MIAMI UNIVERSITY – THE GRADUATE SCHOOL
CERTIFICATE FOR APPROVING THE DISSERTATION
We hereby approve the Dissertation
of
Kata C. Gurski
Candidate for the Degree
Doctor of Philosophy
Mercedes A. Ebbert, Director
Thomas O. Crist, Reader
Dennis L. Claussen, Reader
Marcia R. Lee, Reader
Marjorie M. Cowan, Graduate School Representative ABSTRACT
THE NATURAL PREVALENCE OF TRYPANOSOMATIDS (KINETOPLASTIDA:
TRYPANOSOMATIDAE) IN AQUARIUS REMIGIS (SAY) (HEMIPTERA: GERRIDAE),
AND THEIR EFFECT ON GERRID MORPHOLOGY
By Kata C. Gurski
Insect-parasite interactions and their possible effects on the surrounding community are rarely studied in the context of a natural environment. Typically, studies examine the effect of parasitic infections under controlled conditions often varying one condition at a time with respect to infection. While these studies are important for isolating the effects parasites exert on insects, they may provide only limited insight to the actual host-parasite interaction as they occur in a natural setting when both organisms are exposed to a multitude of environmental stresses. Long- term observational studies of parasite prevalence are a logical starting point for studies of host- parasite interactions. Thus, the purpose of this dissertation was to address the following questions with respect to trypanosomatid parasites of Aquarius remigis hosts. First, what is the natural prevalence of trypanosomatids in gerrids within and among annual field seasons?
Second, how does uninfected gerrid morphology vary spatially and temporally, and with respect to mating status? And third, does trypanosomatid infection affect morphological development of field-caught gerrids?
From 1999 to 2002 I sampled 7633 adult and nymph Aquarius remigis gerrids from eight locations in four streams in Butler County, Ohio. I assayed 6836 gerrids for trypanosomatid infection and measured nine morphological traits of 688 individuals. Trypanosomatids were present in all four streams, and persisted at each site over the course of the study. Prevalence increased with host age, but did not differ between males and females. Patterns were similar among streams and years. Variation within a year was consistent with variation in gerrid mating periods, but not gerrid density. Gerrid morphology varied, but not consistently, between host generations, and among streams and location within stream. Trypanosomatid infections were associated with smaller adult males, but not females, and this effect was variable within and among streams.
I suggest that both variation in parasite prevalence over time and in gerrid morphology over time and space is influenced by fluctuating environmental conditions. This study not only demonstrates the importance of long-term studies of parasite prevalence and host-parasite interactions, but it also suggests that variation in these interactions may be overlooked if only a single population is examined. THE NATURAL PREVALENCE OF TRYPANOSOMATIDS (KINETOPLASTIDA:
TRYPANOSOMATIDAE) IN AQUARIUS REMIGIS (SAY) (HEMIPTERA:
GERRIDAE), AND THEIR EFFECT ON GERRID MORPHOLOGY
A DISSERATATION
Submitted to the Faculty of
Miami University in partial
fulfillment of the requirements
for the degree of
Doctor of Philosophy
Department of Zoology
by
Kata C. Gurski
Miami University
Oxford, OH
2003
Dissertation Director: Dr. Mercedes A. Ebbert TABLE OF CONTENTS
ACKNOWLEDGMENTS.….…………………………………………………….iii
LIST OF TABLES………………………………………………………………....iv
LIST OF FIGURES………………………………………………………………..vi
DEDICATION……………………………………………………………………vii
CHAPTER 1: Dissertation Introduction …………………………………………. 1
CHAPTER 2: Changes in reproductive life history patterns of the gerrid, Aquarius remigis (Say) (Hemiptera: Gerridae), alter trypanosomatid (Kinetoplastida:
Trypanosomatidae) prevalence …………………………………………………...12
CHAPTER 3: Spatial and temporal variation in Aquarius remigis (Say) (Hemiptera:
Gerridae) morphology ……………………………………………………………45
CHAPTER 4: Trypanosomatid (Kinetoplastida: Trypanosomatidae) infection reduces adult Aquarius remigis (Say) (Hemiptera: Gerridae) body size ………...69
CHAPTER 5: Dissertation Conclusion ………………………………………….83
LITERATURE CITED……………………………………………………………88
TABLES…………………………………………………………………………103
FIGURES………………………………………………………………………...146
APPENDIX A……………………………………………………………………176
APPENDIX B……………………………………………………………………198
ii ACKNOWLEDGMENTS
I am grateful for the assistance, encouragement and patience of my advisor, Dr.
Mercedes Ebbert. Thank you to my dissertation committee, Dr. Thomas Crist, Dr.
