularly Canada geese, have become permanent residents and no longer migrate, especially in the mid-Atlantic states, which have experienced significant population increases in recent decades (Caccamise et al,
2000; Canada Goose Committee, 1999; Haramis and Kearns, 2010). An increase in agricultural activity in this region provides an abundant supply of food due to left over grain remaining in fields after harvest, diminishing the need for the birds to migrate
(Canada Goose Committee, 1999; Fox et al., 2005; Haramis and Kearns, 2010). These permanent residents can also have detrimental impacts on migratory waterfowl populations because the former consume the food supply before the migrants arrive, potentially leading to decreases in the migratory populations (Haramis and Kearns, 2010).
Five ponds were sampled in this study that harbored permanent resident waterfowl populations. In these ponds, the prevalence and mean abundance of
Echinostoma spp. metacercariae were significantly greater than the other ponds sampled in the study. The permanence of these birds is advantageous for these trematode parasites and can enhance transmission, at least in comparison with habitats lacking permanent waterfowl. Often the ephemeral nature of migratory waterfowl leads to distinct seasonal patterns of infection resulting in a parabolic trend associated with an early spring recruitment event during bird migration to their breeding grounds (Chapter
108 II). However, if the avian hosts are present yr round, then transmission of parasites may occur consistently throughout the yr. The decreased propensity for birds to migrate in these mid-Atlantic states may also contribute to increased parasitism by Echinostoma spp. in this region, and decreased infection in the southern U.S.
The nature of the complex life cycles of digenetic trematode parasites can potentially play an important role in monitoring the constantly changing environmental landscape’s effect on waterfowl abundance and migration. Nearly all trematode life cycles involve aquatic gastropods as first intermediate hosts. Additionally, Echinostoma spp. and other members of the Echinostomatidae use aquatic snails as both first and second intermediate hosts (Esteban and Munoz-Antoli, 2009). Both the parasites and their snail hosts have a fairly ubiquitous distribution across North America and the low specificity of Echinostoma spp. for their definitive hosts makes them common members of aquatic communities (Johnson and McKenzie, 2009). Although Echinostoma spp. metacercariae tend to accumulate in larger, older pulmonate snail hosts (Morley et al.,
2004; Byers et al., 2008; Detwiler and Minchella, 2009; Chapter VI), these gastropods oftentimes have a 1-yr life cycle, with notable exceptions being larger species, such as
Helisoma trivolvis , in colder climates (Boerger, 1975; Eversole, 1978). This annual life cycle typically culminates in a population wide turnover event, i.e., when the older cohort dies and is replaced by the younger generation of snails, a phenomenon that typically occurs in the early summer mo (Crews and Esch, 1986; Zimmermann et al., 2011). This turnover event essentially resets Echinostoma spp. metacercariae infection annually in the snail populations, allowing for current estimates in the presence and abundance of waterfowl visiting an aquatic habitat.
109 As a result, these parasites can potentially be used not only as a proxy for waterfowl abundance (Smith, 2001; Huspeni and Lafferty, 2004; Hechinger and Lafferty,
2005; Fredensborg et al., 2006; Chapter II), but may also be employed as an indirect measure of avian movement. Other studies have shown that trematode populations in snail hosts can be used as indirect indicators for the status of wetland restoration and ecosystem health due to the high specificity of trematode miracidia for their first intermediate hosts (Huspeni and Lafferty, 2004; Marcogliese, 2005). The abundance of pulmonate snails in aquatic habitats and the ease of parasite detection and enumeration within them, make Echinostoma spp. a potentially useful tool to use in coordination with the current methods of tracking waterfowl migration.
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119 Table I. Bodies of water sampled with permanent year round residents of aquatic waterfowl.
Body of Water City State Latitude Longitude
Campus Pond Charleston IL N 39 28' 42.6" W 88 10' 44.3" Lake Mary Nell Elon NC N 36 06' 23.7" W 79 30' 25.9" Muskingum Lake New Concord OH N 40 00' 24.0" W 81 44' 25.0" Sturtevant Lake New Concord OH N 40 00' 26.2" W 81 45' 17.0" University Lake Bloomington IN N 39 11' 26.5" W 86 30' 08.7"
120 FIGURE 1. Map of the ponds and lakes sampled across the U.S. with the 4 waterfowl flyways. Dark circles denote where Echinostoma spp. metacercariae were present and light circles denote where the parasites were absent.
121 122 FIGURE 2. (A) Prevalence and (B) mean abundance (+/- standard error) of Echinostoma spp. metacercariae infection in pulmonate snails in 2 yr of collection.
123 124 FIGURE 3. (A) Prevalence and (B) mean abundance (+/- standard error) of Echinostoma spp. metacercariae infection in pulmonate snails across 3 waterfowl flyways in 2 yr of collection.
125 126 FIGURE 4. (A) Prevalence and (B) mean abundance (+/- standard error) of Echinostoma spp. metacercariae in collection sites where permanent waterfowl residents were present and absent.
127 128 FIGURE 5. Average precipitation (+/- standard error) between May and July in 2012 and
2013 across 3 U.S. waterfowl flyways based on the standard precipitation index (SPI).
Positive values represent above average precipitation for the region and negative values represent below average rainfall. Data were obtained from NOAA (2012) and NOAA
(2013).
129 130 FIGURE 6. The number of waterfowl found inhabiting the coastal marshes of Louisiana in (A) September, (B) November, (C) December, and (D) January over a 25 yr period with a best fit line (dashed line) indicating the general trends in population numbers.
These data were obtained through aerial surveys conducted by the Louisiana Department of Natural Resources (Louisiana, 2014).
131 132 CHAPTER IV
HABITAT DISTURBANCE AFFECTS THE DISTRIBUTION OF TREMATODE
PARASITES AMONG A PULMONATE SNAIL COMMUNITY
M. R. Zimmermann, K. E. Luth, and G. W. Esch
The following manuscript was submitted for publication in the Journal of Limnology
(2014, In Review) and is reprinted with permission. M. R. Zimmermann performed the study and prepared the manuscript. K. E. Luth aided in sample collection and an editorial
capacity. G. W. Esch acted in an advisory and editorial capacity.
133 ABSTRACT
Habitat disturbance can have a major impact on animal distribution as well as the parasites they harbor. The transmission and infection dynamics of digenetic trematode parasites in their snail first intermediate hosts were examined in a pond that recently underwent habitat disturbance. It was found that the distribution of the parasites was largely tied to changes in host behavior associated with the alterations in microhabitat.
Autogenic species were found further away from disturbance and allogenic parasites were closer to disturbance, probably as a result of habitat preference by their frog and waterfowl definitive hosts, respectively. Additionally, intermediate levels of disturbance at the edges of the disturbed habitat increased parasite transmission due to habitat overlap in the distribution of autogenic and allogenic species hosts.
134 INTRODUCTION
Habitat disturbance can have a major impact on the distribution of animals in both terrestrial and aquatic habitats (Alin et al., 1999; Gill et al., 2001; Beale and Monaghan,
2004; Rodríguez-Prieto and Fernández-Juricic 2005). Microhabitat changes caused by either natural or anthropogenic damage can alter the community composition and distribution of plants and animals within it. These alterations can have a major impact not only on the vertebrate fauna, but their parasites as well (Medeiros and Maltchik, 1999;
Lafferty and Kuris, 2005; Urban, 2006). Disturbance can condense animal populations, consequently increasing parasite transmission (Kristofferson, 1991; Lafferty and Kuris,
2005; Urban, 2006). This situation can be extremely important for host species with low vagility via enhancement of the efficiency of parasite transmission by increasing proximity and encounter rate of parasites with their hosts.
Digenetic trematodes can be significantly influenced by disturbance due to their complex life cycles (Lafferty and Kuris, 2005). Multiple hosts involved in these life cycles result in a greater potential for at least 1 of them to be affected by the changes in microhabitat. Most trematode life cycles require 3 hosts, which may be influenced by these changes (Lafferty and Kuris, 2005; Urban, 2006). Although a wide array of host species are utilized by digenetic trematodes for life cycle completion, each parasite falls into 1 of 2 categories. Autogenic parasites have life cycles in which all hosts are present within a single aquatic system, unable to move between bodies of water, while allogenic parasites have life cycles in which at least 1 host is capable of moving between aquatic habitats (Esch et al., 1988). As a result, parasites in these 2 life-cycle categories often
135 have distinct distributions not only between bodies of water, but within them as well
(Esch et al., 1988; Criscione and Blouin, 2004; Fellis and Esch, 2005;).
One commonality between autogenic and allogenic life cycles is the first intermediate hosts. Almost exclusively, molluscs are extremely important in the distribution and transmission dynamics of trematode parasites (Esch and Fernandez, 1994;
Sapp and Esch, 1994; Detwiler and Minchella, 2009). Changing microhabitat may not only affect the distribution of aquatic snails, but may also indirectly affect the encounter rate of the gastropods with the definitive hosts (Urban, 2006). Consequently, the frequency of snails encountering eggs or miracidia of trematode parasites that infect them may be altered. It was predicted that habitat disturbance would affect the distribution of vertebrate hosts and their trematode parasites infecting gastropod first intermediate hosts due to changes in the habitat structure surrounding the pond.
136 MATERIALS AND METHODS
Three species of pulmonate snails were collected from Coon Lake, a 1.5-ha pond located in the Piedmont region of North Carolina (35 o59’50” N, 80 o24’31” W), between
May and July 2013. Although located within a public park, Coon Lake is isolated from public access and is mostly surrounded by dense wooded vegetation. The western edge of the pond is cleared of all vegetation due to the construction of a cart path associated with a public golf course and a new drainage system that was installed in the summer of
2012. Although stocked with largemouth bass and bluegill sunfish, the pond is rarely visited by the public. Abundant numbers of Rana spp. frogs and visiting waterfowl frequent the pond.
The western half of the pond was divided into 40, 5-m transects, each of which was put into 1 of 3 categories, i.e., open, wooded, or edge habitat. The pond was partitioned so that there was an equal number of open and wooded transects for sampling.
Open habitat was devoid of trees and vegetation and was located exclusively on the western edge of the pond, wooded habitat was covered in dense vegetation and trees, and edge habitat was characterized by transects that were within 5 m of the edge of the tree line. All snails were sampled by hand or dip netting within 2 m of the shoreline and snail density was estimated by counting all snails present within a 0.05-m 2 rectangle prior to the collection of snails in each transect. The snails were then returned to the lab, isolated in 55-mm finger bowls for 48 hr, and checked for emerging cercariae. Each individual was then measured to the nearest 0.05 mm, painted, numbered with indelible ink, and returned to the lake in the same transect from which it was collected. Recaptured snails were rare and were excluded from the analyses.
137 A total of 2,798 pulmonate snails was collected encompassing 3 different species,
Lymnaea columella (N = 680), Menetus dilatus (N = 21), and Physa gyrina (N = 2,097).
A subset of 150 snails, 50 from each habitat category, was necropsied each mo for an estimation of how many snails had pre-patent infections, i.e., infected snails possessing asexually reproducing sporocysts and rediae, but not yet shedding cercariae, and to provide a better estimation of the infection dynamics of the trematode parasites. All shed trematodes were isolated, identified, and then sub-categorized into autogenic and allogenic species.
The population was described in terms of prevalence and intensity according to the terminology of Bush et al. (1997). Generalized additive models (GAMs) were used to determine if the habitat disturbance caused by the golf course and drain installation affected the population structure and distribution of parasites within the pond. A centralized point in the disturbed habitat was marked, and distance from this location was used to determine the influence of the disturbance on the spatial patterns of the trematode parasites. This central disturbance point was equidistant from the wooded habitat on either side of the pond. The GAMs used prevalence of autogenic and allogenic parasites and distance from maximum disturbance in the construction of the models. Additionally,
1-way ANOVA was used to compare snail densities and lengths between sites and Chi square analysis was used to compare prevalence of parasites between the 3 habitat categories. All models and statistical analyses were performed using R (R Development
Core Team, Vienna, Austria).
138 RESULTS
Snail density was greatest in the edge habitat (15.8 ± 1.7 snails per m 2), followed by the open (15.7 ± 0.8 snails per m 2) and wooded habitats (14.8 ± 1.1 snails per m 2), but density did not differ significantly between habitat types (ANOVA, P = 0.3566).
Additionally, snail density did not differ between mo of collection (ANOVA, P = 0.3236), nor was there a difference in snail length between habitat categories (ANOVA, P =
0.2413).
A total of 10 species of trematode parasites was collected from the snails in Coon
Lake, i.e., 3 autogenic species and 7 allogenic species (Table I). All of the autogenic species use Rana spp. frogs as definitive hosts, while the allogenic species employ aquatic waterfowl, both of which were common inhabitants or visitors of the pond.
Allogenic parasites (5.7%) were more abundant than autogenic species (2.1%), with a majority of the parasites occurring in single infections (210 of 220). Dissections revealed that parasite prevalence was underestimated by 20.3% in May, 9.4% in June, and 5.8% in
July due to pre-patent infections of parasites. However, no significant differences in the underestimation of trematode parasites were observed between life cycle strategy or habitat classification in any given mo ( P > 0.05) . The distribution of all trematode parasites, both autogenic and allogenic, showed equal distributions in both the open
(7.1%) and wooded (7.0%) transects, but a significantly higher prevalence was observed in the edge habitat (11.9%; χ 2 = 631.23, P < 0.0001) (Fig. 1A).
Both categories of parasites had distinct distribution patterns across transect and habitat types. Autogenic species had the highest prevalence of infection in snails inhabiting transects in the densely wooded vegetation (5.0%). The prevalence in the
139 wooded transects was significantly greater than both the edge (3.1%) and open habitats
(0.7%; Chi squared test, P < 0.0001) (Fig. 1B). Additionally, the generalized additive model showed that the further away from the central disturbance point, the higher the prevalence of infection of trematode parasites with autogenic life cycles (Generalized
Additive Model, P < 0.0001, R 2 = 0.91) (Figs. 2, 3). Allogenic parasites showed the opposite trend and were significantly more likely to occur in the open (6.5%) and edge
(8.7%) habitats than in the wooded habitat (2.1%; Chi-squared test, P < 0.0001) (Fig. 1B).
Furthermore, the generalized additive model indicated that the closer the collected snails were to the central disturbance point, the higher the prevalence of infection by allogenic parasites (Generalized Additive Model, P = 0.0016, R 2 = 0.68) (Figs. 2, 3).
140 DISCUSSION
Allogenic species outnumbered autogenic parasites in terms of both diversity and prevalence of infection in this system. Definitive hosts of autogenic parasites, such as the
Rana spp. in Coon Lake, are restricted to a single body of water, due to their reliance on an aquatic habitat, while definitive hosts of allogenic parasites can move between aquatic habitats. As a result, a lake may be visited by not only multiple individuals of potential allogenic parasite hosts, but a wide variety of species as well, consequently increasing the diversity of allogenic parasites. This allows for the potential to introduce more allogenic parasite species to an aquatic system.
The underestimates of parasite prevalence are not uncommon among these types of studies (Goater et al., 1989). Most trematode species have pre-patent periods of 4-8 wk, indicating that a majority of the parasites in Coon Lake were recruited in March and
April due to the high degree of underestimation of infection in May and nearly a 4-fold reduction by July. These are common findings among trematodes in the Piedmont region of North Carolina (Fernandez and Esch, 1991; Sapp and Esch, 1994; Negovetich and
Esch, 2007; Chapter II). Snails are in torpor and largely inactive between November and
March before they emerge from the substrata. This emergence coincides with waterfowl migrating to and inhabitating this region of North Carolina, resulting in high recruitment of allogenic parasites during this time period (Chapter II). Parasite recruitment begins to taper off as summer approaches and the migratory waterfowl travel north to their breeding grounds and the residual effects of the parasites begin to decline (Chapter II).
Autogenic species however, are present yr round and could potentially induce infection in the snail intermediate hosts throughout the collection season. This can occur
141 among trematode species that use fish as definitive hosts, since they are active and feeding year round (McDaniel and Bailey, 1974). However, all of the autogenic parasites in Coon Lake cycle through Rana spp. Neither the largemouth bass nor the bluegill sunfish harbored adult trematode parasites with autogenic life cycles using snails as intermediate hosts (K. Luth, pers. comm.). Since Rana spp. were the only hosts for autogenic parasites, comparable patterns in the initial infection dynamics of parasites were observed between the autogenic and allogenic species. Similar to the snail hosts, frogs typically go through a period of hibernation and inactivity in the winter mo
(Holenweg and Reyer, 2000; Regosin et al., 2003). This results in parallel timing of infection and pre-patent periods between the autogenic and allogenic species in the spring.
The lack of differences between the different types of parasites, and the various habitat classifications, allows all the data between mo to be combined.
The distribution of both the autogenic and allogenic parasites in Coon Lake appears to be the by-product of the habitat disturbance caused by the construction of the new drainage system on the golf course. During the construction of the drainage system, the water was reduced to only one-third capacity for 2 mo before being refilled to normal water levels. In addition to draining the water, the western bank was rebuilt for stability and better drainage of run-off from the golf course into the pond. As a result, the habitat on the western bank was devoid of any vegetation aside from the grass that was planted to prevent soil erosion. These changes affected the distribution of the definitive hosts, both visiting and permanent.
Three Rana spp. have been frequently observed at Coon Lake, Rana pipiens, R. catesbeiana, and R. palustris . All of these species are ambush predators and inhabit the
142 shallow regions of the pond and the bank surrounded by dense and emergent vegetation.
These densely vegetated habitats offer the frogs protection from predators as well as an abundant supply of insects that also use the habitat for refuge. Frogs were rarely observed in open habitats, presumably due to the risk of predation and a less abundant food supply.
Parasites employing allogenic life cycles in Coon Lake all used aquatic waterfowl as definitive hosts. Their behavior and habitat preference were opposite those of the frog species. The birds preferred the open habitat more than the densely wooded areas.
Waterfowl were frequently observed inhabiting and grazing in the thick grass filled patches adjacent to the golf course and the wooded areas. The area closest to the golf course where the drainage system was installed has an extremely steep drop-off into the pond, with limited to no littoral zone. The open edges adjacent to the woods, however, offered a flat bank with an extensive littoral zone; these areas have high traffic from the waterfowl entering and exiting the pond, which probably contributed to the high prevalence of allogenic parasites in the edge regions of this system.
Not only has the habitat disturbance affected the distribution of the vertebrate definitive hosts, but this trickles down to affect the parasites and their distribution as well.
As a result of the frog hosts inhabiting the dense and emergent vegetation, this area is where the autogenic parasites infect the snail intermediate hosts (Fig. 1B). This results in the autogenic parasites being distributed further away from central disturbance point
(Figs. 2 , 3). In this particular case, snails distributed deeper in the woods are more likely to be infected by an autogenic parasite. The opposite trend was observed among the allogenic species that use waterfowl as the definitive host. The avian hosts inhabit the
143 regions of open and edge habitats due to the abundance of food and ease of access to enter and exit the pond. This results in the allogenic parasites being distributed closer to the point of maximum disturbance and an increased likelihood of being present in the open and edge areas.
The changing distribution of the hosts, and consequently their parasites, as a result of microhabitat changes can be important to the dynamics of the inhabitants of an aquatic ecosystem. Due to an overlap in the distribution of the parasites in the edge habitat, increased parasite transmission occurs at these locations. Parasite prevalence in the edge habitat is significantly greater than that of the open or wooded habitats because of compound effects due to the overlap in definitive host distribution. Diversity and abundance of parasite species are greatest at intermediate levels of disturbance in this system, a phenomenon known as the intermediate disturbance hypothesis (Hutchinson,
1961; Grime, 1973; Connell, 1978).
Habitat disturbance can have an enormous impact on the distribution and transmission patterns of parasites. By changing the landscape, and potentially the distribution of the hosts, the location of their parasites can change extensively as well.
This can create compounding effects and increased transmission at intermediate levels of disturbance, significantly shaping the distribution and transmission dynamics of the parasites. The effect of habitat disturbance on parasites and their hosts can lead to increased parasite transmission and potentially serve as indirect markers of definitive host behavior and inhabitation in and around aquatic environments.