Dennis Claussen, Dr. Marcia Lee, and Dr. Marjorie Cowan. Thanks to Kelly Buchanan,
Jennifer Avondet, Ben Knopp, Sarah McMasters, and Dan Thomas for their field and laboratory support. Funds for this project were provided by Miami University.
iii LIST OF TABLES
1. Breakdown of gerrids caught, assayed and analyzed………………………..…103
2. Experimental design……………………………………………………………104
3. Collection dates and gerrid density…………………………………………….105
4. Monthly average for gerrids caught and analyzed…………………………..…109
5. Average monthly and seasonal adult and nymph density………………………110
6. ANOVA of gerrid density………………………………………………………112
7. Contingency table for gerrid location paired analysis………………………….113
8. Contingency table for gerrid mating status paired analysis…………………….114
9. Contingency table for gerrid sex paired analysis……………………………….115
10. Contingency table for gerrid overwintering paired analysis……………………116
11. Contingency table for gerrid stage paired analysis…………………………….117
12. Yearly and monthly trypanosomatid prevalence in adults per stream…………118
13. Yearly and monthly trypanosomatid prevalence in nymphs per stream……….121
14. Monthly and seasonal trypanosomatid prevalence…………………………….124
15. Chi-square analysis of prevalence……………………………………………...126
16. MANOVA of stream location and adult morphology………………………….127
17. Summary statistics for stream location and adult morphology………………...128
18. MANOVA of generation, sex and stream on adult morphology………………129
19. Summary statistics for generation and adult morphology……………………..130
20. Summary statistics for generation, stream and adult morphology……………. 131
21. ANOVA of stream location and fluctuating asymmetry.………………………132
iv 22. MANOVA of mating status and adult morphology……………………………133
23. ANOVA of mating status and fluctuating asymmetry…………………………134
24. MANOVA of nymph morphology……………………………………………..135
25. Summary statistics for stream location and nymph morphology………………136
26. Summary statistics for sex, age and nymph morphology………………………137
27. MANOVA of infection, stream location and adult male morphology…………138
28. MANOVA of four-way interaction on adult morphology……………………..139
29. MANOVA of infection, stream and adult G1 male morphology………………140
30. Summary statistics for infection, stream and adult G1 male morphology……..141
31. MANOVA of infection and adult morphology………………………………...142
32. Summary statistics for infection, stream and adult male morphology…………143
33. ANOVA of infection, stream location and fluctuating asymmetry…………….144
34. ANOVA of infection, stream and fluctuating asymmetry……………………..145
v LIST OF FIGURES
1. Host-parasite interactions………………………………………………………..146
2. Study area map…………………………………………………………………..148
3. Precipitation……………………………………………………………………...150
4. Temperature……………………………………………………………………...152
5. Nymph density…………………………………………………………………...154
6. Adult density……………………………………………………………………..156
7. Trypanosomatid prevalence and density correlation…………………………….158
8. Gerrid mating activity……………………………………………………………160
9. Trypanosomatid prevalence in nymphs year 2000………………………………162
10. Trypanosomatid prevalence in adults year 2000………………………………..164
11. Trypanosomatid prevalence in adults year 2001………………………………..166
12. Trypanosomatid prevalence in adults year 2002………………………………..168
13. Trypanosomatid prevalence in adults year 1999………………………………..170
14. Hypothetical host-parasite interactions…………………………………………172
15. Adult body length and fluctuating asymmetry correlation……………………...174
vi Dedicated to the memory of my dad, Dr. Richard Joseph Gurski
vii CHAPTER 1
DISSERTATION INTRODUCTION
1 Parasites play an important role in the biology of animals. They are a significant source of host mortality and thus indirectly influence the interactions of populations within a community (Fuxa and Tanada, 1987; Thompson, 1994; Freeland, 1983; Price et al., 1986; Price et al., 1988; Minchella and Scott, 1991; Combs, 1995; Combs, 1996; Hudson and Greenman,
1998). The impact of parasites can be more subtle and complex than directly causing host mortality. For example, parasites can affect host interactions with the environment: when stressed by limited food availability or cold environments, the negative effects of parasites on their hosts may be enhanced (Spence, 1986). Sublethal infections may influence host population structure in this manner (e.g., Klingenberg et al., 1997) and thus, indirectly shape the structure of the surrounding community. Although ecologists acknowledge the importance of parasites in communities, their effects remain under-investigated (Thomas et al., 2000).
Parasites can also alter, directly or indirectly, host life-history traits, such as fecundity, growth or survival (review in Michalakis and Hochberg, 1994). Changes in host traits result in variation among both individuals and populations and can affect species coexistence (Thomas et al., 2000; Poulin, 1999). For example, parasites can affect the developmental rate of hosts and thus cause temporal segregation between competitors. Parasites that affect the dispersal ability of hosts cause them to become more geographically isolated or widespread. Parasites could also structure host communities by affecting competing hosts unequally and thus altering the competitive ability of one or both host species (review in Hudson and Greenman, 1998).
Parasites can also play a role in structuring parasite communities within an animal host
(Poulin, 1999). Direct interactions between parasite species include predation (Sousa, 1993,
1992) and competition (Dobson, 1985; Kuris and Lafferty, 1994), while indirect interactions may occur when one parasite alters the host and thus alters the environment for other parasites.
2 For example, a parasite may alter host phenotype or immune defenses making the host more or less suitable for other parasites. Additionally, one parasite may alter a host in such a way that makes the host more or less susceptible to predation, altering transmission opportunities for other parasites (Thomas et al., 1997; Thomas et al., 1998).
Although parasitism is present in all natural systems, only a few studies have included parasites in the food webs of whole communities (reviews in Marcogliese and Cone, 1997;
Mouritsen and Poulin, 2002). Poulin (1999) provides one of the only empirical examples of parasitism structuring not only the free-living animal community, but also parasite communities.
The first step in understanding the ecological and evolutionary importance of insect parasites in a particular community is to collect information on prevalence. Prevalence is defined as the percentage of infected individuals in a population and represents the net effect of a complex of host-parasite interactions (Figure 1; Fuxa and Tanada, 1987). Prevalence depends on the duration of infection and the incidence of new infections. Transmission to new hosts increases with (1) opportunities for host-parasite contact (influenced by factors such as host density and host behavior), and (2) establishment of the parasitic infection (which depends on host susceptibility and tolerance). Both contact and establishment may be affected by host sex, age, or environmental stresses, such as food availability and temperature. As a result of this complexity, accurate descriptions of prevalence patterns require large sample sizes.
Insect pathogens are ideal model systems for such studies, but few long-term studies of parasite prevalence in natural populations have been undertaken. In a 15-year study of caddisflies and their microsporidian parasites, Kohler and Hoiland (2001) observed large fluctuations in prevalence both within and among caddisfly generations. The authors conclude that the highly pathogenic parasite appears to be responsible for regulating the caddisfly
3 population in a delayed density-dependent manner. In an 18-year study of a mosquito- microsporidian-copepod system, Andreadis (1999) observed low microsporidian prevalence in the mosquito (<10%) and concluded that the pathogen minimally impacted the mosquito population, although the population density of neither host was examined.
Schmid-Hempel (review in 2001) has extensively investigated the prevalence of Crithidia bombi trypanosomatids in bumblebees. Although this host is a social insect, and its interaction with parasites is expected to vary significantly from non-social insects, infection experiments yield large differences in prevalence among colonies inoculated with the same parasite species.
Fuxa et al. (2000) sampled southern green stink bugs (Nezara viridula) for the presence of trypanosomatids. Multiple samples over two consecutive years showed that prevalence differed significantly between two habitats. Studies of trypanosomatids in water striders (Gurski and
Ebbert, 2003; Tiezsen and Molyneux, 1989) demonstrated that prevalence varied during a field season, but similar patterns were observed among seasons. Additionally, Gurski and Ebbert
(2003) sampled trypanosomatids in a nested design of eight populations of hosts demonstrating that within a watershed, prevalence of parasites did not differ among host populations.
These studies demonstrate the need for documenting parasite prevalence over time in natural environments as the host-parasite system is exposed to variable biotic and abiotic conditions. These studies not only reveal how insect-parasite interactions vary over time, but they could also serve to expose natural host variation in susceptibility and tolerance to parasites
(review in Schmid-Hempel, 2001). For example, most insect trypanosomatids are considered non-pathogenic (Schaub, 1994), but the majority of these species have only been investigated under optimum laboratory conditions. Pathogenic effects of parasites may not be detected until hosts are exposed to stressful environments (e.g. Schmid-Hempel, 2001). Schaub (1992)
4 summarizes the negative effects on hosts infected with trypanosomatids and subjected to stressors, such as temperature and starvation.
Most of the trypanosomatids studied in detail are heteroxenous; that is, their life cycles include both a vertebrate and an invertebrate host. Although many homoxenous trypanosomatids (those infecting a single host) have been identified, the effects that members of this group exert on their hosts are only well documented for two species, Blastocrithidia triatomae in the terrestrial hemipteran Triatoma infestans (Schaub, 1992) and Crithidia bombi in bumblebees (review in Schmid-Hempel, 2001). In an aquatic environment, trypanosomatid effects on hosts have only been investigated in water striders and in only a few studies
(starvation resistance and motility: Arnqvist and Maki, 1990; mating success: Arnqvist, 1992; development: Klingenberg et al., 1997).
I have chosen undertake a multi-year study the prevalence of protozoan parasites
(trypanosomatids, Kinetoplastida: Trypanosomatidae) in natural populations of stream-dwelling insects (water striders, Hemiptera: Gerridae). These hosts are ideal subjects because they are abundant, colonies can be maintained in the laboratory, and infection status is easily assayed. A few studies have documented prevalence of these parasites in pond-dwelling gerrids and the effects they have on gerrid life history characters. However, neither trypanosomatid prevalence nor trypanosomatid effects on life history has been explored in stream-dwelling gerrids. Work on this host-parasite association, therefore, has the potential to yield insights into both gerrid fitness and the importance of the pond and the stream habitat in gerrid-trypanosomatid interactions.
Gerrid ecology
5 Water striders (subfamily Gerrinae) inhabit all types of water surfaces. In Northeastern
North America, Aquarius remigis is the most abundant and most widely distributed gerrid species (Calabrese, 1977). This species prefers smaller streams (Spence, 1981) and cooler habitats than those preferred by most other gerrid species (Calabrese, 1977).