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147 Table I. Trematode parasite species and their snail hosts, definitive hosts, life cycle types, and prevalence in Coon Lake.
Parasite Species Snail Host Definitive Host Life Cycle Type Prevalence Glypthelmins quieta P. gyrina Rana spp. Autogenic 2.4%
Haematoloechus complexus P. gyrina Rana spp. Autogenic 0.5%
Megalodiscus temperatus P. gyrina Rana spp. Autogenic 0.4%
Australapemonus burtii L. columella Waterfowl Allogenic 0.8%
Australapemonus canadiensis L. columella Waterfowl Allogenic 0.2%
Echinoparyphium recurvatum L. columella Waterfowl Allogenic 0.3%
Notocotylus attenuatus P. gyrina Waterfowl Allogenic 5.0%
Plagiorchis sp. P. gyrina Waterfowl Allogenic 1.8%
Tylodelphis sp. P. gyrina Waterfowl Allogenic 0.3%
unknown strigeid M. dilatus Waterfowl Allogenic 4.7%
Total Prevalence 7.8%
148 FIGURE 1. Prevalence of (A) all parasite species combined and (B) autogenic and allogenic parasite species in the 3 habitat categories in Coon Lake
149 150 FIGURE 2. Prevalence of autogenic and allogenic parasites in relation to the distance from the point of maximum disturbance in Coon Lake
151 152 FIGURE 3. The generalized additive models (GAMs) of the prevalence of (A) autogenic parasites and (B) allogenic parasites in relation to the distance from the central point of disturbance.
153 154 CHAPTER V
DIFFERENCES IN MICROHABITAT AFFECT PARASITE TRANSMISSION IN A
PULMONATE SNAIL, HELISOMA ANCEPS
The following manuscript was submitted for publication in Freshwater Science (2014, In
Review) and is reprinted with permission. M. R. Zimmermann performed the study and prepared the manuscript. K. E. Luth aided in sample collection and an editorial capacity.
G. W. Esch acted in an advisory and editorial capacity.
155 ABSTRACT
Habitat selection as a major indicator of species distribution has been examined extensively in both aquatic and terrestrial habitats, but little attention has been given to how this affects the transmission of their parasites. The infection dynamics of both digenetic trematode parasites and a nematode parasite infecting a pulmonate snail,
Helisoma anceps , were observed in a small North Carolina lake using 3 different classifications of substratum type based on percent coverage by leaves and debris.
Although no differences in the distribution patterns of H. anceps were observed in correlation with the substratum type, small-scale differences in microhabitat impacted the parasites’ ability to find and infect their snail hosts, in particular, increased leaf litter and debris resulted in decreased infectivity by the parasites.
156 INTRODUCTION
Habitat selection is a major indicator of species distribution in both terrestrial and aquatic habitats. Resource availability and competition are both major factors that have been extensively scrutinized in regards to where certain animals choose to live
(Rosenzweig, 1981; Pulliam and Danielson, 1991; Elith and Leathwick, 2009), but, to a parasite, a host represents a habitat patch by providing the metabolic resources necessary for host survival (Sousa and Grosholz, 1991). Habitat structure not only influences the distribution of hosts, but may also affect the rate of transmission of their parasites.
Physical features of habitat, ranging from other living organisms to inanimate objects, may influence both host and parasite movement, as well as their infection dynamics
(Sousa and Grosholz, 1991).
Pulmonate snails are common inhabitants of freshwater lakes and ponds throughout North America, typically occupying the benthic regions of the littoral zone.
Moreover, among pulmonate snail families, there is a high degree of habitat differentiation among species in the gastropod fauna (Harman, 1972; Dillon, 2000).
Their selection of habitat appears to be driven based on their diets and preferential food choices, but it may be more complicated than originally assumed (Harman, 1972; Dillon,
2000).
Helisoma anceps is a planorbid snail with a cosmopolitan distribution across the
U.S. and Canada. These snails tend to inhabit areas possessing leaf litter, decaying logs, and detritus in the littoral benthos of lakes and ponds (Malek 1958; Harman, 1972; Pip,
1987; Dillon, 2000). Feeding primarily on the latter, as well as macrophytes and bacteria, these snails are considered to be ‘area restricted searchers’ and mo ve very little
157 over the course of their lifetime (Charnov et al., 1976; Thomas, 1987; Dillon, 2000). As a result, these snails offer a useful model system for studying parasite transmission in varying habitats, because they would presumably exhibit little movement between them.
These snails are also hosts to a variety of trematode parasites (Esch and
Fernandez, 1994; Negovetich and Esch, 2007; Chapter II). These parasites have complex life cycles, most of which include a short-lived and free-swimming larval stage (a miracidium) that hatches from an egg. Hosts for miracidia are almost exclusively molluscs, most often snails. Some trematode species, such as Echinostoma spp., not only use gastropods as a first intermediate host, but as a second intermediate host as well
(Esteban and Munoz-Antoli, 2009). Once a miracidium infects a snail, it sheds its sternal plates, develops into a sporocyst, followed by the production of mother and daughter rediae. Both sporocysts and rediae are pathogenic to their gastropod hosts, frequently consuming gonadal tissue, effectively castrating their host snail (Sullivan et al., 1985;
Esch and Fernandez, 1994; Sorenson and Minchella 1998). These rediae then reproduce asexually, resulting in hundreds of larval clones, known as cercariae. Cercariae, also a free-swimming larval stage, are then released from the snail into the water column, where they then locate a gastropod, which they then use as a second intermediate host. On penetration of the snail host, cercariae lose their tails and encyst in the kidney region of the snail, becoming metacercariae (Esteban and Munoz-Antoli, 2009).
Although trematodes commonly parasitize snails, nematodes can, in a very limited way, infect these hosts as well. Daubaylia potomaca (Rhabditida) is a nematode parasite that inhabits all tissues and blood spaces of their gastropod host (Chernin, 1962;
Zimmermann et al., 2011a). It is unique, however, in having a direct life cycle via
158 planorbid snails in which the adult female is the infective stage, capable of exiting snail hosts and infecting new hosts (Chernin, 1962; Zimmermann et al., 2011a). Adult females are most often shed immediately preceding host death, but are not capable of swimming and must migrate via the substratum in order to locate a new snail host
(Zimmermann et al., 2013). These nematode parasites produce pathology to their host via tissue migration and negatively impact snail survival (Zimmermann et al., 2011b).
The ability to locate a suitable host is a problem encountered by all parasites.
Vagility of both hosts and parasites is a key determinant in the transmission success in host-parasite systems. However, the snail-parasite system studied here involves organisms with low mobility and, presumably, low encounter rates between the host and their parasites. The goal of the present study was to determine if microhabitat structure plays a role in the transmission of trematode parasites, as well as the infection dynamics of the nematode parasite D. potomaca . If habitat structure is a good predictor of host location, or the parasites are fairly mobile, then habitat structure may facilitate host finding. However, if microhabitat is a poor indicator of host presence or interferes with parasite mobility, host finding may be impaired (Sousa and Grosholz, 1991). This hypothesis was tested through the examination of snail host distribution and parasite transmission in 3 different substrata types of a small North Carolina lake.
159 MATERIALS AND METHODS
Study site
Mallard Lake is 4.9 ha body of water located in Forsyth County, North Carolina
(36 o00’03” N, 80 o24’07” W). Situated in a public park, the eastern bank of this lake is bordered by a public golf course and park, resulting in little, to no, vegetation in this portion of the pond. The entire west bank is wooded, with an abundance of hardwood trees. The southern and southeastern banks are covered intermittently with hardwood trees; a small island in the southern end of the pond is also covered by vegetation. As a result of the heterogeneous distribution of vegetation around the lake, the substrata also vary considerably. The pond was divided into 148, 10-m transects, each of which were placed into 1 of 3 classifications. Clay/silt substratum was defined as having at least 2/3 of the transect free of leaves and debris and the bare substratum exposed. Leaf litter substratum was defined as having at least 2/3 of the transect covered in leaves and debris from the surrounding trees and the mixed substratum was defined as a mixture of the 2 aforementioned classifications in which approximately half the substrata was free of leaves and debris and half was covered.
Snail density
Density measurements of H. anceps inhabiting the various substrata were made during each collection. Before each sampling, a 1-m 2 ring was randomly tossed into the transect and all snails within the ring were collected and counted. The prevalence, mean abundance, and mean intensity of H. anceps in each of the substratum types was calculated to determine if the snails preferred 1 microhabitat over another.
Snail collections and necropsy
160 A total of 4,218 H. anceps was collected from Mallard Lake by hand collections and dip-netting. Of these, 1,122 were sampled from clay/silt substrata, 1,677 from leaf substrata, and 1,420 from mixed substrata. The snails were returned to lab and isolated in
55-mm fingerbowls to check for the presence of cercariae. After 48 hr, the snails were crushed and necropsied. The following information was recorded for each snail: substratum type from which the snail was obtained, shell length (to the nearest 0.05 mm), trematode parasites present, number of Echinostoma sp. metacercariae, and number of D. potomaca . The recruitment of miracidia was determined by the presence/absence of sporocysts/rediae in the snail host. Similarly, cercariae recruitment was quantified by the presence and number of metacercariae in H. anceps .
Statistical analysis
The parasite populations were described in terms of prevalence and mean intensity according to the terminology of Bush et al. (1997). Prevalence of snails inhabiting different substrata were compared using Chi square analysis and the mean abundance and intensity of H. anceps were compared using 1-way ANOVA; Tukey-
Kramer honestly significant difference (HSD) post-hoc tests were used to reveal differences between groups. Mixed effects regression models were evaluated to determine if there were differences in the prevalence and intensity of the parasite species between the substratum types. The transects were used as random effects, while the substratum types and the prevalence and intensity of various parasites were used as predictors. Chi square analysis and Steel-Dwass post-hoc tests were used to compare the prevalence and intensities of the parasites between the various substrata. All models and
161 statistical analyses were completed using R (R Development Core Team, Vienna,
Austria).
162 RESULTS
Snail density
There were no statistically significant differences in the prevalence of H. anceps in the 3 substratum types (Chi square test, P = 0.6636). Furthermore, no significant trends in either mean abundance (ANOVA, P = 0.9641) or mean intensity (ANOVA, P =
0.8690) existed across the different substrata.
Trematode miracidia infection
The prevalence of trematode miracidia infection showed statistically significant differences between the 3 substrata (Mixed Effects Model, P < 0.0001). Prevalence was highest in snails in the clay/silt substrata (18.6%), with mixed(12.4%) and leaf substrata having significantly lower prevalence of miracidia infection (Chi square test, P < 0.0001;
10.4%) (Fig. 1).
Echinostoma sp. cercariae infection
Statistically significant differences in the prevalence of Echinostoma sp. cercariae infection were evident among the various substrata (Mixed Effects Model, P = 0.0062).
The highest prevalence of cercariae infection was in snails inhabiting the clay/silt substrata (37.3%), followed by the leaf litter substrata (32.8%) and the mixed substrata
(28.4%; Fig. 1). Similarly, distinct trends in the mean intensity of metacercariae infection were also found (Mixed Effects Model, P = 0.0021). The highest intensity was in snails from the clay/silt substrata (124.0 ± 9.6), followed by snails from mixed (85.1 ±
7.9) and leaf substrata (62.9 ± 5.9), all of which differed significantly from each other
(Steel-Dwass, P < 0.05; Fig. 2A).
163 Daubaylia potomaca infection
There were no statistically significant differences in the prevalence of D. potomaca between the different substrata types (Mixed Effects Model, P = 0.8081) (Fig.
1). However, there were significant differences in the mean intensity of infection, with the highest intensity observed in clay/silt substrata (31.5 ± 4.5), followed by mixed (28.5
± 2.7) and leaf substrata (22.2 ± 1.9) (Mixed Effects Model, P = 0.0326; Fig. 2B). There was no statistical difference in D. potomaca intensity between clay/silt and mixed substrata (Steel-Dwass, P > 0.05), but the mean intensities of the nematode parasite in both these substrata were greater than the leaf substrata (Steel-Dwass, P < 0.05).
164 DISCUSSION
There were no differences in prevalence, abundance, or intensity of H. anceps inhabiting the 3 substrata, showing that the variation in microhabitat was not great enough to affect the snail’s distribution patterns. Habitat structure was a poor indicator of host presence in Mallard Lake as the snails exhibited a ubiquitous distribution throughout the substratum types. This suggests that microhabitat has a much stronger effect on parasites and their transmission to their snail hosts than it does on the gastropods themselves.
All parasite species in the study showed similar trends in their infection dynamics with respect to the different substratum types. The highest prevalence and intensity of parasite infection was observed in the clay/silt substrata and lowest in the leaf substrata, with mixed substrata usually showing intermediate levels of infection (Figs. 1, 2). Given the behavior of these parasites, these findings are not surprising.
Miracidia are ciliated life stages of digenetic trematodes capable of swimming in aquatic environments. The transmission success of this stage of the parasite is often affected by various components of the aquatic environment, such as leaf litter and other debris, water currents, and other biota, as well as the spatial distribution of their hosts
(Sousa and Grosholz, 1991; Smith, 2001). Equipped with special sensory cells, miracidia can respond to a variety of environmental stimuli, e.g., light, current, temperature, and gravity (Ulmer, 1971; Chernin, 1974; Nollen, 1994; Stabrosky and Nollen, 1995), and are often found swimming just above the bottoms of lakes and ponds in search of their first intermediate hosts. First intermediate hosts for digenetic trematode parasites are almost exclusively molluscs, and most often gastropods, that inhabit the benthic environment.
165 Additionally, miracidia are capable of detecting mucus trails of their gastropod hosts and use these cues to aid in host location (Haas et al., 1995; Haberl et al., 2000). The mucus trails and other environmental stimuli would presumably be easier to detect on the bare clay/silt substratum than trying to follow them through the labyrinth of leaf clutter. As a result, it would be suspected that transmission of miracidia to H. anceps occurs in the bare substratum because of the ease in locating the snail host in comparison with the other substrata types.
Echinostoma sp. not only utilize H. anceps as a first intermediate host, but as a second intermediate host as well. Similar to miracidia, Echinostoma sp. cercariae also use environmental and chemical cues to aid in locating snails to infect as second intermediate hosts (Whitfield et al., 1977; Smyth and Halton, 1983; Haas et al., 1995;
Haberl et al., 2000). A non-feeding stage of the trematode life cycle, most cercariae must locate a snail host within 24-36 hr of emergence, before its energy stores are expended
(Lo and Cross, 1975; Fried and Bennett, 1979). This provides only a short transmission window and should be a good indicator of how the different substrata influence their success in finding an intermediate host. Similar to miracidia, Echinostoma sp. cercariae have greater transmission success in terms of both prevalence and intensity of infection in clay/silt substrata compared to leaf litter (Figs. 1, 2A).
Daubaylia potomaca utilizes H. anceps for its entire life cycle and , on reaching sexual maturity, can either leave in search of a new snail host, or remain and continue to reproduce within the same snail (Zimmermann et al., 2011a; Zimmermann et al., 2013).
This parasite also differs from trematode parasites in that the nematodes are not active swimmers and, instead, employ a ‘questing’ behavior in the substratum. The route of
166 infection is presumably through the foot of the snail host, but the mechanism is unknown.
Unlike the digenetic trematodes, there was no difference in the prevalence of infection between the different substrata (Fig. 1). However, there was a statistically significant relationship between the intensity of D. potomaca infections and substrata types. Snails in the bare clay/silt substrata revealed significantly higher mean intensities of nematode infection than snails in the leaf substrata (Fig. 2B).
When D. potomaca are shed from their snail hosts, they tend to be shed in large numbers over a short period of time, typically just prior to host death (Zimmermann et al.,
2013). Accordingly, the transmission probability for a single nematode from among hundreds infecting a host is relatively high. This may account for the lack of difference observed in the prevalence of D. potomaca infection in the various substrata since only a single nematode was required to indicate transmission success. Mean intensity of the nematode parasite is a better indicator of substrata differences and showed that transmission success was greater in the absence of leaf litter. Similar to the trematode parasites, the less debris and obstacles D. potomaca encounters when searching for a snail host, the better the transmission success.
Past studies have shown that habitat choice and differences in habitat selection by hosts can drive differential success in parasite transmission, typically by placing hosts and parasites in close proximity (Marcogliese et al., 1990; Sousa and Grosholz 1991;
Zelmer et al., 1999; Smith, 2001). Sapp and Esch (1994) even showed that parasite transmission in snails may be affected by small scale differences in depth and distance from shore of the hosts. The current study emphasizes that small-scale differences in
167 habitat structure can affect the infection success and that substratum properties are an important factor in parasite transmission dynamics.
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172 FIGURE 1. Prevalence of parasites infecting H. anceps in 3 different substratum types.
173 174 FIGURE 2. Mean intensity (+/- standard error) of (A) Echinostoma sp. metacercariae and (B) D. potomaca infecting H. anceps in 3 different substratum types.
175 176 CHAPTER VI
TRANSMISSION PATTERN DIFFERNCES OF MIRACIDIA AND CERCARIAE
LARVAL STAGES OF DIGENETIC TREMATODE PARASITES IN RELATION TO
SNAIL SIZE AND DENSITY
The following manuscript was submitted for publication in the Comparative Parasitology
(2014, In Review) and is reprinted with permission. M. R. Zimmermann performed the study and prepared the manuscript. K. E. Luth aided in sample collection and an editorial
capacity. G. W. Esch acted in an advisory and editorial capacity.
177 ABSTRACT
Some digenetic trematode species have 2 larval stages capable of infecting snails as intermediate hosts. Miracidia use snails as a first intermediate hosts, with cercariae using gastropods as second intermediate hosts. Significant differences in the infection patterns of these parasitic stages regarding host size and density were observed in 2 separate field studies. The prevalence of miracidia and mean intensity of Echinostoma spp. cercariae infection were positively correlated with host size, while the prevalence of
Echinostoma spp. cercariae infection was positively correlated with host density.
Miracidia and cercariae utilize different chemical cues to aid in host location and also have varying degrees of host specificity, both of which may contribute to the transmission differences. Larger snails within a given species tend to be older and produce more distinct chemical cues that could lead to the correlations between snail size and parasite infection.
178 INTRODUCTION
Digenetic trematode parasites are characterized in having complex life cycles in which multiple hosts are required for completion. These life cycles employ an array of strategies to assure that the larval stages progress through the life cycle to ultimately reach adulthood. In nearly all trematode life cycles, aquatic molluscs, namely gastropods, play an integral role, commonly serving as first intermediate hosts. Additionally, some trematode species utilize aquatic snails as second intermediate hosts (Kuris and Warren,
1980; Morley et al. 2004; Faltynkova et al., 2007a, 2007b; Esteban and Munoz-Antoli,
2009). Although both miracidia and cercariae infect gastropods as hosts in their life cycles, they accomplish this end using very different strategies.
Both trematode larval stages rely on chemical cues produced by molluscs to aid in
‘host locating’ processes, and then utilize different abiotic signals to find them (Haas et al., 1995; Haberl et al., 2000). Miracidia detect high molecular weight glycoproteins, called miracidia-attracting-glycoproteins (MAGs), that are released in mucous trails left by molluscan hosts (Haberl et al., 2000; Kalbe et al., 2000), while cercariae respond to amino acids, urea, ammonia, and other peptides released by gastropods (Haberl et al.,
2000; Haas, 2003). Additionally, miracidia typically respond to highly specific MAGs produced by a small number of host species, which explains the strong affinities exhibited by some trematodes and their snail first intermediate hosts. Cercariae, however, employ comparatively non-specific host finding devices and respond to a larger array of chemical cues, creating less specificity for second intermediate hosts (Haas, 2003).
The infection dynamics of these trematode parasites in the snail first and second intermediate hosts are important to understanding the life history and transmission
179 patterns of these parasites. The infection dynamics of digenetic trematodes may also have a seasonal component in which recruitment is highest in periods when definitive hosts are most abundant, but they are also affected by other factors such as host ecology, abundance, and exposure time (Fernandez and Esch, 1991; Sapp and Esch, 1994;
Detwiler et al., 2009).