Aquarius remigis are scavengers and predators feeding on insects trapped on the water’s surface. Gerrids have many natural predators including spiders (Zimmerman and Spence,
1989), backswimmers (Lowe, 1994), dragonfly naiads (Spence, 1986, Van Buskirk, 1992), fish
(Cooper, 1984), and birds and frogs (Callahan, 1974; Fairbairn, 1993).
Aquarius remigis gerrids are primarily wingless and fewer than 1% are winged or partially winged in most populations (Calabrese, 1979). The degree of wing reduction in insects is often associated with habitat stability and thus dispersal capability (Calabrese, 1979;
Fairbairn, 1985; Kaitala, 1988; Kaitala and Dingle, 1992). In general, gerrids inhabiting unstable habitats will produce more winged individuals and dispersal will correlated with seasonal habitat loss. Conversely, wingless morphs are more often found in permanent habitats where dispersal is probably less crucial (Fairbairn, 1988a). For A. remigis, environmental condition is the primary determinant of wing morphology. Under experimental and field conditions, longer day lengths and warmer temperatures produce increasingly more winged individuals regardless of parental phenotype (Fairbairn, 1985, 1986). Although patterns of wing reduction are correlated with habitat stability in many gerrid species, dispersal in A. remigis is limited, even for winged morphs (Fairbairn, 1986). Gurski (2000) marked and released gerrids in a 1999 trypanosomatid prevalence study, and all recaptured gerrids were found within the same site at which they were released. Of the 1832 adult gerrids caught in the 1999 study, only
9 were fully winged. Thus A. remigis dispersal by flight is unlikely.
6 Aquarius remigis reproduce either once (in cooler climates, e.g., Quebec: Fairbairn,
1985) or twice (in warmer climates, e.g., Pennsylvania: Firko, 1986; New Jersey: Rubenstein,
1989) each year. In warmer climates, such as southern Ohio, A. remigis post-diapause adults mate in early spring and produce the first generation of nymphs that begin hatching in May
(personal observation). The post-diapause adults die by summer, and the first generation nymphs mature through five instars before becoming adults. If food levels are high, part of the first generation population will reproduce the same summer (giving rise to a second generation) then die by winter (Blanckenhorn, 1990). Unmated individuals of the first generation and individuals of the second generation that molt to adults enter diapause, and those that survive the winter emerge to reproduce in the spring. Low food levels can force all first generation gerrids to delay reproduction until the following spring (Blanckenhorn, 1990), and approximately 75% of adult A. remigis die during diapause (Fairbairn, 1985; Blanckenhorn,
1994; Preziosi and Fairbairn, 2000).
Aquarius remigis adults begin mating as soon as they emerge from diapause, and both males and females mate many times during their lifetime. After a short duration (1-30 seconds) of pre-mating struggles, mating individuals remain in copula for an average of three hours with some matings lasting up to 12 hours (Clark 1988; Wilcox, 1984; Fairbairn 1988b; Sih et al.,
1990; Weigensberg and Fairbairn, 1996). Males often remain in tandem with females without copulating for the purpose of mate-guarding. Copulations lasting less than 15 minutes result in little transfer of sperm and are generally unsuccessful (Rubenstein, 1989; Weigensberg and
Fairbairn, 1994). The longer a mating pair remains in copula, the more sperm of previous mating is displaced in the female (Rubenstein, 1989). Additionally, 65% of a female’s eggs are
7 fertilized by the last male to mate with her. Thus, males benefit from both prolonged mating and post-copulatory mate guarding.
Aquarius remigis females lay three to five eggs at a time several times per day for two to three months (Weigensberg and Fairbairn, 1996). Females attach the eggs to submerged substrates (usually rocks: Haskins, 1997) and first instar nymphs will hatch in two to three weeks (Spence et al., 1980) depending on temperature (Vepsalainen, 1973). In the laboratory, newly hatched nymphs remain submerged without eating for one to two days before rising to the water surface (personal observation). Once breaking the water surface, nymphs retreat close to shore near emergent vegetation. Here they are segregated from the older nymphs and adults and typically remain there for the first few weeks (Vepsalainen and Nummelin, 1986), presumably for protection from predators, including older gerrids.
Gerrid nymphs grow through five molts in 40-60 days before becoming adults (Krupa and Sih, 1993; Spence and Andersen, 1994). Development time varies inversely with temperature (Vepsalainen, 1973; Blanckenhorn, 1991a, 1994) and food supply (Blanckenhorn,
1994). Mortality during the juvenile stages is high (up to 80%) and depends on food availability
(Blanckenhorn, 1994). When molting, nymphs crawl onto emergent substrate and shed their exuvium, including the gut lining, by crawling out head first (personal observation). The molting process is completed within 24 hours (personal observation), and within 24 hours of molting, the new exoskeleton of the nymph (or adult) is sclerotized (Arnqvist and Thornhill,
1998).
Trypanosomatid ecology in gerrids
Trypanosomatid flagellates are common in the digestive tracts of many insects (Wallace,
1979). In gerrids, trypanosomatids pass through the foregut and midgut, attach to the hindgut
8 and reproduce there by binary fission (Tieszen and Molyneux, 1989). Three genera of trypanosomatids infect water striders: Blastocrithidia, Crithidia, and Leptomonas. Mixed infections are common (Wallace et al., 1960; Wallace et al., 1965), and the first two species can coexist in A. remigis (Tieszen and Molyneux, 1989).
Gerrids infected with trypanosomatids have been reported from a variety of locations for almost a century (summary in Wallace, 1966). Although more is known about them than most insect trypanosomatids (Klingenburg et al., 1997), only four population-level studies have been reported (Gurski and Ebbert, 2003; Tieszen and Molyneux, 1989; Arnqvist and Maki, 1990;
Arnqvist, 1992).
As with most one-host, horizontally transmitted insect trypanosomatids, trypanosomatids infect gerrids when the host feeds. In the only study undertaken to elucidate trypanosomatid transmission in gerrids, Tieszen and Molyneux (1989) demonstrated that Blastocrithidia gerridis and Crithidia flexonema experimentally infect the gerrid Gerris lacustris via host water intake.
Transmission via cannibalism (Patton, 1908) and coprophagy (Porter, 1909) have been suggested, but no experimental investigation has definitively demonstrated these mechanisms in gerrids. However, both methods have been documented in other trypanosomatid hosts. The reduviid bug Triatoma infestans is the most closely related Hemipteran to gerrids for which a substantial amount of information regarding trypanosomatid infections is known. Transmission of Blastocrithidia triatomae occurs via both mechanisms in its reduviid host Triatoma infestans
(Schaub et al., 1989), and Crithidia fasciculata is transmitted from adult mosquitos (Culiseta incidens) to larvae as well as from larvae to adults via coprophagy (Clark et al., 1964).
Carvalho and Deane (1974) suggest that transmission of Blastocrithidia in another reduviid
9 host, Zelus leucogrammus, probably occurs via coprophagy and cannibalism, but present no evidence on this question.
Transmission between generations via eggs (transovarian) does not occur in Gerris lacustris and is considered an unlikely transmission mode of trypanosomatids in gerrids
(Tieszen and Molyneux, 1989). No infected gerrid embryos have been found (Porter, 1909;
Tieszen and Molyneux, 1989), nor have laboratory-inoculated eggs been able to sustain the parasite (Wallace et al., 1960). In addition, eggs from infected females do not develop into infected nymphs or adults (Tieszen and Molyneux, 1989). Under laboratory conditions, trypanosomatids harvested from gerrid fecal samples are capable of surviving in sterilized water for 48 hours, and those derived from culture can survive up to eight days (Tieszen and
Molyneux, 1989).
Gerrids and mosquitoes are the only two aquatic insects known to naturally harbor trypanosomatid parasites. The aquatic larvae of Culiseta incidens infected with Crithidia fasiculata (Clark et al., 1964) may therefore provide additional opportunities for transmission of trypanosomatids to gerrids if they feed on infected larvae or adult mosquitoes.
The life histories and developmental stages of gerrid trypanosomatids have not been clarified (Tieszen et al., 1983). Mixed infections of two or more trypanosomatid species within one host were originally identified as being different developmental stages (Wallace et al.,
1965). Tieszen et al. (1983) identified three morphs of trypanosomatids in Gerris odontogaster infected with Blastocrithidia gerridis, but could not definitively identify all three forms as B. gerridis. Further, they did not attempt to determine the life cycle of this trypanosomatid species.
10 My goal is to investigate the natural prevalence of trypanosomatid parasites of gerrid hosts and their effects on gerrid development in local streams. I address the following three questions: 1) What is the natural prevalence of trypanosomatids in gerrids within and among annual field seasons? 2) How does uninfected gerrid morphology vary spatially, temporally and with respect to mating status? 3) Does trypanosomatid infection affect morphological development of field-caught gerrids? I address these questions using Aquarius remigis gerrids collected in Butler County, Ohio streams.
11 CHAPTER 2
CHANGES IN REPRODUCTIVE LIFE HISTORY PATTERNS OF THE GERRID,
AQUARIUS REMIGIS, ALTER TRYPANOSOMATID PREVALENCE
12 Temperate, non-migrating insects with a short lifespan are faced with the problem of fitting their life cycle into a given season length. Insects must complete reproduction in order to produce a viable successive generation before winter, otherwise suffer zero fitness. Voltinism, the number of generations an insect species can complete in one season, depends on the length of the reproductive season relative to offspring development time. Univoltine populations have one generation per year, while bivoltine populations have two.
Local fluctuations in season length, and other environmental factors, such as temperature, photoperiod, moisture, and food abundance, affect the number of generations these insects complete in one season (Tauber et al., 1986; Danks, 1987). Insects must rely on these environmental cues to anticipate future environmental conditions and cease or delay reproduction in time for their offspring to reach the overwintering developmental stage (Ludwig and Rowe, 1990; Rowe and Ludwig, 1991).