Additionally, both snail density and shell length are important factors in the structuring of trematode infections in their gastropod first intermediate hosts. Density tends to be negatively correlated with miracidia infection due to limited numbers of the larval stage in a particular habitat, but the effects on snail density have yet to be investigated in the field. Snail size has also been shown to be an important factor in the infection patterns of trematode parasites, oftentimes used as a proxy for encounter rate since larger animals tend to be more frequently and heavily infected with parasites
(Fernandez and Esch, 1991; Jokela and Lively, 1995; Sorensen and Minchella, 1998;
Sandland et al., 2001; Byers et al., 2008; Detwiler et al., 2009). Larger snails, within a given species, also tend to be older and, with age, also develops a potentially increased exposure time to the larval stages of trematode parasites, leading to increases in parasite infection (Evans et al., 1981; Fernandez and Esch, 1991; Jokela and Lively, 1995; Morley,
Crane, and Adam, 2004).
Although trematode acquisition by snails has been well documented in natural systems (Fernandez and Esch, 1991; Jokela and Lively, 1995; Sorenson and Minchella,
1985; Byers et al., 2008), the recruitment patterns of cercariae by molluscan hosts have been a far less well researched phenomenon (McCarthy, 1990; Evans et al., 1981; Morley,
Crane, and Adam, 2004). In this regard, Echinostoma spp. can serve as a useful model
180 for cercariae recruitment because they are able to employ snails as both first and second intermediate hosts (Morley, Crane, and Lewis, 2004, 2004b; Esteban and Munoz-Antoli,
2009). It was hypothesized that snail density and shell size would have an impact on the infection patterns of both miracidia and Echinostoma spp. cercariae. This hypothesis was investigated in 2 separate field studies using several species of pulmonate snails.
181 MATERIALS AND METHODS
Snail collections
Mallard Lake : Mallard Lake is a 4.9 ha pond located in the Piedmont region of
North Carolina (36 o00’03” N, 80 o24’07” W). A total of 4,218 Helisoma anceps snails was collected by hand and dip-netting from various portions of the lake between
September 2008 and March 2010. The snails were returned to lab, isolated for 48 hr, and necropsied. Each snail was measured to the nearest 0.05 mm and the presence, patency, and species of digenetic trematode were recorded. In addition, the number of
Echinostoma sp. metacercariae was noted.
Samples sites across the United States : A total of 9,498 pulmonate snails was collected from 139 lakes and ponds across the United States, east of the Rocky
Mountains, between June and August 2012 and July and August 2013. Five snail species were examined, including Helisoma anceps (N = 1,065), H. trivolvis (N = 1,360),
Lymnaea columella (N = 781), Physa acuta (N = 4,500), and P. gyrina (N = 2,792).
Prior to each collection, snail density was estimated by collecting all snails within a randomly placed 0.05-m 2 rectangle. Snails were preserved in alcohol after collection and each snail was measured to the nearest 0.05 mm; the trematode species present were identified, and the number of echinostome metacercariae was recorded. Echinostome metacercariae were identified by counting the number of collar spines present. Only those belonging to the revolutum -group, possessing 37 collar spines, were used in the study.
182 Statistical analysis
The parasite population was described according to the terminology of Bush et al.
(1997). A linear mixed effects model was performed on the prevalence of miracidia and cercariae infection as well as the intensity of metacercariae of the snails in Mallard Lake.
For this model, length was used as a predictor and month of collection was used as a random effect. Linear mixed effects models were also performed on the prevalence of miracidia infection and the prevalence and intensity of Echinostoma spp. cercariae infection using snail shell length, lake surface area, and snail density as predictors, and the collection site as a random effect for the sample sites across the U.S. These models were performed on each snail species separately. An analysis of covariance (ANCOVA) was performed to determine which predictors best explained the observed trends. All statistics were performed using the R software (R Development Core Team, Vienna,
Austria).
183 RESULTS
Mallard Lake
A statistically significant positive correlation was found for shell length and the prevalence of infection by trematode miracidia, indicating that larger snails were more likely to be infected by the larval stage of the parasite (Mixed Effect Model, P < 0.0001;
Fig. 1A). This trend was not observed for the prevalence of infection by Echinostoma sp. cercariae (Mixed Effect Model, P = 0.3181; Fig. 1B); however, larger snails harbored greater intensities of echinostome metacercariae (Mixed Effect Model, P < 0.0001; Fig.
1C).
Sample sites across the U.S.
In all snail species studied, larger snails were significantly more likely to be infected by miracidia of trematode species (Mixed Effect Model, P < 0.05; Fig. 2). Both
H. anceps (P = 0.0052) and L. columella (P = 0.0174) showed negative correlations with density and prevalence of infection, but, in all snail species, shell length was the only predictor that was statistically significant (ANCOVA, P < 0.05). This trend was not observed for the infection by Echinostoma spp. cercariae. None of the snail species exhibited a significant trend with respect to parasite infection and shell length (Mixed
Effect Model, P > 0.05; Fig. 3). However, there was a significant positive correlation between snail density and the prevalence of infection by echinostome cercariae, indicating that a greater density of snails increases the prevalence of infection for all species tested (Mixed Effect Model, P < 0.05; Fig. 4). Furthermore, snail density was the only significant predictor for the prevalence of metacercariae among all snail species sampled (ANCOVA, P < 0.05). The infection patterns for the intensity of Echinostoma
184 spp. metacercariae resembled those of the trematode miracidia in that larger snails harbored greater parasite loads across all species (Mixed Effect Model, P < 0.05), with shell length being the only statistically significant predictor for each of the snail species tested (ANCOVA, P < 0.05).
185 DISCUSSION
Both the survey conducted in Mallard Lake and the sampling studies from across the U.S. showed similar trends regarding the infection patterns of both miracidia and cercariae larval stages of trematode parasites in their gastropod hosts. Snails in Mallard
Lake were collected yr round and revealed seasonal differences in parasite transmission
(Chapter II). Higher prevalence and intensities of Echinostoma sp. infection in the summer mo could possibly skew the correlations of infection with snail size. However, the effect of snail shell length on the transmission patterns of the trematodes was independent of the seasonal component, indicating that snail size is a strong predictor of the infection patterns of miracidia prevalence and echinostome metacercariae intensity, but not metacercariae prevalence. Even though trematode miracidia and Echinostoma spp. cercariae both utilize pulmonate snails as hosts in completing their life cycles, they rely on completely different factors to accomplish these ends. Miracidia are short-lived stages that infect snails as first intermediate hosts. As a non-feeding stage, miracidia can typically survive 12-24 hr in the water column before exhausting their energy stores
(Anderson et al., 1982; Muñoz-Antoli et al., 2000; Muñoz-Antoli et al., 2002).
Consequently, they must locate their hosts quickly, accomplishing this step via the detection of chemical cues produced by the snails (Haberl et al., 2000; Kalbe et al., 2000).
Miracidia typically detect MAGs, chemical agents that are present in the mucus of snails, which promote the very high host specificity (Kalbe et al., 2000; Haas, 2003)
In both the Mallard Lake study site and the sample sites from across the eastern
2/3 of U.S., larger snails were more likely to become infected by miracidia of various trematode species. Snail size is often used as a proxy for encounter rate (Detwiler and
186 Minchella, 2009) and it has been demonstrated that larger snails are more likely to become infected by miracidia (Fernandez and Esch, 1991; Jokela and Lively, 1995;
Sorensen and Minchella, 1998; Byers et al., 2008). Additionally, larger snails produce more distinct signals for the miracidia to follow while locating and infecting snail hosts, i.e., larger snails produce more mucus. Neither lake surface area nor snail density were significant indicators of miracidia infection. Fenton et al. (2002) suggested that miracidia transmission success is not dependent on host density, at least in terms of prevalence of infection, because they infect a single snail and inhabit that host for the duration of their life. In other words, there is a threshold for the number of snails that can be infected by miracidia, due to the limited number of eggs deposited in an aquatic system by a definitive host, reducing the impact of snail density on miracidia infection (Johnson et al.,
2012). Free-living miracidia have limited dispersal ability and infect snails in close proximity to the point of egg release by the definitive host; accordingly, the density of snail hosts does not change infection probabilities (Fenton et al., 2002). In fact, increasing snail density may actually decrease prevalence since a single miracidia can only infect 1 snail host. This was observed in both H. anceps and L. columella in the current study. Both species showed a negative correlation between the prevalence of miracidia infection and the density of snails. Limited numbers of miracidia that can infect only a single host result in decreased prevalence when the overall abundance of hosts increase (Fenton et al., 2002).
A similar trend was observed for the intensity of infection by Echinostoma spp. metacercariae. Larger snails accumulated significantly more metacercariae than smaller individuals of the same species. Given that larger snails within a species tend to be older
187 and have had more time to accumulate parasite infections, this trend is not surprising
(Sandland et al., 2001; Morley, Crane, and Adam, 2004; Detwiler and Minchella, 2009;
Byers et al., 2008). Neither lake surface area nor snail density showed consistent trends with the intensity of infection by echinostome cercariae, while length, a proxy for exposure time, was the best predictor in both Mallard Lake and all snail species collected throughout the more widely scattered sample sites.
The prevalence of infection by Echinostoma spp. cercariae did not follow the same trend as the prevalence of miracidia or intensity of cercariae infection in the snail hosts. Thus, no correlation was observed for shell size and the likelihood of being infected by Echinostoma spp. cercariae, indicating that snail size is not a good predictor of cercariae infection. Cercariae respond to different chemical cues than miracidia and exhibit far less host specificity (Haas, et al., 1995; Haberl et al., 2000). These cues include amino acids, urea, ammonia, and various peptides, all of which are produced by snail hosts of different sizes and species (Haas, 2003). As a result, size is much less of an influencing factor in host location by cercariae in comparison with miracidia. However, echinostome cercariae tend to accumulate in hosts with increased exposure time in the larger, older snails, resulting in greater intensities of this larval stage (Morley, Crane, and
Adam, 2004; Detwiler and Minchella, 2009).
Although shell length did not reveal a significant correlation in the prevalence of cercariae infection, snail density did. The greater the density of snails, the more likely an individual was to become infected across all study sites and species. Cercariae are similar to miracidia in that, while usually mobile, they are limited in their dispersal ability.
They can only survive for 24-48 hr and need to find a host quickly before losing their
188 energy stores since they are a non-feeding larval stage of the parasite (McCarthy, 1999;
Toledo et al., 1999; Muñoz-Antoli et al. 2002). Unlike miracidia, cercariae production results in an abundant supply of parasites able to infect the snails since they are larval clones. Densely populated snails offer a multitude of available hosts for the cercariae to infect and since size discrimination does not occur, this can significantly increase the prevalence of infection because cercariae are not required to travel far to find a host.
Other studies have found similar results regarding the infection patterns of echinostome cercariae (McCarthy, 1990; Morley, Crane, and Adam, 2004); however, there are a few key differences between those studies and the present one. The latter authors found that larger snails had greater intensities of echinostome metacercariae, similar to the present study. They also reported an extremely high prevalence of metacercariae infection at their sample site, coupled with much smaller sample sizes than those of the current study. Unfortunately, these factors do not give a clear picture of the infection dynamics of metacercariae prevalence regarding snail density or size. The intermediate levels of prevalence of echinostome metacercariae in the present study indicate that snail density is a more important factor than size, with respect to cercariae infection patterns. McCarthy (1990) also examined the effect of snail density as a factor for cercariae transmission. He found that as density increased, so did the prevalence and intensity of infection. A similar trend was found in regards to prevalence of echinostome metacercariae in the present study, but not intensity. One key difference between the current study and McCarthy et al. (1990) is that the latter used snails that were all the same size and tested snail density without accounting for the effect of size discrepancies observed in nature. The present study shows that the effects of snail size and age that
189 allow for the accumulation of parasites outweigh the effects of density in nature, in regards to the intensity of Echinostoma spp. metacercariae infection.
In conclusion, miracidia and cercariae stages of digenetic trematodes have varying levels of host specificity and very different chemotactic properties. As a result, these larval stages show varying patterns of infection among their snail hosts in natural populations. Miracidia are dependent on larger snails that produce more mucus and presumably stronger signals of the host-specific MAGs, while cercariae can detect a variety of chemical cues commonly produced by snails of varying sizes and species.
Cercariae infection is not influenced by the size of the snail hosts, but their density.
Accumulation of these parasites, however, is affected by snail size due to larger snails, within a given species, tending to be older and having longer exposure times in which to accumulate metacercariae. These findings lend important information regarding the role of host specificity and the factors involved in the transmission patterns of the larval stages of digenetic trematode parasites to gastropod first intermediate hosts.
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194 FIGURE 1. The (A) prevalence of miracidia infection, (B) prevalence of Echinostoma sp. cercariae infection, and (C) mean intensity (+/- standard error) of Echinostoma sp. metacercariae in H. anceps snails divided into 1-mm size classes from Mallard Lake.
195 196 FIGURE 2. Prevalence of trematode miracidia infection in 5 pulmonate snails species divided into 1-mm size classes from sample sites across the U.S. (A) H. anceps , (B) H. trivolvis , (C) L. columella , (D) P. acuta , (E) P. gyrina .
197 198 FIGURE 3. Prevalence of Echinostoma spp. metacercariae infection in 5 pulmonate snail species divided into 1-mm size classes from sample sites across the U.S. (A) H. anceps ,
(B) H. trivolvis , (C) L. columella , (D) P. acuta , (E) P. gyrina .
199 200 FIGURE 4. Mean intensity (± standard error) of Echinostoma spp. metacercariae infection in 5 pulmonate snail species divided into 1-mm size classes from sample sites across the U.S. (A) H. anceps , (B) H. trivolvis , (C) L. columella , (D) P. acuta , (E) P. gyrina .
201 202 CHAPTER VII
AUTO-INFECTION BY ECHINOSTOMA SPP. IN NATURE
203 ABSTRACT
Auto-infection is a life history strategy used by many parasitic organisms, including digenetic trematodes. The process of auto-infection most frequently involves the transfer of a life cycle stage of the parasite from 1 site to another inside the same host, usually accompanied by morphological transformation. Moreover, among trematodes, the stage being transferred may also move from 1 host to another in completing the life cycle, i.e., an indirect cycle. Echinostoma spp. parasites offer the opportunity to study auto-infection because they utilize gastropods as both first and second intermediate hosts.
In 2 separate field studies, significant increases in both prevalence and intensity of
Echinostoma spp. metacercariae were observed in Helisoma anceps snails harboring rediae compared to those lacking the larval stage. This indicates that auto-infection does indeed occur among echinostome parasites in nature. Furthermore, this strategy could potentially have many life history consequences for both the parasite, e.g., increased infection success and a potential fitness advantage, and the host, e.g., increased pathogenicity and decreased fitness.
204 INTRODUCTION
Auto-infection, or the transfer of a life cycle stage from one site to another in the same host, is well known among nematode (Neva, 1986), cestode (Garcia et al., 2003), monogenean (Kaneko, II et al., 2006), protistan (MacKenzie, 1981; Tzipori, 1988), and trematode parasites (Olsen, 1937; Rankin, 1944; Sullivan and Byrd, 1970; Bolek and
Janovy, 2008). However, most studies, particularly among digenetic trematodes, have focused on transmission between vertebrate intermediate and definitive hosts, with just a few comparing auto-infection and transmission between an invertebrate first and second intermediate host (McCarthy and Kanev, 1990; Morley, Crane, and Lewis, 2004, 2004b).
Some digenetic trematodes have a series of larval stages that employ polyembryony and exogenous budding to produce hundreds to thousands of clones
(called cercariae), each capable of infecting new hosts (Bush et al., 2001). Auto-infection as a life cycle strategy would ensure that a large number of these clones successfully develop into the subsequent larval stage by infecting the same individual for use as the next intermediate host. Success of cercariae finding the next host in the life cycle is low in nature and may, at least in part, explain such high reproductive rates, i.e., type 3 survivorship curves among the digenetic trematodes (Whittington, 1997). In most cases, cercariae can only survive for 24-36 hr outside of a snail host (Pechenik and Fried, 1995), and must find a host before they use all of their energy reserves, since these stages are non-feeding. A notable exception to this rule are trematodes with specialized cercariae, e.g., cystopherous cercariae of Halipegus spp. (Shostak and Esch, 1990), which do not actively seek out molluscan intermediate hosts; rather, they sink to the bottom of a body of water and wait for the host to find them. Auto-infection can significantly increase
205 infection success of the short-lived cercariae and, consequently, fitness. In contrast, non- motile Halipegus spp. cercariae are relatively long-lived and remain infective for 19-21 days.
Auto-infection, and its persistence within a system, raises several ecological and evolutionary questions. There could be many potential advantages to auto-infection in digenetic trematodes that exhibit the typical 3-host life cycle. For example, the phenomenon of host location can be relatively complicated. Encounter rate appears to be a major factor in the success of parasite transmission (Dybdahl and Lively, 1998), particularly among the echinostomes (Detwiler and Minchella, 2009). Host density
(Detwiler and Minchella, 2009) and vagility (Bush et al., 2001; McCoy et al., 2003) may also be major factors affecting encounter rate. For example, auto-infection ensures success since searching for a new host becomes unnecessary. Additionally, the fitness and life history attributes of parasites that are capable of auto-infection are immediately improved.
However, not all aspects of auto-infection are positive. For example, parasitism induces high levels of stress in an infected host. When a host is continuously infected by parasites, chronically high stress levels may negatively impact host survivorship.
Echinostomes are known to have negative effects on their hosts when infected by miracidia, or cercariae, or both (Kuris and Warren, 1980). Additionally, while echinostomes are hermaphroditic, they will often outcross to obtain a supply of sperm with which to fertilize their ova (Trouve et al., 1996). Auto-infection by many clones may increase the chances of inbreeding, which could negatively affect the parasites’ fitness in the definitive host.
206 Another potential downside to auto-infection is placing a majority of the offspring and reproductive resources into a single host. Although auto-infection may potentially increase the fitness of a single clone by accumulating genetically identical metacercariae within a single host, if the individual is not consumed by a definitive host, the parasite’s fitness would decline to 0 in the absence of a successful transfer to a definitive host.
Auto-infection as the sole means of dispersal and infection strategy is clearly a risk.
The general life cycle of Echinostoma spp. parasites has been well documented
(Esteban and Muñoz-Antoli, 2009). Although host specificity is very high in the first intermediate host, it is markedly decreased in the second intermediate host. Second intermediate hosts range from invertebrates, e.g., molluscs and planarians, to vertebrates hosts, e.g., fish, frogs, and salamanders (Esteban and Muñoz-Antoli, 2009; Johnson and
McKenzie, 2009). Although it is known that cercariae are capable of auto-infecting their gastropod hosts (Detwiler, 2010; Morley, Crane, and Lewis, 2004, 2004b), the propensity for auto-infection in a natural setting has rarely been documented (McCarthy and Kanev,
1990; Morley, Crane, and Lewis, 2004, 2004b).
It was predicted that auto-infection by Echinostoma spp. occurs in nature, particularly in low density snail populations where finding another gastropod host may be difficult. The aim of the present study was to determine the extent to which auto- infection occurs among echinostome parasites and the snail species they infect and to better understand what impact this life history strategy might have on these parasites and their hosts.
207 MATERIALS AND METHODS
Study sites
A total of 4,218 Helisoma anceps snails was collected in Mallard Lake (36 o00’03”
N, 80 o24’07” W), a 4.9 ha impoundment in the Piedmont region of North Carolina, between September 2008 and March 2010. Physa acuta , Lymnaea (Pseudosuccinea ) columella , and Menetus dilatus snails were also present in the lake, but were far less abundant and were not collected extensively. Snails were isolated and dissected within 1 wk of collection. The following information was recorded for each snail: date of collection, shell length (to the nearest 0.05 mm), trematode species present, patency of trematode infection, number of echinostome metacercariae, and the number of Daubaylia potomaca (a rhabditidan nematode). Relative density measurements of all snail species were calculated between April and September 2009 by randomly throwing a 1-m 2 ring into the pond and collecting all snails, regardless of species, within it.
In addition to the Mallard Lake study, 461 H. anceps were collected from 10 ponds and lakes in 5 states (WV, VA, IN, IL, and KY) in the mid-Atlantic and
Midwestern United States in June through August 2012. The following information was recorded for each snail: shell length (to the nearest 0.05 mm), trematode species present, and number of echinostome metacercariae.
Statistical analysis
The population structure was described in terms of prevalence and intensity according to the terminology of Bush et al. (1997). Chi square analysis was used to compare the prevalence of metacercariae infection in snails that were infected with echinostome rediae and snails not infected with rediae in both Mallard Lake and from the
208 5 states previously indicated. Student’s t-tests were used to compare mean intensities of metacercariae infection between snails containing Echinostoma spp. rediae, other digenetic trematodes, D. potomaca , and the patency of infection. Student’s t-tests were also used to compare the size of snails. Analysis of covariance (ANCOVA) was used to compare snail size and the presence of rediae with the intensity of metacercariae infection.
209 RESULTS
Mallard Lake
Of the 4,218 H. anceps collected, 1,457 (34.5%) were infected with at least 1 stage of Echinostoma spp. Of these, only 453 (10.7%) of the snails possessed echinostome sporocysts/rediae, and 1,366 (32.4%) snails harbored infections of metacercariae alone. There were 362 (8.6%) snails with co-infections of both sporocysts/rediae and metacercariae. The mean intensity of metacercariae in the Mallard
Lake population was 87.9 ± 2.5. No significant difference in prevalence (Chi square test;
P = 0.4194) (Fig. 1A) or intensity (Student’s t-test, P = 0.2851) (Fig. 1D) of Echinostoma spp. metacercariae was observed between snails infected with digenetic trematodes other than Echinostoma spp. and completely uninfected snails. Thus, these groups were combined for subsequent analyses.
When rediae were present, the prevalence of metacercariae was 80.0%, which was significantly higher than snails not possessing the larval stage (24.3%; χ2 = 507.17, P
<0.0001; Fig. 1 A). The mean intensity of Echinostoma spp. metacercariae infection when rediae were present (195.2 ± 10.5) was significantly higher than when the latter larval stage was absent (47.6 ± 1.6; Student’s t-test, P <0.0001) (Fig. 1D). Patent snails had a sign ificantly higher prevalence of infection with metacercariae (84.6%) in comparison with pre -patent snails (60.9%; χ2 = 30.69, P < 0.0001) (Fig. 1B); patent snails also exhibited a higher mean intensity of metacercariae infection (201.5 ± 13.0) than pre -pate nt snails (180.1 ± 17.8), although this difference was not statistically significant (Student’s t-test, P = 0.21) (Fig. 1E). In snails with the nematode parasite, D. potomaca , there was no difference in prevalence of Echinostoma spp. metacercariae ( Chi 210 square test, P = 0.2739) (Fig. 1C). However, a significantly higher mean intensity of metacercariae was found in snails not harboring the nematode parasite (99.3±4.5) in comparison to when D. potomaca was present (69.3 ± 2.5; Student’s t- test, P = 0.0003)
(Fig. 1D).
Helisoma anceps infected with rediae were significantly larger (7.4 ± 0.3 mm) than snails uninfected by any digenetic trematodes (7.0 ± 0.1 mm; Student’s t-test, P
<0.0001) (Fig. 2A). There was also no significant difference in size between snails that harbored only Echinostoma spp. rediae (7.4 ± 0.4 mm) and snails that had co-infections of rediae and metacercariae (7.4 ± 0.6 mm; Student’s t-test, P = 0.1384). Similarly, there was no difference between snails harboring metacercariae (7.0 ± 0.2 mm) and snails not infected with metacercariae (7.0 ± 0.1 mm; Student’s t-test, P = 0.159) (Fig. 2A).
Helisoma anceps with patent infections of Echinostoma sp. were significantly larger (7.6
± 0.4 mm) than pre- patent snails (7.2 ± 0.5 mm; Student’s t-test, P = 0.0026) (Fig. 2B).
Additionally, snails infected with D. potomaca were significantly larger (7.5 ± 0.2 mm) than uninfected H. anceps (6.7 ± 0.2 mm; Student’s t-test, P <0.0001) (Fig. 2C).
Comparison of snail size and the presence of Echinostoma spp. rediae revealed that the intensity of Echinostoma spp. metacercariae was better explained by the presence of rediae (ANCOVA, P = 0.0124, F = 270.0) than by host size (ANCOVA, P = 0.0633, F =
25.2).
Eastern U.S.
A total of 461 H. anceps was collected, with 130 (28.2%) of these snails harboring Echinostoma spp. infections. Of these, 43 (9.3%) were infected with rediae,
104 (22.6%) were infected with metacercariae, and 17 (3.7%) possessed co-infections of
211 both larval stages. The mean intensity of echinostome metacercariae across all of the sample sites for H. anceps was 7.9 ± 1.5.
When rediae were present, the prevalence of metacercariae was 39.5%, which was significantly greater than in snails lacking the larval stage (20.8%; Chi square test, P =
0.0092) (Fig. 3A). Additionally, the mean intensity of Echinostoma spp. metacercariae, when rediae were present (21.3 ± 6.5), was significantly higher than when the latter larval stage was absent (5.3 ± 0.6; Student’s t-test, P = 0.0118) (Fig. 3B). Snails infected with rediae were significantly larger (9.2 ± 0.4 mm) than uninfected snails (8.2 ± 0.1 mm;
Student’s t-test, P = 0.0015). No difference in size was observed in snails infected by metacercariae (8.1 ± 0.2 mm) and uninfected snails (8.2 ± 0.1 mm; Student’s t-test, P =
0.3350). As in the Mallard Lake study, the presence of rediae (ANCOVA, P = 0.0033, F
= 19.3) was a better explanatory variable for Echinostoma spp. metacercariae than snail size (ANCOVA, P = 0.7159, F = 0.3).
212 DISCUSSION
In both Mallard Lake and the 5 eastern U.S. states, H. anceps snails harboring
Echinostoma spp. rediae had significantly higher prevalence and intensity of metacercariae than snails lacking this larval stage ( P < 0.05; Figs. 1, 3). This correlation clearly supports the contention that auto-infection of Echinostoma spp. is occurring in these hosts. The significantly elevated prevalence of metacercariae in snails shedding cercariae observed in Mallard Lake (Fig. 1C) further supports this assertion.
Snails harboring Echinostoma spp. rediae were significantly larger than uninfected snails in both studies ( P < 0.05), suggesting that larger snails are more likely to harbor these trematode infections, a phenomenon that is not uncommon among digenetic trematodes (Sousa, 1983; Esch and Fernandez, 1994; Kuris and Lafferty, 1994;
Jokela and Lively, 1995; Detwiler and Minchella, 2009). Logically, it would seem that miracidia are better able to find larger snails, presumably due to a higher encounter rate
(Sandland et al., 2001), or more pronounced chemical cues created by larger snails (Haas et al., 1995; Haas and Haberl, 1997).
Conversely, when echinostome metacercariae in the second intermediate host were considered, no significant difference in the size of infected versus uninfected snails was observed in either the Mallard Lake site or the eastern U.S. study sites ( P > 0.05).
This suggests that snail size does not play as large a role in locating a second intermediate host in this parasite system (Chapter VI). This was further supported by the fact that the presence of rediae was a better explanatory variable (ANCOVA, P <0.05) for the intensity of Echinostoma spp. metacercariae infection than host size (ANCOVA, P >
0.05).
213 Cercariae are much larger, have greater energy stores, different chemical cues
(Haas et al., 1995; Haas and Haberl, 1997; Haberl et al., 2000), and much lower host specificity than miracidia (Esteban and Muñoz-Antoli, 2009), thus making the search for second intermediate hosts much different than finding a first intermediate host. Similar to miracidia, cercariae exhibit relatively low dispersal and are viable for only a few hr, making quick location of a new host imperative for survival (Lo and Cross, 1975; Fried and Bennett, 1979). This fact suggests that auto-infection would be a helpful strategy for better ensuring that developmental stages find their next host.
Helisoma anceps with patent infections of Echinostoma spp. in Mallard Lake were significantly larger than pre-patent snails. This is most likely due to a 6 wk pre- patent period in the snails as the miracidium develops into sporocysts, mother and daughter rediae, and finally cercariae, which are then released to search for a second intermediate host (Esteban and Muñoz-Antoli, 2009). Helisoma anceps show considerable growth in a 6-wk-period (Negovetich and Esch, 2008) and this is the probable cause for the discrepancy in size of pre-patent and patent snails, not that larger snails are more likely to exhibit auto-infection.
Another parasite, D. potomaca , was found to commonly infect (39.1%) H. anceps in Mallard Lake. This nematode inhabits all tissues and blood spaces of the snail
(Chernin, 1962; Zimmermann et al., 2011a, b). Snails infected with D. potomaca were significantly larger than uninfected individuals, a pattern similar to gastropods infected with digenetic trematodes as first intermediate hosts. The presence of the nematode parasite in the snail host had no impact on the prevalence of Echinostoma sp. metacercariae, similar to what was observed with other digenetic trematode species at the
214 Mallard Lake study site. This indicates that snails parasitized by other, non-echinostome parasites are not more susceptible to infection by Echinostoma spp. cercariae. In terms of mean intensity, however, there did appear to be a significant impact of the presence of D. potomaca such that when the nematode was present in a host, there were significantly fewer metacercariae present in the same individual. This was attributed to the negative interactions of Echinostoma spp. rediae and the nematode parasite and that the 2 were rarely found co-habiting the same snail in Mallard Lake (Zimmermann et al., 2011c).
This was also compounded by the observation that many of the snail hosts harboring rediae were also heavily infected with metacercariae.
McCarthy and Kanev (1990) and Detwiler and Minchella (2009) reported that auto-infection did not commonly occur in their studies of echinostome parasites.
However, there are some key differences between their work and data presented here.
First, both of the former studies were focused on echinostome parasites infecting exclusively lymnaeid snails as the first intermediate host; the current study focused on echinostomes infecting planorbid snails. Lymnaeids exhibit lower susceptibility in experimental infections by echinostomes than planorbids (Evans and Gordon, 1983;
Detwiler and Minchella, 2009). Moreover, McCarthy and Kanev (1990) studied
Pseudechinoparyphium echinatum , an echinostome that exclusively infects L. stagnalis as the first intermediate host, which was rarely infected with not only Ps. echinatum cercariae, but also Echinoparyphium recurvatum cercariae (Evans and Gordon, 1983), suggesting that it may not be a highly susceptible host for echinostome cercariae.
Lymnaeid snails suffer far greater mortality when infected with digenetic trematodes than planorbids (Sorenson and Minchella, 2001) and should, therefore,
215 experience declines in density more rapidly. The total density of snails found by
Detwiler and Minchella (2009) was similar to that of the Mallard Lake site; however, another difference between their study and the current one was the relative densities of the snail species observed. Approximately half of the snails collected by Detwiler and
Minchella (2009) were P. gyrina , and the remainder were H. trivolvis , and L. elodes . The current study found that 72.4% of the snails in Mallard Lake were H. anceps and 24.0% were P. acuta , with the remaining snails being M. dilatus (3.4%) and L. columella (0.2%).
Detwiler and Minchella (2009) reported that various ecological factors and host availability can mask compatibility in nature. However, in the Mallard Lake study, unlike what Detwiler and Minchella (2009) found, the most abundant species was also the most susceptible to infection. Auto-infection occurred in H. anceps because they were the most abundant species, highly susceptible to cercariae penetration and encystment, and relatively successful in terms of surviving infection by miracidia and the subsequent larval stages.
There are ecological and evolutionary advantages of using auto-infection as a life history strategy. Specifically, auto-infection ensures that a larger number and percentage of clones progress to the next stage of the life cycle. This could potentially increase the fitness of the individual responsible for auto-infection by ensuring a large number of the metacercariae present in a particular host develop into adults when consumed by the definitive host. Additionally, the dispersal ability and survival time of Echinostoma spp. cercariae are low (Lo and Cross, 1975; Fried and Bennett, 1979), and auto-infection should negate these factors.
216 Auto-infection as the sole infective strategy poses many potential consequences.
Infecting the same host continually may accelerate the parasite’s impact on the survival of the host (Kuris and Warren, 1980). Additionally, investing all clones in a single host is a risky strategy in that if the infected host is not consumed by a suitable definitive host, then these reproductive and infection efforts are wasted. It is likely best to employ a combination of auto-infection and the release of cercariae to infect other hosts. This would ensure the maintenance of high infectivity, but also spread the infection among multiple hosts. Preliminary work by Detwiler (2010) showed this to be the case, but the degree to which this occurs in nature has not yet been determined. Elucidation of the infection patterns of echinostome cercariae could produce interesting results regarding the population genetics of the asexually reproducing stages of the parasites. In conclusion, host size may not play nearly as large of a role in the infection patterns of
Echinostoma sp. cercariae as once thought as auto-infection is a very real phenomenon in this system.
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222 FIGURE 1. Prevalence (A – C) and mean intensity (+/- standard error) (D – E) of infection by Echinostoma spp. metacercariae in Mallard Lake (* Represents a statistically significant relationship [ P < 0.05]).
223 224 FIGURE 2. Comparisons of mean shell length (+/- standard error) of H. anceps snails from Mallard Lake of (A) Echinostoma spp. infections of various life stages, (B) patency of Echinostoma spp. infection, and (C) D. potomaca infection. Like letters indicate a lack of statistically significant differences ( P < 0.05).
225 226 FIGURE 3. (A) Prevalence and (B) mean intensity (+/- standard error) of infection by
Echinostoma spp. metacercariae in H. anceps in the presence and absence of rediae.
Both prevalence and intensity yielded statistically significant results ( P < 0.05).
227 228 CHAPTER VIII
DIFFERENCES IN SNAIL ECOLOGY LEAD TO INFECTION PATTERN
VARIATION OF ECHINOSTOMA SPP. LARVAL STAGES
The following manuscript was submitted for publication in Acta Parasitologica (2014, In
press) and is reprinted with permission. M. R. Zimmermann performed the study and prepared the manuscript. K. E. Luth aided in sample collection and an editorial capacity.
G. W. Esch acted in an advisory and editorial capacity.
229 ABSTRACT
The infection patterns of parasites are often tied to host behavior. Although most studies have investigated definitive hosts and their parasites, intermediate host behavior may play a role in shaping the distribution and accumulation of parasites, particularly the larval stages. In an attempt to answer this question, more than 4,500 pulmonate snails were collected from 11 states in the mid-Atlantic and Midwestern United States in the summer of 2012. These snails were necropsied and echinostome metacercariae were commonly observed infecting the snails as 2 nd intermediate hosts (20.0%). The snails included species of 3 genera with distinct differences in the infection patterns of
Echinostoma spp. metacercariae among them. Physa spp. (comprising of P. acuta and P. gyrina ) snails exhibited a significantly higher prevalence of infection (23.5%) than both
Lymnaea columella (11.6%) and Helisoma spp. (comprising of H. anceps and H. trivolvis )
(14.2%; P < 0.05), with no difference in prevalence observed between the latter 2 genera
(P > 0.05). The intensity of metacercariae within the snail hosts was significantly different between the 3 genera ( P < 0.05), with L. columella having the highest intensity
(24.3 ± 5.6), followed by Physa spp. (15.2 ± 1.5) and Helisoma spp. (5.0 ± 0.9).
Differences in prevalence and intensity were also observed when the different snail families co-habited the same body of water. The disparities in infection patterns are likely due to distinct differences in the behavioral and feeding ecology of the snail hosts.
230 INTRODUCTION
Parasite distribution and accumulation in natural populations of animals is a well- documented phenomenon. Frequently, host behavior has been a major factor determining the distribution of parasites among the hosts in both terrestrial and aquatic systems
(Freeland, 1976; Moller et al., 1993; Loehle, 1995; Ezenwa, 2004; Loys et al., 2010). In most of these investigations, the focus has been placed on vertebrate hosts and their parasites, with little attention given to how the intermediate hosts acquire their parasites and then their resulting distribution. Differences in the patterns of the distribution and accumulation of the parasites in the intermediate hosts may be important in explaining the dispersal of the parasites among the vertebrate definitive hosts.
In an attempt to determine if differences in parasite distribution exist among closely related intermediate hosts, Echinostoma spp. were examined in naturally infected populations of pulmonate snails. Similar to other digenetic trematodes, Echinostoma spp. have a complex life cycle involving 3 hosts. Eggs are passed into water via feces. On hatching, miracidia swim in the water column until they locate and infect a pulmonate snail, typically a planorbid or lymnaeid species in North America (Johnson and
McKenzie, 2009; Detwiler et al., 2012). They penetrate the snail as the first intermediate host and migrate to the gonad where they develop into sporocysts, followed by mother and daughter rediae, all of which are asexually reproducing stages. Rediae produce hundreds of cercariae that are released into the water column, where they search for a second intermediate host (Esteban and Munoz-Antoli, 2009).
Host specificity in the second intermediate host for Echinostoma spp. is much lower than the first intermediate host. A variety of pulmonate snail, amphibian, and fish
231 species, can serve as second intermediate hosts for echinostome parasites (Johnson and
McKenzie, 2009). The cercariae penetrate the second intermediate host and encyst as metacercariae. In both snail and amphibian hosts, the cercariae migrate to the kidney region of the host before encysting. The life cycle is complete when one of a number of vertebrate hosts consumes an infected second intermediate host. More than 40 different definitive hosts are known to harbor infections of adult Echinostoma spp. (Johnson and
McKenzie, 2009).
These echinostome parasites serve as excellent models for the study of differential infection patterns in intermediate hosts because the parasites have a cosmopolitan distribution across North America, are common across this region, and have a wide range of intermediate hosts, many of which are closely related. In the present study, pulmonate snails were selected because they are used as both first and second intermediate hosts.
Additionally, they are widely distributed and abundant in many bodies of water. These snails are also important members in the biotic community of many aquatic habitats due to their propensity for feeding on numerous species of algae, bacteria, and macrophytes.
As a result, they play an important role in structuring the plant and macrophyte communities in their environment (Hunter, 1980; Lowe and Hunter; 1988).
Five species of pulmonate snails representing 3 different families, e.g., Helisoma anceps and H. trivolvis (Planorbidae), Physa acuta and P. gyrina (Physidae), and
Lymnaea ( Pseudosuccinea ) columella (Lymnaeidae), were collected in the present study.
Each of these species exhibit subtle differences in behavioral ecology, feeding ecology, and microhabitat choice, which can be used to detect differences in parasite infection patterns.
232 Planorbid snails typically inhabit regions of lakes and ponds with little, or no, current, and firm mud substrata that are rich in decaying matter. For Helisoma spp., algae, detritus, and bacteria are the main food items, with no preference in particle size
(Calow, 1973, 1974, 1975; Liang 1974). They also employ ‘area restricted searching’ in which they will continually feed on the same patch (Charnov et al., 1976; Dillon, 2000).
Physid snails are often associated with fine substrata (Crowl and Schnell, 1990), with a preference for plants, rocks, and other hard surfaces near the shore (Bickel, 1965;
Clampitt, 1970). Physa spp. use detritus and algae as staples in their diet and have been shown to exhibit extensive grazing pressure, e.g., substantially limiting the growth and production of the plant community, due to their large volume of consumption (Lowe and
Hunter, 1988; Swamikannu and Hoagland, 1989). Physa spp. employ a basic system of
‘give up time’ in which they continually change patches when resources become depleted
(Charnov et al., 1976, Dillon, 2000)
Lymnaeid snails, similar to the other pulmonates, show a dietary preference for algae (Bovbjerg, 1965, 1968, 1975; Hunter, 1980; Reavell, 1980). These snails typically possess sand in their stomachs, which they use to aid in the digestion of more complex food items such as algae and live plant tissue (Storey, 1970; Reavell, 1980). Similar to physids, lymnaeids can also have a large impact on the macrophyte diversity (Hunter,
1980; Cuker, 1983). They use a feeding system of ‘give up time’ (Charnov et al ., 1976;
Dillon, 2000), i.e., when food is abundant, lymnaeids are typically associated with rocks, plants, and other hard substrata, but when food is scarce, they can also be found foraging in the mud substratum (Walter, 1980). Lymnaea columella , as well as a number of other
233 Lymnaea spp., is amphibious and prefers soft, moist substrata on the water’ s edge when not feeding (Foster, 1983; Dillon 2000).