Changes in voltinism patterns alter opportunities for contact between potential insect hosts and the parasites that rely on host interactive behavior for transmission. As a result, prevalence patterns of insect parasites may fluctuate with voltinism. A second mating period will produce a second generation, increasing the density of the population. An increase in mating activity and density increases contact rates among hosts and facilitates parasite transmission resulting in increased parasite prevalence.
Trypanosomatid flagellates are common in the digestive tracts of many insects, and gerrids infected with trypanosomatids have been reported from Europe and North America for almost a century (summary in Wallace, 1966, 1979). The epizooitology of relatively few monoxenous (one host) trypanosomatids have been investigated in any detail; recent exceptions in insects other than gerrids include work on trypanosomatids in bumblebees (Imhoof and
13 Schmid-Hempel, 1998), Drosophila (Ebbert et al., 2001), and southern green stink bugs (Fuxa et al., 2000).
Trypanosomatid infection rates in gerrids have been studied in three natural populations of one gerrid species, the pond-dwelling Gerris odontogaster. Gurski and Ebbert (2003) is the only report on prevalence in stream-dwelling gerrids. Aquarius remigis is the most widely distributed gerrid species in North America (Calabrese, 1974; Fairbairn, 1990). It prefers small streams in cool habitats where it feeds on insects trapped on the water’s surface. A. remigis populations can reproduce once (univoltine) or twice (bivoltine) each year depending on climate. I found high trypanosomatid prevalence (47%) during the gerrid mating period in May that significantly dropped in June (12%) and remained low throughout the summer and fall.
Two of the pond-gerrid studies indicate low early spring infection rates (0-15%) that rapidly increase during the reproductive period to 80-90% (Tieszen and Molyneux, 1989; Arnqvist and
Maki, 1990). Tieszen and Molyneux (1989) report that prevalence decreases after the spring reproductive period throughout the summer to 50-60% in October. The third study sampled three gerrid populations once in early June and found infection rates ranging from 48-98%
(Arnqvist, 1992). Additionally, Klingenberg et al. (1997) studied Gerris buenoi reared in enclosures in their natural pond habitat in Canada and found dramatically lower infection rates
(2% or less) throughout the season. This low prevalence is an exception to gerrid trypanosomatid infections.
Several explanations for this seasonal pattern in pond-gerrid prevalence have been proposed. Tieszen and Molyneux (1989) suggest that the presence of infected adult gerrids appearing from diapause indicates that the parasites overwinter with their gerrid hosts, although no studies have followed natural infection rates during gerrid diapause. The rapid increase in
14 spring could be attributed to gerrid behaviors that increase contact rates, such as mating, territoriality and prey sharing, and therefore increase opportunities for transmission by coprophagy. Transmission of trypanosomatids in gerrids is typically by feeding (Wallace, 1966,
1979); vertical transmission is rare or absent in gerrids (Tieszen and Molyneux, 1989). Gerrids are predators and scavengers, and so can consume contaminated material from the feces of other hosts, via cannibalism, or in their prey items. Tieszen and Molyneux (1989) suggest that a combination of transmission mechanisms occurs in natural gerrid populations. The low infection rates during the summer and fall could be due to the decrease in gerrid interactive behavior as the new generation of gerrids prepares for diapause (Tieszen and Molyneux, 1989).
Decreased host-to-host contact allows fewer opportunities for parasite transmission.
It is a common epidemiological assumption that increasing host density increases contact between potential hosts, and therefore produces increased prevalence (Fuxa and Tanada, 1987).
Trypanosomatid prevalence in gerrids has been explained by gerrid-gerrid contact rates, and these rates are highest during the mating season. Arnqvist (1992) sampled three populations of
Gerris odontogaster with differing population densities and found that infection rates increase with increasing population density (5.5, 8.1, 31.3 individuals per square meter of pond surface).
The proportion of mating males in these populations ranged from 31-38%, while infection rates ranged from 48-95%. Apparently, there was no relationship between the number of mating males and prevalence. Their data suggest that additional factors must be responsible for trypanosomatid transmission in the absence of high mating contact rates.
Tieszen and Molyneux (1989) found no significant difference between average infection rates of males and females (54 vs. 53%) in Gerris odontogaster collected over three years.
Infection rates were lower in nymphs, as 24-25% of third-fifth instars and 3% of first-second
15 instars were infected. The authors attributed these prevalence differences to smaller sample sizes for the nymphs (n=123) than for the adults (n=551), which could bias prevalence estimates.
I found no difference in prevalence between male and female Aquarius remigis (Gurski and Ebbert, 2003). I did find that adults were more likely to be infected than nymphs and attributed this difference not to sample size, but to behavioral differences between the two age groups. Nymphs typically segregate themselves from adults by remaining close to stream edges, while adults are often found foraging in the more open areas of the stream.
In this study I test the following hypotheses.