The goal of the present study was to determine if the recruitment patterns and infection dynamics of Echinostoma spp. parasites differed among closely related intermediate hosts in nature and to determine if the natural history of these hosts could lend explanation for the patterns observed.
234 MATERIALS AND METHODS
In total, 4,538 pulmonate snails were collected from 71 bodies of water in the mid-Atlantic and midwestern United States from June through August 2012. In all, 11 states (NC, WV, VA, MD, PA, OH, MI, IN, IL, KY, and TN) were sampled and 5 different species of snails from 3 different families were collected, including H. anceps
(N = 664) and H. trivolvis (N = 632) (Planorbidae), P. acuta (N = 1,696) and P. gyrina
(N = 1,139) (Physidae), and L. columella (N = 407) (Lymnaeidae). Collections were made by hand and dip-netting, with a total of 100 snails obtained from each body of water or 1 hr of search time, whichever came first. Prior to each collection, a density measurement was obtained by counting all snails within a randomly placed 515.6 cm 2 rectangle. The snails were then necropsied and the following information was collected for each snail, i.e., shell length (to the nearest 0.05 mm), trematode species present, and number of Echinostoma spp. metacercariae. The number of collar spines was also counted to confirm the genus of the echinostome parasites (Morgan and Blair 1995).
Although several genera of echinostome metacercariae were found, only those belonging to the revolutum -group, which are characterized by 37 collar spines, were included in the study. No further morphological or molecular effort was made to identify the parasites to the species level. The GPS coordinates, elevation, and surface area were recorded for each body of water. Additionally, the microhabitat of each snail species was also identified.
The population structure of the echinostome parasites was described in terms of prevalence and mean intensity according to the terminology of Bush et al. (1997). The prevalence and intensity of Echinostoma spp. metacercariae were assessed in each snail.
235 A mixed-effects model was conducted to determine if differences in Echinostoma spp. prevalence or intensity existed between snail species using site as a random effect, due to variations in sample size between them, and snail species, shell length, and lake/pond area were used as predictors. Once site effects were shown to have no influence, chi square analysis was used to compare prevalence of Echinostoma spp. infection; Steel-
Dwass post-hoc tests were used to compare mean intensities of Echinostoma spp. between week of collection, snail species, and snail families. Logistic regression was used to establish if there were any relationships between prevalence and intensity of
Echinostoma spp. metacercariae and lake area or elevation. Moran’s I spatial autocorrelation was used to determine if there was a geographic pattern in the distribution of Echinostoma spp. in the bodies of water sampled.
236 RESULTS
Snail microhabitat
Physa acuta , P. gyrina , and L. columella were found very close to the water’s edge associated with vegetation and other hard substrata such as rocks and timber. These snails were typically found within 5 cm of the bank in less than 5 cm of water.
Frequently, L. columella was found just outside of the water on the bank 1-2 cm emerged from the water. Helisoma anceps and H. trivolvis typically inhabited slightly deeper water than the other pulmonates and were frequently found up to 1 m from the shoreline in >20 cm of water. Occasionally, the distribution of H. anceps and H. trivolvis would overlap P. acuta , P. gyrina , and L. columella , but the microhabitat of these pulmonates appeared to be fairly distinct. Lymnaea columella occurred in ponds with the highest average snail density (10.5 ± 5.5), followed by Helisoma spp. (10.3 ± 5.1) and Physa spp.
(9.4 ± 5.5), however, density did not vary between ponds containing the various snail genera (ANOVA, P = 0.9691).
Population biology of Echinostoma spp. metacercariae
A total of 71 ponds and lakes was sampled, with 32 (45.1%) having infections of
Echinostoma spp. metacercariae. Of the 4,538 snails examined, 2,951 came from ponds with confirmed infections of Echinostoma spp.; accordingly, these snails will be the focus of the remainder of the analyses. Altogether, 590 (20.0%) of the snails from the echinostome-infected sites possessed metacercariae, with a mean intensity of 13.9 ± 0.6.
No statistically significant associations were found between either prevalence or intensity with lake surface area, elevation (logistic regression, P > 0.05), or date of collection
237 (Steel-Dwass, P > 0.05). There was also no discernible pattern in the geographic distribution of Echinostoma spp. infection across the study sites (Moran’s I, P = 0.3385).
Infection patterns in snails
There were no significant differences in prevalence or intensity of Echinostoma spp. metacercariae between snail species within the same genus ( P > 0.05); therefore, both species of Helisoma were combined for analyses, as well as both species of Physa
(Table I). Statistically significant differences in Echinostoma spp. metacercariae between genera were found (Mixed Effects Model, P = 0.0426), but no correlation between shell length or collection site ( P > 0.05). The physids exhibited a significantly higher prevalence (23.5%; χ 2 = 27.1, P < 0.0001) than both Helisoma spp. (14.5%) and L. columella (11.7%), which were not significantly different from one another (χ 2 = 1.0, P =
0.3245; Fig. 1A).
Statistically significant differences in mean intensity of Echinostoma spp. were also observed (Mixed Effects Model, P = 0.0152), but no differences in infection due to shell length or collection site were found ( P > 0.05). Lymnaea columella had the highest intensity of metacercariae (24.3 ± 5.6), followed by Physa spp. (15.2 ± 1.5) and Helisoma spp. (5.0 ± 0.9), all of which were significantly different from each other (Steel-Dwass, P
< 0.05; Fig. 1B). Lymnaea columella harboring Echinostoma spp. rediae (94.0 ± 26.6) had a significantly greater mean intensity of metacercariae than uninfected snails (14.7 ±
2.7; Student’s t-test, P = 0.0295).
The number of snail species present in a particular body of water affected the infection pattern of each snail family differently. Neither prevalence nor intensity of
Echinostoma spp. metacercariae in Helisoma spp. was affected by the presence of other
238 snail species ( P > 0.05; Fig. 2A). However, metacercariae infection in Physa spp. showed significantly higher prevalence of infection (χ 2 = 72.2, P < 0.0001) when individuals from multiple pulmonate snail families were present and a significantly higher intensity when individuals from all 3 snail families were present (Steel-Dwass, P <
0.05; Fig. 2B). The opposite trend was seen in L. columella , where prevalence (χ 2 = 3.92,
P = 0.0470) and intensity (Steel-Dwass, P < 0.05) of infection significantly decreased when more snail families were present in the pond/lake.
239 DISCUSSION
The differences observed in both the prevalence and intensity of Echinostoma spp. infection can be attributed to distinct disparities in snail behavior. For example,
Helisoma spp. not only had the lowest prevalence of infection, but also the lowest intensity (Fig. 1). These planorbid snails prefer a habitat with a firm substratum and large quantities of decaying matter (Malek, 1958). Primarily feeding on algae and decaying matter in the benthos, Helisoma spp. do not move extensively and are known to feed on the same patch for comparatively longer periods of time (Clampitt, 1975; Boss et al., 1984). The low vagility of snails in this genus should act to substantially decrease their encounter rate and, subsequently, parasite recruitment, by free-swimming cercariae of Echinostoma spp. Moreover, because these cercariae rely so heavily on their chemotactic abilities to locate potential hosts, planorbids inhabiting the benthos, should confer the advantage of decreased encounter rate. Helisoma spp. tend to inhabit areas with extensive leaf litter and debris which would likely result in cercariae having increased difficulty finding the next intermediate host (Chapter V).
In contrast, physid snails are voracious feeders, and their high impact on aquatic macrophyte communities necessitates increased vagility in this family (Boss et al., 1984;
Sheldon, 1987; Swamikannu and Hoagland, 1989), relative to the other snail species in the study. The increased movement should increase their encounter rates with echinostome cercariae, which, at least in part, explains the differences in prevalence and intensity between the physid and planorbid snails.
Lymnaeid snails, especially L. columella , differ greatly from species in the other 2 families in terms of behavior and feeding ecology. The feature that sets this species apart
240 from the other pulmonates in this study is its amphibious tendency. Lymnaea columella spends a majority of its time on the moist banks at the water’s edge, and prefers a muddy substratum to a flocculent one (Harris and Charleston, 1977; Foster, 1983). However, lymnaeid snails rely on the aquatic environment for food, and L. columella frequently returns to the water to forage. Their feeding behavior is similar to that of the physid snails, which have a huge impact on plant and macrophyte communities (Hunter, 1980) and exhibit significantly greater vagility than Helisoma spp. (Boss et al., 1984).
Additionally, L. columella feeds on algae and macrophytes on hard surfaces (Walter,
1980; Dillon, 2000), similar to Physa spp., suggesting extensive movement between patches is required to obtain an adequate food supply. However, since L. columella is amphibious and spends substantial time on the moist bank outside of the water, their exposure to echinostome parasites may be substantially less than that of the physids, leading to a significantly lower prevalence of infection (Fig. 1A).
The intensity of infection in the lymnaeid snails was significantly greater than both of the other snail families in the study. One reason for the high intensity, relative to the planorbids, is the similarity in the feeding ecology of Physa spp. and L. columella .
All physid and lymnaeid species exhibit extensive vagility while foraging in order to acquire sustainable food resources (Hunter, 1980; Lowe and Hunter, 1988). In North
America, lymnaeid snails are more likely to harbor Echinostoma spp. as first intermediate hosts than Physa spp. (Johnson and McKenzie, 2009; Detwiler et al., 2009).
Additionally, autoinfection by the echinostome parasites has been shown to occur in nature (Morley et al ., 2004; Detwiler, 2010).
241 In the present study, L. columella infected with Echinostoma spp. rediae had a significantly higher mean intensity of metacercariae than snails of the same species without the former larval stage. If L. columella infected with Echinostoma spp. rediae are removed from analysis, then there is no difference in mean intensity of metacercariae between physids and lymnaeids (Student’s t-test, P = 0.2113). These data suggest that autoinfection of Echinostoma spp. may be contributing to the differences in mean intensities of the physid and lymnaeid snails, but further work is required to support this contention. Similar to L. columella , both H. anceps and H. trivolvis can serve as first intermediate hosts for Echinostoma spp. parasites (Sapp and Esch, 1994; Schmidt and
Fried, 1997; Fried et al ., 2007; Griggs and Belden, 2008) and can also be subject to autoinfection (Chapter VII). However, only a single snail was infected with rediae of
Echinostoma spp., so autoinfection was not a factor for Helisoma spp. in this study.
In a similar study, no differences were found in the infection patterns of echinostome metacercariae between the same 3 pulmonate snail families (Detwiler and
Minchella 2009). However, the latter investigation was conducted in a single ephemeral pond in Indiana (USA) as opposed to the 32 ponds and lakes across a wider range of the
U.S. Similar to the present study, however, Detwiler and Minchella (2009) hypothesized that encounter rate played a large role in the infection patterns of echinostome parasites, based mainly on snail density and availability. Here, we extend this hypothesis to also include snail behavior as a key factor driving both the rate of encounter and recruitment of Echinostoma spp. Detwiler and Minchella (2009) also showed that encounter rate was able to cover up discrepancies in host susceptibility in the field. This same trend was also observed in the current study, i.e., the most susceptible species ( Helisoma spp.) had the
242 lowest intensities and the least susceptible species ( L. columella ) had the highest infection intensities.
Differing trends in the patterns of prevalence and intensity of Echinostoma spp. infection were observed for the 3 pulmonate snail families when the number of snail families present in the body of water varied (Fig. 2). Helisoma spp. were unaffected by the presence of other species in the pond. As indicated previously, these planorbids tend to be rather sedentary in terms of foraging behavior and move very little during the course of their lifetime (Clampitt, 1975; Boss et. al., 1984). Moreover, they have a completely different habitat preference than the other pulmonate snail families. This was observed in the field when these snails tended to be in deeper water further from the bank associated with softer benthic substrata as opposed to shoreline. No significant difference was observed in either prevalence or intensity of echinostome metacercariae infection (Fig. 2).
In Physa spp., the more diversity that was present in terms of snail families, the greater the prevalence of echinostome infection (Fig. 2A) and intensity (Fig. 2B).
However, physids typically do not serve as first intermediate hosts for Echinostoma spp. in North America, while Helisoma spp. and L. columella are known to host a number of different echinostome genera and species (Johnson and McKenzie, 2009). The presence of the other 2 snail families is paralleled by an increase in the prevalence and intensity of infection of Echinostoma spp. in the physid snails.
In contrast to Physa spp., L. columella harbored significantly more Echinostoma spp. metacercariae in terms of both prevalence and intensity when they were the only snail family present in a pond/lake (Fig. 2). Lymnaea columella was never found to co-
243 occur with only Helisoma spp., so it appears that their interactions with Physa spp. may be influencing the change in parasite load. Since Physa spp. are not typical first intermediate hosts of Echinostoma spp., their presence in a pond does not enhance the presence of Echinostoma spp. cercariae in a pond/lake, but does increase the population of snails susceptible to infection by echinostome cercariae. Increasing host availability in a habitat essentially dilutes the intensity of infection in lymnaeid snails by giving
Echinostoma spp. other sources for cercariae recruitment (Hall et al., 2009; Johnson and
Thiltges, 2010). Therefore, the more hosts that are available in a system, the lower the intensity of infection of Echinostoma spp. in L. columella . Field observations revealed frequent microhabitat overlap between Physa spp. and L. columella , suggesting that there may be interactions between these snail species.
In summary, snail feeding ecology and habitat preference appear to be important factors in the infection dynamics of Echinostoma spp. cercariae among the snail second intermediate hosts, with distinct differences in the infection patterns among the 3 snail genera. Additionally, the presence of other snail species from different families appears to impact the infection dynamics of each group differently, mainly due to differences in habitat and foraging preference and specificity of Echinostoma spp. for snail first intermediate hosts. These patterns may have implications in the transmission from the second intermediate host to the definitive host. The snails that harbor the most infections,
Physa spp. and L. columella , inhabit microhabitats nearest the shoreline. This makes them more susceptible for consumption by some of the more common hosts for
Echinostoma spp., i.e., aquatic waterfowl. Presumably, snails closer to the shoreline, or even just outside of the water, as for L. columella , are easier to find since they are in
244 shallow water. Although further research is needed in this area, these snails may be at a higher risk of predation than snails in deeper water further from the shoreline such as
Helisoma spp. and may be more advantageous for Echinostoma spp. in the completion of their life cycle.
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250 Table I. Shell length (+/- standard error) of each snail species in the study both infected and uninfected and the prevalence and intensity of Echinostoma spp. metacercariae.
Snail Species Snail Size Echinostoma spp. Infected Uninfected Average Size Range Prevalence Intensity Helisoma anceps 8.08 ± 1.86 mm 8.18 ± 2.07 mm 8.15 ± 2.03 mm 2.60 - 13.60 mm 16.4% 5.1 ± 1.0 Helisoma trivolvis 12.01 ± 2.61 mm 13.19 ± 3.27 mm 12.18 ± 3.23 mm 3.00 - 19.00 mm 12.0% 3.8 ± 0.8 Physa acuta 7.21 ± 1.90 mm 6.01 ± 1.35 mm 6.28 ± 1.57 mm 3.05 - 13.85 mm 25.6% 13.3 ± 2.1 Physa gyrina 6.42 ± 1.37 mm 6.54 ± 1.56 mm 6.51 ± 1.52 mm 3.10 - 17.0 mm 21.4% 17 ± 3.5 Lymnaea columella 5.08 ± 1.40 mm 6.72 ± 1.55 mm 6.53 ± 1.62 mm 2.80 - 11.60 mm 11.7% 24.3 ± 5.6
251 FIGURE 1. The prevalence and mean intensity (± standard error) of Echinostoma spp. metacercariae in 3 pulmonate snail genera.
252 253 FIGURE 2. The prevalence and intensity (± standard error) of Echinostoma spp. metacercariae in the presence of other pulmonate snail families.
254 255 CHAPTER IX
THE DILUTION EFFECT IN ECHINOSTOMA SPP.: EXAMPLES FROM FIELD
COLLECTED DATA
256 ABSTRACT
Rapid losses of biodiversity due to the changing environmental landscape have spurred increased interest in the role of species diversity and disease risk. A leading hypothesis for the importance of biodiversity in disease reduction is the dilution effect, which suggests that increasing species diversity within a system decreases the risk of disease among the organisms inhabiting it. This hypothesis was investigated using field studies from sites across the U.S. to examine the role of snail diversity in both first and second intermediate larval stages of Echinostoma spp. parasites. The prevalence of
Echinostoma spp. miracidia infection was not affected by increases in snail diversity, but significant negative correlations with metacercariae prevalence and intensity and snail species richness were observed. Additionally, varying effectiveness of the diluting hosts was found, i.e., snail species that were incompatible first intermediate hosts for
Echinostoma spp. were more successful at diluting the echinostome parasites in the focal species than snail species that can harbor the first intermediate larval stages. These findings have important implications not only on the role of species diversity in reducing disease risk, but the success of the parasites in completing their life cycles and maintaining their abundance within an aquatic system.
257 INTRODUCTION
Rapid losses of populations and species of organisms associated with environmental change has led to a number of studies focusing on emerging disease as a result of decreased species diversity (Mitchell et al., 2002; Begon, 2008; Lootvoet et al.,
2013; Vanesky et al., 2013). The ‘dilution effect’ hypothesis suggests that increas es in biodiversity within a system reduce the risk of disease among organisms inhabiting it and that infection risk should vary inversely with host diversity (Ostfeld and Keesing, 2000a,
2000b; LoGuidice et al., 2003; Keesing et al., 2006). Both field and lab studies have suggested that species-rich communities support more low compatibility hosts for the pathogens, leading to wasted transmission events and decrease disease risks (Keesing et al., 2006; Perkins et al., 2006; Johnson and Thieltges, 2010).
To date, a majority of studies have focused on the transmission of vector-borne diseases with only recent attention given to organisms with complex life cycles, i.e., those involving multiple hosts and larval stages for completion (Johnson et al., 2012;
Lootvoet et al., 2013; Vanesky et al., 2013). However, parasites employing complex life cycles are likely to respond strongly to changes in species composition or the diversity of the host community, particularly the larval stages. Transmission of these parasites can be significantly altered by dilution in the host community (Johnson and Thieltges, 2010).
Several mechanisms for the reduction of disease risk through increased biodiversity have been proposed, including decreased encounter rate between parasites and their hosts, decreased infection after encounter, increased host recovery from infection, increased host mortality, or decreased density of potential hosts (Keesing et al., 2010). However, changes in community composition tend to either reduce the encounter rate of pathogens
258 with their hosts or reduce the availability of susceptible hosts through dilution (Johnson and Thieltges, 2010).
There are 2 categories of organisms that lead to wasted transmission events, i.e., completely unsuitable hosts that produce a decoy effect or alternative hosts that exhibit lower compatibility (Johnson and Thieltges, 2010). Both decoy and alternative hosts have been shown to significantly reduce infection levels in target hosts in both lab and applied field studies (Upatham and Sturrock, 1973; Thieltges et al., 2009; Lootvoet et al.,
2013; Venesky et al., 2013). The abundance of focal hosts relative to potential diluting organisms is another factor that may affect the strength of the dilution effect. Parasite loads in target hosts tend to decrease with increased numbers of diluting organisms
(Johnson and Thieltges, 2010). Additionally, the diluting capacity of individual species varies widely, ranging from no effect (Upatham and Sturrock, 1973; Johnson et al., 2009) to almost completely stopping transmission to a focal host (Thieltges et al., 2008). This means that the physical and behavioral traits, ecology, and density of diluting organisms must be considered when examining the capacities of dilution for different species, a challenge that is difficult in field studies (Johnson and Thieltges, 2010).
Another factor that needs to be considered when studying the dilution effect of various pathogens is host specificity, which is defined as the extent to which a pathogen is restricted to establish infection in a range of host species (Combes, 2001). Organisms with complex life cycles often have varying degrees of host specificity, depending on the life stage of the organism. For instance, digenetic trematodes exhibit very high specificity for their first intermediate hosts and less in their second and definitive hosts, potentially making the dilution effect much more evident early in the life cycle (Johnson
259 et al., 2012). Investigations into the role of gastropods and the dilution effects on miracidia are extensive, particularly on schistosome parasites (Upatham and Sturrock,
1973; Laracuente et al., 1979; Combes and Mone, 1987; Johnson et al., 2009; Johnson et al., 2012). However, investigations regarding snails being used as second intermediate hosts could have important implications in the spread of parasites to the definitive host communities, as well as transmission to amphibian second intermediate hosts, and have yet to be considered.
Echinostoma spp. are digenetic trematodes that have complex life cycles using pulmonate snails as both first and second intermediate hosts before infecting a wide range of aquatic waterfowl and mammals as definitive hosts (Esteban and Munoz-Antoli, 2009;
Johnson and McKenzie, 2009). Specificity is high in the first intermediate hosts, but decreases substantially in subsequent hosts, e.g., more than 60 species of birds and mammals have been reported as definitive hosts (Johnson and McKenzie, 2009).
Additionally, these parasites can also infect amphibian hosts and have been suggested to be contributing to their declines in North America (Schotthoeffer et al., 2003; Johnson and McKenzie, 2009). This ‘model ’ parasite offers a unique opportunity to examine the dilution effect on both miracidia and cercariae infection among pulmonate snail communities. Both Helisoma spp. and Lymnaea spp. are suitable first intermediate hosts for Echinostoma spp., while Physa spp. are not capable of harboring first intermediate host infections of these parasites (Johnson and McKenzie, 2009; Detwiler et al., 2012).
However, each of these snail genera harbor metacercariae, a larval stage in second intermediate hosts, of Echinostoma spp. parasites with differing degrees of susceptibility to the cercariae infection (Detwiler and Minchella, 2009).
260 It was predicted that the addition of other snail species into an aquatic system would reduce parasite infection in a focal host, and that more active snails would be more successful at diluting Echinostoma spp. This was examined in a focal species for
Echinostoma spp., i.e., one that can harbor both first and second intermediate larval stages of the parasite, as well as if the addition of other snails into the aquatic system impacts the infection dynamics of miracidia and cercariae on the focal species.
Additionally, comparisons of the impacts of the diluting snail species were observed to determine if differences in the diluting properties of various hosts occur and the possible impacts that these differences may have on the transmission of these parasites to definitive hosts.
261 MATERIALS AND METHODS
A total of 14,798 pulmonate snails was collected from 139 ponds and lakes across the U.S., east of the Rocky Mountains. Five species of pulmonate snails were collected, including, Helisoma anceps (N = 5,283), H. trivolvis (N = 1,360), Lymnaea columella (N
= 781), Physa acuta (N = 4,500), and P. gyrina (N = 2,792). All snails were collected by hand or dip-netting and either necropsied within 48 hr of collection or preserved in 95% alcohol until they were dissected. Snail density measurements were obtained by collecting all snails within a randomly placed 0.05-m 2 rectangle. Each snail was measured to the nearest 0.05 mm, the trematode species present were identified, and the number of Echinostoma spp. metacercariae were counted. Echinostoma spp. metacercariae were identified by counting the number of collar spines and only those belonging to the revolutum -group, i.e., those possessing 37 collar spines, were included in this study.
Using the terminology of Bush et al. (1997), the population of Echinostoma spp. metacercariae was described in terms of prevalence and mean intensity. Helisoma anceps was used as the focal species in the current study because it can harbor both first and second intermediate larval stages of Echinostoma spp. parasites in North America.
Additionally, this snail species was abundant in both number and space across the sample sites. The density of H. anceps in relation to varying levels of snail species richness were compared using one-way ANOVA and the relative abundance of was compared using
Chi squared analysis. A linear mixed effects model was performed on the prevalence of both Echinostoma spp. miracidia and cercariae infections, as well as the intensity of
Echinostoma spp. metacercariae in H. anceps . Snail density, shell length, and the number
262 of snail species present in a body of water were used as predictors, while the site of collection was treated as a random effect in each model. For a snail species to be considered present and relevant within an aquatic system, the relative abundance of the snail species had to be greater than 5.0% of the total snails collected within the body of water. Chi square analysis was used to compare the prevalence of metacercariae infection when H. anceps was the only snail found in a lake to the prevalence when other snail species were present; Kruskal-Wallace tests were used to compare the mean intensities of Echinostoma spp. metacercariae in the same conditions, since the metacercariae populations were overdispersed. Additionally, Chi square analysis was used for pairwise comparisons of Echinostoma spp. metacercariae infection in H. anceps and other species present in the ponds. Kruskal-Wallace tests were also used to compare mean intensity of the same pairwise comparisons. All statistical analyses were performed using the R software (R Development Core Team, Vienna, Austria).
263 RESULTS
Physa acuta were the most frequently found snail species across the study sites, followed by H. anceps (Fig. 1A). Despite varying species compositions and densities of snail hosts in the 139 sampled lakes and ponds, no significant differences in the density of H. anceps were observed in relation to varying levels of snail diversity (ANOVA, P =
0.7496; Fig 1B). However, the relative abundance of the focal species decreased significantly as snail species richness increased (Chi square test, P < 0.0001).
There were no significant correlations between the prevalence of Echinostoma spp. miracidia infection and snail density ( P = 0.9399) or the number of snails species present in the ponds ( P = 0.4876), but there was a significant positive correlation with shell length in H. anceps (Mixed Effects Model, P = 0.0088 ; Fig. 2). There were also no statistically significant correlations between the prevalence of Echinostoma spp. metacercariae infections and snail density ( P = 0.4570) or shell length ( P = 0.7939), but there was a significant negative correlation with the number of snail species present in the pond (Mixed Effects Model, P = 0.0200; Fig. 4A). Additionally, a statistically significant negative correlation was found between the number of snail species and the intensity of metacercariae (Mixed Effects Model, P = 0.0010), with both snail density ( P
= 0.7361) and shell length ( P = 0.1209) failing to produce significant trends (Fig. 4B).
Helisoma anceps showed a significant reduction in the prevalence of Echinostoma spp. miracidia infection in the presence of all other snail species ( P < 0.0016) with the exception of H. trivolvis (Chi square test, P = 0.0820; Fig. 3). Furthermore, a significant reduction in the prevalence of Echinostoma spp. metacercariae in H. anceps in the presence of all other snail species was observed ( P < 0.0001), except when in the
264 presence of H. trivolvis , when there was a significant increase in metacercariae prevalence in the focal species (Chi square test, P = 0.0240; Fig. 5A). The presence of other snail species led to a significant reduction in the mean intensity of echinostome metacercariae in H. anceps for all species tested (Kruskal-Wallace, P < 0.0001; Fig. 5B).
Additionally, when P. acuta or P. gyrina were present in an aquatic system, each of these species garnered a significantly higher prevalence of infection (Chi square test, P <
0.0100) than H. anceps , while H. trivolvis (Chi square test, P = 0.2994) and L. columella
(Chi square test, P = 0.8965) showed no difference in prevalence between the 2 snail species (Chi square test, P = 0.2994; Fig. 4A). Both P. acuta (P = 0.0071) and P. gyrina
(P = 0.0002) were found to acquire significantly more metacercariae than H. anceps when they cohabited the same pond, while the focal host recruited more parasites than both H. trivolvis (Kruskal-Wallace, P = 0.0008) and L. columella (Kruskal-Wallace, P =
0.0071) when they inhabited the same body of water (Kruskal-Wallace, P = 0.0008; Fig.
5B).
265 DISCUSSION
The so- called ‘ dilution effect ’ is focused on the idea that increased biodiversity reduces disease risk by decreasing the relative abundance of target hosts (Ostfeld and
Keesing, 2000b). Although the ponds varied in both geographic range and snail species composition, there was no difference in the density of H. anceps in relation to snail species richness. However, the abundance of the focal species decreased significantly with increased gastropod diversity. Additionally, the transmission success of
Echinostoma spp. larval stages also showed differences relating to snail diversity.
There was not a significant correlation between the prevalence of Echinostoma spp. miracidia infection and the number of snail species in the pond with H. anceps , indicating that diluting the abundance of H. anceps did not decrease the chances of the snails becoming infected with this larval stage of the parasite. Although decreases in the prevalence of miracidia infection were observed with increases in snail diversity, these differences were probably attributed to varying levels of miracidia infection at the different samples sites, a variable that cannot be controlled for in field studies, and decreases in the relative abundance of focal hosts. Johnson et al., (2012) observed that the prevalence of miracidia infection was not related to the number of snail species in a body of water, due to decreases in the density of competent hosts. The same reduction in compatible host density was not evident in the current study, but significant decreases in the relative abundance of H. anceps in relation to other snail species was observed, possibly contributing to the lack of correlation
These significant reductions in H. anceps relative abundance are most likely attributed to the poor competitive ability of the planorbid snails in the presence of
266 lymnaeid and physid snail species (Brown, 1982; Osenburg, 1989; Chase et al., 2001;
Chase, 2003). Additionally, a significant correlation between snail size and prevalence of infection by miracidia was evident, indicating that larger snails were more likely to become infected. This trend has been shown in several studies, which indicate that snail size is a good measure for encounter rate (Fernandez and Esch, 1991; Byers et al., 2008;
Detwiler and Minchella, 2009). Despite species diversity not being a good indicator of miracidia infection, significant decreases in the prevalence of infection of this larval stage in H. anceps were observed with all snail species pairings except H. trivolvis . This supports the idea of the decoy effect, in which miracidia infection is diluted by incompatible hosts (Laracuente et al., 1979; Combes and Mone, 1987; Johnson and
Thieltges, 2010).
There was a significant correlation between the prevalence of Echinostoma spp. metacercariae and snail diversity in these aquatic habitats. The presence of all snail species, with the exception of H. trivolvis, led to significant reductions of metacercariae prevalence. Helisoma trivolvis is also employed as a first intermediate host for the echinostome parasite and occupies overlapping habitat and a similar niche to its congener
(Dillon, 2000). Instead of diluting the potential host species, H. trivolvis may actually be enhancing infection since they are also capable of harboring Echinostoma spp. as first intermediate hosts and are potentially adding cercariae to the population.
A dilution effect was also illustrated by the mean intensity of Echinostoma spp. metacercariae. There was a significant negative correlation between the mean intensity of the echinostome parasites and the number of snail species present in a pond. The addition of 1 other pulmonate snail host into the system led to a 61.9% reduction in the
267 mean number of metacercariae in H. anceps and a second added snail species resulted in a 56.1% reduction in parasite infection (Fig. 4B). Other studies have shown similar parasite reductions in a focal species with the introduction of alternative hosts (Orlofske et al., 2012; Johnson et al., 2009; Thieltges et al., 2009; Venesky et al., 2013). This can have immense impacts on the parasite distribution in the snail communities as well as other second intermediate hosts, and may be of particular interest among amphibian hosts.
Some hosts are effective at diluting parasite transmission (Hopper et al., 2008;
Johnson et al., 2008; Thieltges et al., 2008; Venesky et al., 2013), while others show little additive effects (Upathum and Sturrock, 1973; Johnson et al., 2009; Johnson and
Thieltges, 2010). The strength of the dilution effect is dependent on the abundance and ecology of the diluting organisms, not just their presence (Loguidice et al., 2008; Johnson and Thieltges, 2010; Venesky et al., 2013). Ecological similarity in hosts can enhance transmission and lead to further dilution in target hosts (Cooper et al., 2012; Lootvoet et al., 2013). Additionally, the use of alternative hosts can be favored for certain species and are not just the result of random infection (Lootvoet et al., 2013).
Significant differences in the effects of various pulmonate snail species were observed in the present study. Physa acuta and P. gyrina were the most successful species for diluting Echinostoma spp. cercariae. When these snails were present, significant decreases in the prevalence and intensity of Echinostoma spp. metacercariae in H. anceps were observed, coupled with increased frequency and accumulation of metacercariae in both P. acuta and P. gyrina compared to the focal host. Both Physa spp. are active feeders on aquatic macrophytes and algae, and frequently move between food patches to keep up with their high metabolic demands (Boss et al., 1984; Sheldon, 1987;
268 Swamikannu and Hoagland, 1989). Increased movement should increase their encounter rate with Echinostoma spp. cercariae, which may, in part, explain the differences in prevalence and intensity observed (Zimmermann et al., 2014).
Additionally, Physa spp. do not serve as first intermediate hosts for Echinostoma spp. and, as a result, accumulate cercariae infections without adding cercariae to a population (Johnson and McKenzie, 2009; Zimmermann et al., 2014). One difference between the current model and previous work regarding the dilution effect is that all of the pulmonate snails are susceptible and viable hosts for the echinostome metacercariae; they are not alternative hosts with reduced fitness or decoy hosts, which do not become infected at all (McCarthy, 1999; Johnson and Thieltges, 2010). Although there are varying levels of susceptibility of these pulmonate snails to Echinostoma spp. cercariae infection, this is often masked by the relative abundances of snail species in natural settings (Detwiler and Minchella, 2009). Additionally, there are little, or no, adverse effects for the parasites using Physa spp. instead of H. anceps as a second intermediate host (McCarthy, 1999).
In contrast, H. trivolvis did not dilute infrapopulations of echinostome parasites for H. anceps nearly as well as Physa spp. Neither H. anceps nor H. trivolvis move extensively through the benthic habitat, preserving low encounter rates with echinostome cercariae (Clampitt, 1975; Boss et al., 1984; Zimmermann et al., 2014). Although the mean intensity of metacercariae in H. anceps decreased significantly in the presence of H. trivolvis , the former harbored 3 times more metacercariae than the latter species when they were present in the same pond. Additionally, prevalence of Echinostoma spp. miracidia infection in H. anceps did not change significantly in the presence of H.
269 trivolvis, and the prevalence of cercariae infection increased significantly when the latter host was present, showing that it actually enhanced infection in H. anceps and had poor dilution effects on Echinostoma spp.
The presence of L. columella co-inhabiting the same body of water as H. anceps showed significant reductions in both prevalence and intensity of echinostome metacercariae infection in the focal species. However, the prevalence of metacercariae infection in H. anceps was not significantly different from L. columella when they were found in the same pond. Similar to co-habitation with H. trivolvis , the mean intensity of metacercariae in H. anceps was significantly greater than L. columella when they cohabited a body of water. Both H. trivolvis and L. columella can serve as first intermediate hosts for Echinostoma spp. trematodes in addition to H. anceps (Johnson and McKenzie, 2009). In addition to the latter snail species adding cercariae to the pond is the phenomenon of auto-infection, which can alter the dilution effects of both H. trivolvis and L. columella (Morley, Crane, and Lewis, 2004; Morley, Crane, and Adam,
2004; Chapter VII). These variables are difficult to account for in field studies since all of these snail species can potentially be infected by Echinostoma spp. miracidia. Further testing needs to be performed in a controlled setting to elucidate the diluting properties of
H. trivolvis and L. columella on Echinostoma spp. metacercariae infection in H. anceps .
Parasite fitness is maximized by using alternative hosts, thus reducing intraspecific competition in the principle host (Poulin, 1998; Emelianov, 2007; Lootvoet et al., 2013). This may be important in the Echinostoma spp. system. The snails that were the most successful at diluting Echinostoma spp. miracidia and cercariae infection were the species that do not serve as first intermediate hosts for the parasites, i.e., P.
270 acuta and P. gyrina . When these snail species are present, although they may decrease the relative abundance of H. anceps through interspecific completion (Brown, 1982;
Osenburg, 1989; Chase et al., 2001; Chase, 2003; Johnson et al., 2012), they can also decrease the parasite burden for the focal host. When a single snail species is found by itself in an aquatic system, metacercariae are highly overdispersed and auto-infection by
Echinostoma spp. becomes a likely occurrence (Morley, Crane, and Lewis, 2004, b;
Chapter VII). By diluting the snail population, parasite intensity is reduced in focal hosts.
This not only enhances the survival of the focal host species, it spreads the parasite to multiple host species at lower levels of infection, successfully dispersing the parasites.
This also may serve as an important evolutionary strategy in that parasites may infect multiple hosts in order to ‘hedge their bets’ and not put all their offspring into a single source, but instead spread their genes to multiple hosts in case of a localized extinction
(Bush and Kennedy, 1994; Koh et al., 2004; Lootvoet et al., 2013).
Infecting multiple snail species can heighten life cycle completion by increasing the odds of transmission to the definitive host. Each of these pulmonate snail species in the present study are commonly consumed by various mammals and waterfowl species that can serve as definitive hosts, with little preference for the snail species they consume
(Swanson and Meyers, 1977; Swanson et al., 1985; Benoy et al., 2003; Detwiler et al.,
2012). Furthermore, H. anceps is a poor competitor in the presence of more actively feeding snails such as physids or lymnaeids and often exhibit significant reductions in their relative abundance (Brown, 1982; Osenburg, 1989; Chase et al., 2001; Chase, 2003;
Johnson et al., 2012). As a result, low specificity by Echinostoma spp. cercariae would
271 be extremely beneficial for enhancing transmission by utilizing other snail species that may be more abundant in an aquatic system.
In conclusion, significant decreases in the prevalence and mean intensity of
Echinostoma spp. metacercariae were found as snail diversity increased, showing that the dilution effect is involved in echinostome cercariae infection. However, no correlations existed with respect to the prevalence of Echinostoma spp. miracidia infection in relation to snail diversity, only correlations with host size. Additionally, other snail species showed varying effects of dilution on Echinostoma spp larval stages. Physa acuta and P. gyrina were both successful in diluting the infection of Echinostoma spp. larval stages in
H. anceps , while H. trivolvis and L. columella showed mixed effects of dilution, most likely because they are suitable first intermediate hosts for the parasite. Low host specificity by Echinostoma spp. cercar iae may be beneficial for the parasites’ survival by spreading to multiple hosts which increases host survival, negates the effects of host competition, and potentially enhances the transmission of these parasites to the definitive hosts.
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279 FIGURE 1. The (A) frequency of pulmonate snail species present in the aquatic systems
(larger circles indicate greater frequency, (B) the density of Helisoma anceps per 0.05-m 2, and (C) the relative abundance of H. anceps in the presence of varying levels of pulmonate snail species richness from 139 sample sites across the U.S.
280 281 FIGURE 2. Prevalence of Echinostoma spp. miracidia infection in H. anceps in the presence of varying numbers of snail species. Like letters indicate a lack of statistical significance.
282 283 FIGURE 3. Prevalence of Echinostoma spp. miracidia infection in H. anceps in the presence of other pulmonate snail species. (*) Represents a significant difference from the focal species, Helisoma anceps.
284 285 FIGURE 4. The (A) prevalence (± standard error) and (B) mean intensity (± standard error) of Echinostoma spp. metacercariae infection of H. anceps snails in the presence of varying numbers of snail species. Like letters indicate a lack of statistical significance.
286 287 FIGURE 5. The (A) prevalence (± standard error) and (B) mean intensity (± standard error) of Echinostoma spp. metacercariae infection in H. anceps (gray bars) and other pulmonate snail species (white bars) when co-inhabiting the same body of water. (*)
Represents the focal species is significantly different from the other snail species inhabiting the same bodies of water.
288 289 CHAPTER X
SUMMARY
290 The present dissertation focused on the population biology and ecology of digenetic trematode parasites infecting gastropods hosts, with emphasis on Echinostoma spp. Pulmonate snails were collected in 3 separate studies to elucidate some of the infection patterns and transmission dynamics of both miracidia and cercariae larval stages in these gastropods, focusing on (1) life cycle variation among the trematode parasites and patterns of infection, (2) host ecology and behavior and their effect on the transmission dynamics to snail intermediate hosts, (3) microhabitat differences and its effect on trematode distribution and infection patterns, (4) infection pattern differences among trematode larval stages, and (5) the presence and abundance of other potential hosts in relation to the dilution effect and wildlife disease.