Hypothesis
Hypothesis: Prevalence varies with host age distribution; therefore temporal factors are important for trypanosomatid transmission because contact among gerrids is largely a function of their age. The age distribution of gerrids varies during and among annual field seasons, but is similar in all populations within a watershed in a given month. Additionally, mating activities bring male and female gerrids into close and frequent contact while nymphs segregate themselves from the adult population.
If prevalence varies temporally, then I predict that prevalence will not differ among streams and locations within streams, but will differ among months and among annual field seasons. Additionally, I predict that prevalence will be lower in nymphs than in adults because contact among nymphs, and between nymphs and adults, is limited. However, I predict prevalence will not differ between males and females because both sexes are engaged in mating activity.
16 METHODS
General
Study sites
I collected gerrids in the Four Mile Creek watershed near the cities of Oxford and
Darrtown in Butler County, Ohio. During the study, I made collections among nine locations within four streams. Eight of the sites were collection sites for the prevalence study, whereas one site was used to obtain gerrids for rearing and experimental purposes. These streams were small, shallow, 1st or 2nd order streams (stream classification methods in Strahler, 1952) that flowed throughout the summer except in dry years when a few dried up into pools.
Capture
I randomly caught gerrids using a dip net. To prevent transmission of trypanosomatids among individuals, I isolated each gerrid in a marked vial plugged with a cotton ball soaked in stream water (to prevent dehydration) immediately after capture. I stored the live insects for at least nine days at 8°C until processing. Nine days is the minimum amount of time required to detect trypanosomatids in a gerrid hind-gut smear once the parasites are ingested (Tieszen and
Molyneux, 1989).
Host to host encounter rate is a direct measure of risk of trypanosomatid infection, and therefore, I chose to estimate encounter rate with gerrid density. I calculated a relative density estimate for each of the gerrid age groups within each population by calculating catch per unit effort (CPUE). I caught gerrids randomly with respect to age. To calculate catch per unit effort,
I divided the total time spent collecting at a site into both the number of adults and the number
17 of nymphs. I used these catch per unit effort values to compare relative gerrid density by age and by site.
Laboratory assessment
I sexed each adult gerrid and estimated the age of nymphs using keys from Calabrese
(1974). I assayed for trypanosomatid infection using either or both of two methods. A nondestructive technique was used occasionally to obtain live infected bugs for culturing purposes. Each gerrid was made to exude a fecal sample onto a microscope slide by exposing it to a steady stream of carbon dioxide for 5-10 seconds. For all other samples, I dissected gerrids by first removing the head, legs and wings (if present), then making lateral incisions along the abdomen, removing the dorsum, and exposing the digestive tract. The gut was removed and smeared in a drop of phosphate buffered saline on a slide. Smears were allowed to dry before fixation with methanol and Giemsa staining (Lacey, 1997). Each slide was examined until trypanosomatids were identified or the entire smear was viewed. Representative trypanosomatid specimens will be placed in the U.S. National Parasite Collection at the
Beltsville Agricultural Research Center in Beltsville, MD. Gerrid specimens will be placed in the Miami University, Department of Zoology insect collection, and I will store remaining trypanosomatid specimens in my personal collection.
Recognizing infection
Each slide was examined under a light microscope at 200X. The trypanosomatid body shape and the presence of two dark staining bodies (the nucleus and kinetoplast) within the cell are distinctive. Trypanosomatid infections were usually heavy (more than 20 individuals per field) which facilitates identification among gut or fecal contents.
18 I observed two morphotypes in these collections: some trypanosomatids were relatively short with thick bodies while others appeared much longer and thinner in shape. Both morphotypes were observed within a single individual, and the two forms most likely represent infection by multiple species of trypanosomatids (Wallace et al., 1960; Wallace et al., 1965).
Since morphological identification of trypanosomatids is unreliable (Wallace, 1966, 1979;
Fernandes et al., 1997; Teixeira et al., 1997; Hollar et al., 1998; Philippe, 1998; Momen, 2001;
Podlipaev, 2001; Momen, 2002), I used the family Trypanosomatidae as the "recognizable taxonomic unit" in this study.
Comparison of assay
Although I made smears of gerrids that died after collection, I excluded these data from my analysis. Intestinal smears of dead gerrids lacked distinguishable gut characteristics and appeared as a clouded smear of bacteria. Trypanosomatid cells initially in the guts of these gerrids could have been decomposed by bacteria and rendered undetectable in the smears. I therefore considered these smears unreliable.
I used two methods to sample infection: fecal and gut smears. Both methods were considered reliable (Gurski, 2000) and gerrids were scored as infected if either one of the assay methods yielded a positive result. Permanent slides were not made for each gerrid caught (due to gerrid death before processing), nor were all gerrids for which slides were made used in the prevalence analyses (due to smears made from dead gerrids and lack of fecal smear) (Table 1).