Variations in life cycle, namely the hosts involved, have led to trematode parasites to be classified into 2 groups, autogenic parasites that have all hosts restricted to a single body of water and allogenic parasites that employ at least 1 host that is capable of moving between aquatic habitats. The differences in these life cycles have major impacts on the infection patterns and transmission dynamics of the larval stages of these parasites in their gastropod first intermediate hosts, which were intimately tied to definitive host behavior. The consistent presence of autogenic hosts, compared to the ephemeral nature of allogenic hosts, led to distinct differences in the infection patterns of these parasites in their snail hosts. The former showed more or less continuous patterns of infection due to the consistent presence of their hosts and were supported by the null model, i.e., fluctuation around a mean value. Allogenic species on the other hand, showed seasonal trends tied to the presence and abundance of their hosts, most often following parabolic trends.
291 Trematode prevalence was also correlated with the prevalence and abundance of definitive hosts, as well as snail size, indicating that various aspects of host biology are important in explaining the infection patterns among trematode parasites. Additionally, habitat partitioning by the definitive hosts relating to anthropogenic disturbance also helped to explain the prevalence and distribution of their parasites. Diet and behavior of definitive hosts led to distinct separations in the areas the trematodes infected their snail intermediate hosts. Allogenic parasites, all of which cycled through waterfowl in this study, were distributed in the disturbed open habitats, while autogenic species, which utilized frogs as definitive hosts, were found in snails adjacent to the densely wooded portions of the pond. These discrepancies in infection also resulted in edge effects where parasite transmission was elevated in areas of host habitat overlap associated with intermediate levels of disturbance.
Similar patterns associated with the microhabitat affecting definitive hosts were seen on a large scale when the relationship between waterfowl definitive hosts was associated with parasite infection in their snail first intermediate hosts. Due to decreasing numbers and less propensity of waterfowl to migrate as far south in North America as in previous decades, Echinostoma spp. were not found below 35 o N latitude, but instead were concentrated in the mid-Atlantic states. Additionally, higher prevalence and abundance of echinostome metacercariae in the Atlantic flyway were attributed increases in permanent resident waterfowl in this region due to increased food supply and warmer climates associated with anthropogenic disturbance. Parasite prevalence in snail first intermediate hosts was closely associated with the waterfowl definitive host behavior, making the parasites potentially useful as an indirect indicator of waterfowl movement
292 and dispersal. Additionally, significant differences in precipitation between yr of collection and flyways may have potentially led to differences in the intensity and abundance of Echinostoma spp. in the snail intermediate hosts.
Trematode transmission was not only affected by the microhabitat surrounding a pond, but differences within it as well. Substratum differences led to distinct patterns of infection among not only trematode larval stages, but also D. potomaca , a nematode parasite that has a direct life cycle through planorbid snails. Comparison of 3 different categories of substratum revealed that the more leaf litter and debris that is present in a particular microhabitat, the lower the infection success of the parasites. Increasing the amount of structure and obstacles the parasites had to overcome to locate their snail hosts, significantly hindered infection by the helminths, which has potentially important implications relating habitat disturbance and wildlife disease.
Echinostoma spp. use pulmonate snails as both first and second intermediate hosts, offering a unique system for studying the infection patterns of multiple trematode larval stages. Distinct differences were observed in the factors that affected the transmission of these parasites, namely snail shell length and host density. Both the prevalence of miracidia infection and the mean intensity of infection by Echinostoma spp. were significantly influenced by the size of the snail hosts, indicating that larger snails produce more distinct chemical cues for the former stages and result in increased exposure time of the latter trematode larval stage. The prevalence of cercariae infection in these echinostome parasites, on the other hand, was not influenced by host size, but rather the density of their gastropod hosts. The more aggregated the snails, the higher the infection success of cercariae since there is little host specificity among these larval stages.
293 Differences in the transmission success of trematode parasites was associated with the behavior of definitive hosts and transmission to their gastropod first intermediate hosts, as well as, transmission from first to second intermediate hosts. The behavior and ecology of aquatic gastropods, which can be utilized as both first and second intermediate hosts by Echinostoma spp., affects the transmission patterns of the cercariae larval stage.
Active snails with high metabolic demands, such as Physa spp. and Lymnaea spp., are infected much more frequently and in higher numbers than Helisoma spp., that employ
‘area -restricted searching’ and exhibit very little vagility over the course of their lifetime.
Additionally, the amphibious nature of L. columella , significantly affected the prevalence of Echinostoma spp. cercariae infection due to significant decreases in exposure time, indicating the importance of host biology and ecology in shaping the transmission patterns of trematodes from first to second intermediate hosts.
As a result of using pulmonate snails as both first and second intermediate hosts, auto-infection, or the transfer of a life cycle stage from one site to another in the same host accompanied by a morphological change, can be an important component in the transmission dynamics of Echinostoma spp. Significant signs of auto-infection among H. anceps populations were observed in 2 separate field studies. Increases in both prevalence and intensity of Echinostoma spp. metacercariae were observed in snails possessing rediae of the echinostome parasite compared to individuals lacking the larval stage. Additionally, a lack of correlation with the presence of other trematode parasites or D. potomaca indicated that the observed trend was due to the presence of the first intermediate host larval stages. Furthermore, the lack of correlation with metacercariae infection and snail size indicated that the presence of Echinostoma spp. rediae was a
294 strong predictor in both the prevalence and intensity of echinostome metacercariae in the snail host, suggesting that auto-infection was taking place within these snail populations.
The presence of other potential snail hosts and species are also important factors shaping the infection dynamics and transmission patterns of Echinostoma spp. parasites.
In a large scale field study of H. anceps , significant differences in the mean intensity of echinostome metacercariae were observed in relation to the abundance and species of other snails in the aquatic habitat. Increased species richness of gastropod hosts significantly decreased the prevalence and intensity of metacercariae in H. anceps . The presence of the other snails effectively diluted the parasite population since the other pulmonate snail species were also capable of harboring Echinostoma spp. metacercariae infections. Additionally, ecological and compatibility differences among the snail hosts affected their diluting properties. Active snails that were not capable of harboring first intermediate larval stages of Echinostoma spp., e.g., Physa spp., were more successful at diluting the trematode parasites than snails that can be infected by Echinostoma spp. miracidia.
The findings presented in this dissertation lend to the importance of understanding snail-trematode interactions to the assimilation of factors regarding host ecology and behavior, microhabitat differences, and disease transmission among digenetic trematode parasites. Echinostoma spp. parasites are implicated to be a considerable cause of amphibian decline in North America. The current study illustrated that habitat disturbance, both within and around the pond, can have a significant impact on the spatial distribution and infection success of the trematode parasites infecting gastropods as both first and second intermediate hosts. These could have important implications on the
295 infection dynamics of Echinostoma spp. in amphibians as second intermediate hosts as well, an area that should be further studied.
Additionally, the dilution effect hypothesis was tested and supported through field studies, allowing important conclusions to be drawn regarding disease risk in natural populations. Increased biodiversity decreased parasite transmission in gastropod hosts, a finding that may be applicable to amphibians as well. Active snail species that are not suitable first intermediate hosts for Echinostoma spp., e.g., Physa spp., were better able to dilute the Echinostoma spp. populations since they were not adding parsites to the population, but just accumulating them. These snails are natural inhabitants of lakes and ponds across North America and could potentially be used as a means to reduce disease among amphibian hosts. Introduction of these snails could reduce the parasite burden of
Echinostoma spp. on amphibian second intermediate hosts and also potentially reduce the abundance of other snail species due to their superior competitive abilities. This could also have implications on the intermediate hosts for other pathogenic parasites, such as
Ribeiroia ondatrae , another parasite responsible for amphibian declines in North
America. The use of gastropods as a control for parasitic disease is another avenue for future research.
A final finding with potentially important implications is the association between definitive hosts and gastropod first intermediate hosts. The complex life cycles exhibited by digenetic trematodes require interactions between theses hosts and as a result, create intimate associations. The infection patterns of trematodes in snails mirror the abundance and distribution of the definitive hosts, lending potential use in both small and large scale studies as indirect indicators of definitive host abundance. More research into this area is
296 needed, but the use of snails as indirect indicators for vertebrate host behavior could potentially provide an inexpensive and easy proxy to monitor the presence, distribution, and behavior of definitive hosts. The role of gastropods in the complex life cycles of digenetic trematodes is often overlooked, but remains integral for the understanding of parasite transmission and needs to be a continuing focus for future parasitological research.
297 APPENDIX
298 Table AI-1. The location, elevation, water surface area, and infection status of
Echinostoma spp. metacercariae from all bodies of water sampled in the study.
Echinostoma spp. Body of Water City State Latitude Longitude Elevation (m) Area (ha) Present Alder Pond Akron OH 41.07664 N 81.45461 W 328.1 2.4 - Bards' Pond Westville IN 41.58597 N 86.89300 W 243.6 0.1 Basil Griffin Lake Bowling Green KY 36.91936 N 86.43925 W 176.9 11.7 Batesville Reservoir Batesville IN 39.27619 N 85.19364 W 116.8 78.1 - Big Creek Lake Polk City IA 41.80990 N 93.74661 W 295.2 331.6 - Bruin Lake Lorman LA 31.95780 N 91.20286 W 27.3 1335.7 - Campus Pond Charleston IL 39.47850 N 88.17897 W 271.0 0.8 Cap Mauzy Lake Morganfield KY 37.61900 N 87.85628 W 160.0 32.8 - Carbondale Reservoir Carbondale IL 37.00040 N 89.23087 W 143.6 48.5 - Cattail Pond Laurel MD 39.06403 N 76.73806 W 35.2 0.7 Cedar Creek Reservoir Waynesville OH 39.48817 N 84.04919 W 263.8 1105.3 Cedar Lake Carbondale IL 37.63975 N 89.27981 W 143.1 650.6 - Charlie's Pond Pine Hall NC 36.28825 N 80.06243 W 223.1 1.7 Cheston Lake Sewanee TN 35.21019 N 85.93131 W 591.7 2.5 Chickasha Lake Chickasha OK 35.13243 N 98.12728 W 371.5 435.9 - Christmas Run Pond Wooster OH 40.80597 N 81.94647 W 276.8 0.1 Coon Lake Clemmons NC 35.99719 N 80.40853 W 224.0 1.5 Corney Lake Summerfield LA 32.90893 N 92.73337 W 25.7 595.6 - Couchville Lake Lavergne TN 36.09656 N 86.54247 W 157.8 46.8 Council Bluff Lake Belgrade MO 37.73210 N 90.93085 W 331.5 109.5 - Crowne Butte Mandan ND 46.86987 N 101.09331 W 588.6 11.3 - Crystal Lake Pelican Rapids MN 46.62000 N 95.96571 W 403.9 41.5 - Dewey Lake Prestonsburg KY 37.70286 N 82.73194 W 199.8 406.0 - Dogwood Lake Montgomery IN 38.54381 N 87.05133 W 144.2 505.0 - Douglas Lake Dandridge TN 35.99861 N 83.38017 W 313.4 10096.7 - Dr. Hilton's Pond West Lafayette IN 40.49642 N 86.88378 W 199.4 0.6 Duncan Lake Golden Pond KY 36.89564 N 88.10036 W 128.0 4.6 Dutchman Lake Bloomfield IL 37.49153 N 88.90147 W 146.5 43.3 Evergreen Lake Bloomington IL 40.64936 N 89.03681 W 237.0 296.5 - Fairfield Lakes Lafayette IN 40.39258 N 86.76106 W 188.0 6.7 - Forbes Lake Kinmundy IL 38.72650 N 88.77386 W 167.2 95.8 - Fort Cobb Lake Carnegie OK 35.17240 N 98.44856 W 419.0 1575.9 - Fort Smith Lake Mountainsburg AR 35.69332 N 94.12026 W 295.5 152.4 - Girl Scout Pond Mocksville NC 35.86314 N 80.21276 W 218.8 1.5 Glen Jones Reservoir Equality IL 37.69207 N 88.38249 W 125.8 39.5 Govenor's Bridge Bowie MD 38.94628 N 76.69844 W 30.2 4.3 Graylyn Pond Winston-Salem NC 36.11769 N 80.28428 W 281.6 0.2 Gull Lake Richland MI 42.40647 N 85.40383 W 285.0 834.3 - Harry S. Truman Warsaw MO 38.28133 N 93.42930 W 224.8 38860.5 He Dog Lake Parmalee SD 43.28915 N 101.06293 W 828.8 18.3 - Hills Pond Mogadore OH 41.06139 N 81.38775 W 325.5 18.0 Horseshoe Pond Montgomery IN 38.56564 N 87.05172 W 151.9 2.3 - Hungry Mother Marion VA 36.87297 N 81.51322 W 688.1 41.1 Indian Lake Cuba MO 38.07904 N 91.44429 W 275.1 114.7 Jamestown Reservoir Jamestown ND 46.97889 N 98.70688 W 446.9 779.0 - Kentucky Lake Aurora KY 36.81356 N 86.14142 W 114.9 21551.7 - Keyhole Lake Sundance WY 44.36161 N 104.76218 W 1270.7 1457.3 - Kingsley Park Rock Hill SC 35.02611 N 80.96544 W 177.4 2.1 - LAK Reservoir Fort Mill WY 43.82442 N 104.10571 W 1349.8 41.1 - Lake #1 Rock Hill SC 34.93306 N 81.00078 W 185.0 3.6 - Lake Ahquabi Warren IA 41.29111 N 93.59009 W 268.4 45.3 Lake Ashtabula Valley City ND 47.03297 N 98.07482 W 396.9 1171.4 - Lake Barkley Cadiz KY 36.85244 N 87.97947 W 121.7 12866.3 Lake Cheniere Monroe LA 32.47732 N 92.20001 W 6.7 1274.6 - Lake Clinton Farmer City IL 40.19167 N 88.74447 W 230.6 1952.9 - Lake Conway Conway AR 35.00495 N 92.38119 W 116.4 777.1 - Lake Crystal Lake Crystal MN 44.10802 N 94.21252 W 316.0 148.8
299 Table AI-1 (Continued)
Echinostoma spp. Body of Water City State Latitude Longitude Elevation (m) Area (ha) Present Lake Dardanelle Dardanelle AR 35.28603 N 93.20539 W 104.4 9749.2 - Lake Eufaula Eufaula OK 35.32781 N 95.53744 W 189.1 35648.6 - Lake Francis Case Chamberlain SD 43.83055 N 99.33900 W 419.4 31399.9 Lake Jordan Wetumpka AL 32.63692 N 86.29832 W 88.1 10237.9 - Lake Junaluska Lake Junaluska NC 35.52614 N 82.97189 W 788.9 80.9 Lake Lanier Buford GA 34.16619 N 83.99380 W 327.9 12771.8 - Lake Mary Nell Elon NC 36.10694 N 79.50792 W 210.3 1.1 Lake Merideth Sanford TX 35.70911 N 101.59325 W 1024.1 2389.7 - Lake Norman Cornelius NC 35.46471 N 80.90060 W 242.5 12608.1 - Lake Okitabbee Collinsville MS 32.49896 N 88.80407 W 124.6 1618.7 - Lake Ouchita Mountain Pine AR 34.61965 N 93.17857 W 200.9 15737.0 - Lake Sharpe Harrold SD 43.81333 N 99.34611 W 426.7 23744.2 - Lake Stanley Draper Oklahoma City OK 35.34977 N 97.35967 W 350.5 864.2 - Lake Tschida Elgin ND 46.60090 N 101.81511 W 633.1 1260.2 Lake Verona Elon ND 36.14110 N 79.51888 W 204.2 1.3 Lake Wylie Rock Hill SC 35.02502 N 81.04395 W 184.8 2522.4 - Lambert Pond Princeton WV 37.39489 N 81.06439 W 810.1 0.4 Lexington City Lake Lexington NC 35.85922 N 80.21590 W 211.2 21.3 Little River Lake Decatur IA 40.75125 N 93.77236 W 318.2 295.5 Longview Lake Kansas City MO 38.90141 N 94.46879 W 281.5 376.4 Mallard Lake Clemmons NC 36.00047 N 80.40206 W 215.8 4.9 McDowell Lake Bismarck ND 46.82696 N 100.63996 W 533.9 21.5 Medipark North Amarillo TX 35.20328 N 101.91463 W 1111.1 2.9 - Melton Hill Lake LeNoir City TN 35.95281 N 84.24325 W 255.1 26792.6 - Middle 3 Lakes Richland MI 37.76958 N 83.66644 W 251.9 14.0 - Mill Creek Lake Slade KY 41.06525 N 81.37386 W 349.9 14.0 Mogadore Reservoir Mogadore OH 40.69297 N 79.63714 W 231.3 9.5 - Montrose Lake Montrose MO 38.31003 N 93.97480 W 228.4 581.4 Mountain Island Lake Mount Holly NC 35.35398 N 80.97250 W 206.8 1011.7 - Murphy's Bottom South Buffalo Township PA 40.00667 N 81.74028 W 292.4 1.5 - Muskingum Lake New Concord OH 40.00725 N 81.75364 W 277.4 6.7 Nealy-Henry Lake Ohatchee AL 33.78566 N 86.06497 W 229.0 7683.2 - New Concord Village Reservoir New Concord OH 37.34633 N 86.12422 W 162.2 1341.1 Nolin Lake Clarkston KY 35.79289 N 89.69283 W 84.1 387.7 Open Lake Ripley TN 37.28300 N 80.46939 W 676.0 3.0 Osage Lake Osage WY 43.97325 N 104.40920 W 1334.2 7.9 Ozark City Lake Ozark MO 35.52355 N 93.86412 W 153.4 165.6 - Pactola Reservoir Rapid City SD 44.06033 N 103.49827 W 1386.4 333.8 - Pandapas Pond Blacksburg VA 40.16922 N 81.19136 W 315.7 968.5 - Plum Orchard Lake Scarbro WV 37.94722 N 81.21928 W 523.5 694.9 Pointe Lake Willmar MN 45.19523 N 95.00781 W 348.7 69.8 - Pond 1 Bowie MD 38.97142 N 76.75672 W 39.7 1.1 - Pond 17 Kearneysville WV 39.35375 N 77.93506 W 146.7 0.1 - Pond 3 Bowie MD 38.99267 N 76.79111 W 47.9 2.8 Poplar Tree Lake Millington TN 35.30478 N 90.06306 W 93.3 40.4 Pound Lake Pound VA 37.11903 N 82.63986 W 521.4 49.3 - R.D. Bailey Lake Justice WV 37.59767 N 81.81825 W 278.1 218.6 - Rathbun Lake Centerville IA 40.86486 N 92.92708 W 282.4 4364.1 - Red Rock Lake Knoxville IA 41.40237 N 93.09352 W 251.9 5570.4 - Reelfoot Lake Hornbeak TN 36.37267 N 89.39156 W 86.3 2399.1 Renee's Pond Galax VA 36.59642 N 80.92550 W 803.2 0.5 Reynolds #12 Clemmons NC 35.99636 N 80.41400 W 220.1 0.1 - Richard B. Russell Reservoir Elberton GA 33.99305 N 82.59446 W 101.5 9680.6 - Robert S. Kerr Reservoir Sallisaw OK 35.31995 N 94.82191 W 139.9 22474.3 Rock Lake Wheatland WY 41.98782 N 105.02654 W 1528.6 13.5 - Roosevelt Lake Portsmouth OH 38.72958 N 83.17939 W 206.5 6.1 - Ross Barnett Reservoir Brandon MS 32.50552 N 89.93045 W 99.5 10431.1 -
300 Table AI-1 (Continued)
Echinostoma spp. Body of Water City State Latitude Longitude Elevation (m) Area (ha) Present Rough River Reservoir Falls of Rough KY 37.60236 N 86.50411 W 163.5 1265.4 Saylorville Lake Polk City IA 41.74787 N 93.70691 W 259.1 2973.1 - SECCA Pond Winston-Salem NC 36.11769 N 80.28428 W 266.1 0.8 - Shadow Lake Monroe MS 32.31030 N 89.68349 W 163.9 50.5 - Shank Lake Findlay OH 41.02117 N 83.68644 W 245.0 3.0 - Shanty Hollow Lake Bowling Green KY 37.14947 N 86.38611 W 165.9 44.2 - Sheppard-Meyers Reservoir West Manheim PA 39.73189 N 76.95558 W 212.3 19.7 - Sheridan Lake Rapid City SD 43.96774 N 103.48494 W 1404.1 149.2 - Skilpot Lake Clemmons NC 35.99767 N 80.41450 W 217.9 1.4 - Sloan Lake Cheyenne WY 41.15708 N 104.82833 W 1872.1 12.2 Smithville Lake Smithville MO 39.39163 N 94.55515 W 272.0 1628.5 - South Holston Lake Abingdon VA 36.60017 N 81.00778 W 537.5 3008.2 Sturtevant Lake New Concord OH 40.00728 N 81.75472 W 293.2 0.6 Swan Lake Richmond VA 37.54039 N 77.47306 W 59.7 5.4 Townsend Park Murraysville PA 40.45953 N 79.50925 W 330.1 0.2 Trinidad Lake Trinidad CO 37.14573 N 104.55772 W 1906.6 323.7 Trout Pond Kearneysville WV 39.35558 N 77.93506 W 137.5 0.6 - Tuscaloosa Lake Tuscaloosa AL 33.29130 N 87.51956 W 75.5 602.6 University Pond Bloomington IN 39.19069 N 86.50242 W 219.8 2.1 Wabash College Lake Linden IN 40.16153 N 86.90589 W 255.3 1.1 - White Oak Lake Prescott AR 33.69048 N 93.11179 W 59.5 14.6 - William E. Towell Lake St. James MO 37.99774 N 91.69172 W 313.0 16.7 - Wingfoot Lake Suffield NC 41.01119 N 81.36597 W 349.9 155.5 Wintergreen Lake Richland MI 42.39767 N 85.38531 W 272.2 14.0 - Woodward Pond Bowie VA 38.95967 N 76.74783 W 54.3 1.9 -
301 Table AII-1. P-values and AIC values of both linear and polynomial regression models in trematode parasites infecting H. anceps in Mallard Lake. ** Represents a statistically significant fit to the regression model.
P -value P -value Best Fit Trematode Species AIC (Linear) AIC (Polynomial) (Linear) (Polynomial) Model Clinostomum marginatum 0.0376 ** 0.1350 23.01 24.95 Linear Cotylurus sp. 0.8182 0.0739 25.27 21.74 Polynomial Echinostoma sp. 0.83790.0480 ** 70.33 66.31 Polynomial Megalodiscus temperatus 0.4038 0.5291 14.33 15.38 Null Uvulifer ambloplitis 0.78770.0160 ** 13.46 5.66 Polynomial
302 Table AII-2. P-values and AIC values of both linear and polynomial regression models and the best fit model in trematode parasites infecting H. anceps in Charlie's Pond over several collection seasons. ** Represents a statistically significant fit to the regression model.
P -value P -value Best Fit Trematode Species AIC (Linear) AIC (Polynomial) (Linear) (Polynomial) Model Diplostomum scheuringi 1988 † 0.8374 0.4199 25.47 23.97 Polynomial 1989 ‡ 0.5665 0.3751 52.57 50.90 Polynomial 1992§ 0.18320.0301 ** 32.44 31.08 Polynomial Echinostoma trivolvis 1989 ‡ 0.5890 0.3162 49.12 47.86 Polynomial 1992§ 0.70570.0090 ** 40.51 30.81 Polynomial 1996 ‖ 0.8657 0.6516 29.98 28.89 Polynomial Halipegus occiduals 1984* 0.7970 0.9631 65.26 67.16 Null 1988 † 0.2400 0.5443 60.67 61.20 Null 1989 ‡ 0.0573 0.1345 60.60 62.60 Null 1992§ 0.9328 0.0034 59.19 52.35 Polynomial 1996 ‖ 0.3190 0.6541 26.88 28.69 Null 2002# 0.0928 0.2858 45.01 46.98 Null 2005# 0.4530 0.6092 38.89 40.61 Null 2006# 0.2404 0.4950 47.66 48.20 Null Megalodiscus temperatus 1988 † 0.47470.0433 ** 31.03 26.29 Polynomial 1992§ 0.45300.0137 ** 44.14 36.01 Polynomial * Data from Crews and Esch (1986) † Data from Williams and Esch (1991) ‡Data from Fernandez and Esch (1991b) § Data from Sapp and Esch (1994) ‖ Data from Schotthoeffer (1996) # Data from Negovetich and Esch (2007)
303 Table AII-3. P-values and AIC values of both linear and polynomial regression models and the best fit model in trematode parasites infecting H. anceps in Charlie's Pond over several collection seasons. ** Represents a statically significant fit to the regression model.
P -value P -value Best Fit Trematode Species AIC (Linear) AIC (Polynomial) (Linear) (Polynomial) Model Echinoparyphium parvum 1996 ‖ 0.70880.0381 ** 17.88 11.66 Polynomial Echinostoma trivolvis 1992§ 0.79950.0132 ** 57.27 48.63 Polynomial Glypthelmins quieta 1991¶ 0.4342 0.4342 52.36 53.11 Null 1992§ 0.0315 ** 0.1200 45.02 46.89 Linear 1996 ‖ 0.88540.0375 ** 46.00 39.37 Polynomial Haematoloechus complexus 1991¶ 0.7009 0.9025 53.82 55.71 Null 1992§ 0.1137 0.3202 36.47 38.44 Null Halipegus eccentricus 1991¶ 0.4132 0.6267 66.03 66.18 Null 1992§ 0.3958 0.6263 56.53 58.08 Null Megalodiscus temperatus 1992§ 0.3573 0.4010 29.92 30.22 Null 1996 ‖ 0.2265 0.4139 22.32 23.49 Null Posthodiplostomum minimum 1996 ‖ 0.25840.0476 ** 53.89 50.02 Polynomial Unknown Strigeid 1992§ 0.61010.0451 ** 55.52 50.75 Polynomial ¶ Data from Snyder and Esch (1993) § Data from Sapp and Esch (1994) ‖ Data from Schotthoefer (1996)
304 FIGURE AI-1. Three species of digenetic trematode cercariae isolated from L. columella in Coon Lake: (A) Australapatemon canadiensis , (B) Tylodelphis sp., and (C)
Australapatemon burti. Scale bars represent 0.2 mm.
305 306 FIGURE AI-2. (A) Echinoparyphium recurvatum cercariae and (B) the oral sucker surrounded by a row of collar spines isolated from L. columella from Coon Lake. The scale bar represents 0.2 mm.
307 308 FIGURE AI-3. Three species of digenetic trematode cercariae isolated from P. gyrina in
Coon Lake: (A) Glypthelmins quieta , (B) Haematoloechus complexus , and (C)
Plagiorchis sp . Inlays are magnified images of the oral sucker and spicule. Scale bars represent 0.2 mm.
309 310 FIGURE AI-4. Notocotylus attenuatus larval stages recovered from P. gyrina in Coon
Lake. (A) Mature rediae with developing cercariae inside, (B) Shed cercariae, (C)
Encysted metacercariae on glass dish. Scale bars represent 0.2 mm.
311 312 FIGURE AI-5. Unknown strigeid nematode recovered from M. dilatus in Coon Lake.
The scale bar represents 0.2 mm.
313 314 FIGURE AI-6. Chaetogaster limnaei limnaei (A) Brightfield, (B) darkfield, (C) and an individual that consumed a larval trematode from Coon Lake. Scale bars represent 0.2 mm.
315 316 Figure AII-1. Seasonal patterns of the prevalence of E. trivolvis infections in H. anceps from various years o f collection in Charlie’s Pond (Fernandez and Esch, 1991b; Sapp and
Esch, 1994; Schotthoefer, 1996). The dashed line represents the best fit regression model.
** Represents a statistically significant fit regression model.
317 318 Figure AII-2. Seasonal patterns of the prevalence of D. scheuringi infections in H. anceps from various years of collection in Charlie’s Pond (Williams and Esch, 1991;
Fernandez and Esch, 1991b; Sapp and Esch, 1994). The dashed line represents the best fit regression model. ** Represents a statistically significant fit regression model.
319 320 Figure AII-3. Seasonal patterns of the prevalence of E. trivolvis infections in P. gyrina from Charlie’s Pond (Sapp and Esch, 1994). The dashed line represents the significantly fit regression model.
321 322 Figure AII-4. Seasonal patterns of the prevalence of E. parvum infections in P. gyrina from Charlie’s Pond (Schotthoefer, 1996). The dashed line represents the significantly fit regression model.
323 324 Figure AII-5. Seasonal patterns of the prevalence of P. minimum infections in P. gyrina in Charlie’s Pond (Schotthoefer, 1996). The dashed line represents the significantly fit regression model.
325 326 Figure AII-6. Seasonal patterns of the prevalence of an unknown strigeid infection in P. gyrina from Charlie’s Pond (Sapp and Esch, 1994). The dashed line represents the significantly fit regression model.
327 328 Figure AII-7. Seasonal patterns of the prevalence of H. occidualis infections in H. anceps from various years of collection in Charlie’s Pond (Crews and Esch, 1986;
Williams and Esch, 1991; Fernandez and Esch, 1991b; Sapp and Esch, 1994;
Schotthoefer, 1996; Negovetich and Esch, 2007). The dashed line represents the significantly fit regression model or the mean prevalence of the parasite if neither regression model were statistically significant. ** Represents a statistically significant fit regression model.
329 330 Figure AII-8. Seasonal patterns of the prevalence of H. eccentricus infections in P. gyrina from various years of collection in Charlie’s Pond (Snyder and Esch, 1993; Sapp and Esch, 1994). The dashed line represents the mean prevalence of the parasite infection since neither regression model was statistically significant.
331 332 Figure AII-9. Seasonal patterns of the prevalence of H. complexus infections in P. gyrina from various years of collection in Charlie’s Pond (Snyder and Esch, 1993; Sapp and
Esch, 1994). The dashed line represents the mean prevalence of the parasite infection since neither regression model was statistically significant.
333 334 Figure AII-10. Seasonal patterns of the prevalence of M. temperatus infections in H. anceps from various year s of collection in Charlie’s Pond (Williams and Esch, 1991;
Sapp and Esch, 1994). The dashed line represents the best fit regression model. **
Represents a statistically significant fit regression model.
335 336 Figure AII-11. Seasonal patterns of the prevalence of M. temperatus infections in P. gyrina from various years of collection in Charlie’s Pond (Sapp and Esch, 1994;
Schotthoefer, 1996). The dashed line represents the mean prevalence of parasite infection since neither model was significantly significant.
337 338 Figure AII-12. Seasonal patterns of the prevalence of G. quieta infections in P. gyrina from various years of collection in Charlie’s Pond (Snyder and Esch, 1993; Sapp and
Esch, 1994; Schotthoefer, 1996). The dashed line represents the significantly fit regression model or the mean prevalence of the parasite if neither regression model were statistically significant. ** Represents a statistically significant fit regression model.
339 340 CURRICULUM VITAE
MICHAEL R. ZIMMERMANN
CONTACT INFORMATION______
Wake Forest University Detent of Biology Winston-Salem, NC 27106
Office: (336) 758-5577; Home: (740) 704-3241 E-mail: [email protected] Date and Place of Birth: January 6, 1986, Akron, OH Married: Abigail E. VanFleet, September 22, 2012
_____EDUCATION______
Wake Forest University Winston-Salem, NC May 2014 Doctor of Philosophy in Biology, with a focus in parasitology. My dissertation, Population biology and landscape ecology of digenetic trematode parasites in their gastropod hosts, with special emphasis on Echinostoma spp., is in progress under the direction of Dr. G. W. Esch with expected completion in May 2014.
Wake Forest University Winston-Salem, NC May 2010 Master of Science in Biology, with a focus in parasitology. My thesis, Population Biology of Daubaylia potomaca (Nematoda: Rhabditida) in Mallard Lake, North Carolina , was under the direction of Dr. G. W. Esch.
Muskingum College New Concord, OH May 2008 Bachelor of Science in Biology. Minor in Chemistry and History
TEACHING EXPERIENCE______
Fall 2013 Elon University Elon, NC I designed all of the lectures and ran laboratories for a course in biodiversity. The course focused on the diversification of life.
Fall 2008 – Spring 2014 Wake Forest University Winston-Salem, NC I independently taught the lab section of a number of courses. My duties included lab preparation, creating and delivering pre-lab lectures, and writing and grading coursework, lab reports, and group projects. In upper level labs, I was solely responsible for designing, teaching, and assessing the lab portion of the course. I have taught the following courses:
Genetics and Molecular Biology (Fall 2012, Fall and Spring 2013, Fall 2014) Ecology and Evolution (Spring 2011, Spring 2012) Biology of Fishes (Fall 2011)
341 General Ecology (Fall 2010) Comparative Physiology (Fall 2008, Spring 2009, Fall 2009, Spring 2010)
SCHOLASTIC AND PROFESSIONAL EXPERIENCE______
Fall 2011 – Spring 2012 Wake Forest University Winston-Salem, NC Biology Tutor I tutored for the Wake Forest University athletic department for student athletes in need of help in classes in the biological sciences. I tutored at least one student athlete in each of the following courses:
Biology and the Human Condition (1 Semester) Comparative Physiology (4 Semesters) Ecology and Evolution (2 Semesters)
Manuscript Referee Freshwater Science, 2012 Marine Environmental Science, 2012 Vector Biology, 2013
HONORS AND AWARDS______
Alumni Student Travel Award, Wake Forest University, 2011, 2012 Grady-Britt Travel Grant, Wake Forest University, 2009, 2010, 2011, 2012, 2013 Vecellio Grant for Graduate Research, Wake Forest University 2011, 2013 Elton C. Cock Travel Grant, Wake Forest University, 2011 Marc Dresden Student Travel Fund, American Society of Parasitologists, 2010, 2011, 2013, 2014 Bradford Colloquium, Best Oral Presentation, Muskingum College, 2008
ORGANIZATIONS______
American Society of Parasitologists . Session Chair, Annual Meeting, 2009, 2010, 2011, 2012, 2013 Southeastern Society of Parasitologists American Malacological Society American Society of Naturalists North American Benthological Society
PUBLICATIONS______
1. Esch, G. W., K. Luth, and M. Zimmermann . 2008. Wincenty Wiśniewski: A retrospective review. Wiadomości Parazytologiczne 54: 279 -284.
342 2. Zimmermann, M. R., K. E. Luth, and G. W. Esch. 2011. Population and infection dynamics of Daubaylia potomaca (Nematoda: Rhabditida) in Helisoma anceps . Journal of Parasitology 97: 384-388.
3. Zimmermann, M. R., K. E. Luth, and G. W. Esch. 2011. The unusual life cycle of Daubaylia potomaca , a nematode parasite of Helisoma anceps . Journal of Parasitology 97: 430-434.
4. Zimmermann, M. R., K. E. Luth, and G. W. Esch. 2011. Complex interactions among a nematode parasite ( Daubaylia potomaca ), a commensalistic annelid (Chaetogaster limnaei limnaei ), and trematode parasites in a snail host (Helisoma anceps ). Journal of Parasitology 97: 788-791.
5. Zimmermann, M. R., K. E. Luth, and G. W. Esch. 2013. Shedding patterns of Daubaylia potomaca (Nematoda: Rhabditida). Journal of Parasitology 99: 966-969.
6. Esch, G. W., K. E. Luth, and M. R. Zimmermann. 2014. The ecology of animal parasites. In Encyclopedia of Food Safety. Y. Motarjemi and L. Gorris (eds.). Academic Press, Oxford, U.K., Vol. 1, p. 31-40.
7. Zimmermann, M. R., K. E. Luth, and G. W. Esch. 2014. Differences in snail ecology lead to infection pattern variation in Echinostoma spp. larval stages. Acta Parasitologica, In press .
8. Zimmermann, M. R., K. E. Luth, and G. W. Esch. 2014. Differences in microhabitat affect parasite infection in Helisoma anceps . Freshwater Science, In review.
9. Zimmermann, M. R., K. E. Luth, and G. W. Esch. 2014. Habitat disturbance affects the distribution of trematode parasites among a pulmonate snail community. Journal of Limnology. In review.
10. Zimmermann, M. R., K. E. Luth, and G. W. Esch. 2014. Differences in transmission patterns of miracidia and cercariae larval stages of digenetic trematodes in nature. Comparative Parasitology. In review.
11. Zimmermann, M. R., K. E. Luth, and G. W. Esch. 2014. Auto-infection by Echinostoma spp. in nature. In preparation .
12. Zimmermann, M. R., K. E. Luth, and G. W. Esch. 2014. The dilution effect in Echinostoma spp.: Examples from field collected data. In preparation .
13. Zimmermann, M. R., K. E. Luth, and G. W. Esch. 2014. Digenetic trematode life cycles shape their infection patterns in their pulmonate snail hosts. In preparation.
14. Zimmermann, M. R., K. E. Luth, and G. W. Esch. 2014. Infection pattern differences of Echinostoma spp. across the North American waterfowl flyways. In preparation .
343 PRESENTATIONS______
Poster Presentations
1. Zimmermann, M. R., and D. Ingold. 2008. Parasite loads in three sunfish species ( Lepomis spp.) in reclaimed surface mine ponds (The Wilds). Ohio Academy of Science. Toledo, Ohio.
2. Zimmermann, M. R., K. E. Luth, J. Rodriguez, L. Camp, and G. W. Esch. 2009. Population and infection dynamics of Daubaylia potomaca (Nematoda: Cephalobidae). American Society of Parasitologists, 84th Annual Meeting. Knoxville, Tennessee.
3. Zimmermann, M. R., K. E. Luth, and G. W. Esch. 2009. Population and infection dynamics of Daubaylia potomaca (Nematoda: Cephalobidae). Prospectives in Biology, 20th Annual Meeting. Winston-Salem, North Carolina
4. Zimmermann, M. R., K. E. Luth, and G. W. Esch. 2010. The unusual life cycle of Daubaylia potomaca , a nematode parasite of Helisoma anceps . Prospectives in Biology, 21st Annual Meeting. Winston-Salem, North Carolina.
5. Zimmermann, M. R., K. E. Luth, and G. W. Esch. 2011. Complex interaction among a nematode parasite, a commensal, and trematode parasite in a snail host. Prospectives in Biology, 22nd Annual Meeting. Winston-Salem, North Carolina.
6. Zimmermann, M. R., K. E. Luth, and G. W. Esch. 2012. Infection pattern differences of Echinostoma spp. cercariae in three natural snail host genera. Prospectives in Biology, 23rd Annual Meeting. Winston-Salem, North Carolina.
Oral Presentations
1. Zimmermann, M. R. 2008. Parasite loads in three sunfish species ( Lepomis spp.) in reclaimed surface mine ponds (the Wilds). Bradford Colloquium, Muskingum College, New Concord, Ohio.
2. Zimmermann, M. R. 2010. Life history of Daubaylia potomaca in Mallard Lake. Departmental Seminar, Wake Forest University, Winston-Salem, North Carolina.
3. Zimmermann, M. R., K. E. Luth, and G. W. Esch. 2010. Infection dynamics of Daubaylia potomaca (Nematoda: Rhabditida) in Helisoma anceps . American Society of Parasitologists, 85th Annual Meeting. Colorado Springs, Colorado
4. Zimmermann, M. R., K. E. Luth, and G. W. Esch. 2011. Complex interactions among a parasitic nematode ( Daubaylia potomaca ), a commensalistic annelid (Chaetogaster limnaei limnaei ), and various trematode species in an aquatic
344 snail host ( Helisoma anceps ). American Society of Parasitologists, 86th Annual Meeting. Anchorage, Alaska.
5. Zimmermann, M. R., K. E. Luth, and G. W. Esch. 2012. Population genetics of Echinostoma revolutum along the major North American flyways. American Society of Parasitologists, 87th Annual Meeting. Richmond, Virginia.
6. Zimmermann, M. R., K. E. Luth, and G. W. Esch. 2013. Differential infection patterns of Echinostoma spp. in three different snail genera. American Society of Parasitologists, 88th Annual Meeting. Quebec City, Quebec, Canada.
7. Zimmermann, M. R. 2014. Snail trematode interactions: Transmission and disease. Departmental Seminar. Wake Forest University, Winston-Salem, North Carolina.
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