Prevalence Study
I surveyed eight sites in 2000 and 2001 (September and October only) and four sites (a subset of the eight) in 2002. Sites surveyed in these years were the same locations sampled in
1999 (Gurski and Ebbert, 2003). Paired sites on each of four tributaries of Four Mile Creek
19 which included Harker’s Run, Collin’s Run, Coulter’s Run, and Darr’s Run (Figure 2) were surveyed in 2000 and 2001. In 2002 the only paired site was in Harker’s Run while the remaining two sites were downstream locations in Collin’s and Darr’s Runs. I also sampled a fifth site in July in Harker’s Run at a location between the upstream and downstream sites. I chose this distribution of sites to determine patterns in prevalence among the infected gerrid populations in the watershed and as an extension to a 1999 study (Gurski and Ebbert, 2003).
I sampled gerrids once per month at each of the sites from March-October (except
August). Depending on the relative abundance of gerrids at each site, I attempted to collect 50 adults and 50 nymphs at each location. I collected only third-fifth instar nymphs as younger nymphs are difficult to find and handle due to their small size. The number collected at each site was constrained by the amount of time required to process the gerrids in the lab and sample sizes needed for statistical analyses.
I chose a sample size of 50 adult gerrids per site per collection date based on the following considerations. First, I planned to compensate for the possibility that I would not find gerrids at each time and place of collection. I therefore chose to collect at seven times (March,
April, May, June, July, September, and October), in four streams, each with a pair of upstream- downstream sites, for a total of 56 collections. Second, I calculated the sample size I would need assuming I could process (make and view) 500 slides among collections. Preliminary data suggested prevalence varied between 10 and 20%. To distinguish between these two values, I needed a sample size of 50. Given this number, the binomial error on an estimate of 10% is
4.2%, and on an estimate of 20% is 5.7%. A sample size of 20 would give overlapping errors
(10 ± 6.7% and 20 ± 8.9%), and a sample size of 80 would only marginally improve my estimate (giving errors on 10% of ± 3.4% and on 20% of ± 4.5%). I expected to collect fewer
20 nymphs than adults, so for planning purposes, I chose a goal of 30 nymphs. Based on these calculations, if I were able to collect 50 adult and 30 immature gerrids at each site in seven months, I would have 640 slides to process each month, for a total of 4480.
Experimental design
I organized data according to the presence or absence of trypanosomatid infection
(dependent variable) in relation to factors potentially related to prevalence (Table 2).
My experimental design for this dissertation was modified from my previous design, which I summarize briefly here. At the start of the study (1999), I considered the following possibilities for the spatial structure of prevalence.
a. Infection could be present or absent depending on stream.
b. Infection could be present at all streams, but the streams could differ in prevalence.
c. Depending on the site within a stream, infection could be present or absent.
d. Infection could be present at both sites, but the locations could differ in prevalence.
e. Infection could be explained by distance along a streambed.
f. Infection could be explained by distance across land among streams.
I found option (b) to be consistent with my data. Based on these data and a mark-recapture study, I concluded that gerrids rarely move among streams within a season (Gurski and Ebbert,
2003). Based on available literature, I assumed that gerrids are the primary source of trypanosomatid inoculum at our sites.
Among years, gerrids could move among streams, other sources of inoculum could become available, and local conditions (predators, water levels, etc.) could interact with
21 infection rates. I assumed that prevalence would vary significantly, and with factors I did not plan to measure, across years. I therefore planned to compare across years qualitatively.
My experimental design for the current study is based on the idea that the streams provided four opportunities per year to document whether and how a given starting prevalence changed over a season. I expected that contact rates could vary depending on stage, sex, and density of the population at each site. I expected all these factors could vary from month to month. The main goals of my study concerned these possible changes in prevalence over time.
My goals are:
1. To test whether prevalence changed within a stream over the course of the season.
2. To test whether, within a month, prevalence varied with factors that could affect contact
rates: gerrid stage, sex or density of the population.
3. To suggest whether any changes over time in prevalence might be due to parallel changes
in potential contact rates between the sexes, stages or with population density.
My data allowed me to consider two preliminary hypotheses concerning the fitness of infected individuals. I reasoned that hosts may differ from uninfected gerrids in being more or less likely to survive the winter or to mate. I used post-hoc tests to determine whether my data supported a hypothesis that prevalence was correlated with either of these events (mating or surviving the winter).
Density is potentially an important factor in prevalence. I therefore planned an Analysis of Variance (ANOVA) to test for the effect of the factors (Table 2) on density with the null expectation that none would have a significant effect on density.
22 Statistical analyses
I conducted statistical analyses by year. I used a log linear model to test whether prevalence varied with stream or month nested within stream (Sokal and Rohlf, 1981). This analysis was reported on earlier for 1999 (Gurski and Ebbert, 2003), and in this dissertation for
2000, 2001 and 2002. I found similar patterns in prevalence across the streams in 1999, 2000 and 2001 and so dropped one stream from the design in 2002.
For the remaining categorical factors (stage, location and sex), I used several statistical approaches. Because very few nymphs were infected, including stage in the log linear model made it difficult to interpret, as many of the stream by month counts for infected nymphs were zero. I therefore used non-parametric paired sign tests to test for differences in prevalence related to gerrid stage. Paired sign tests reduce data for a given month and stream to a single observation recorded with one of three “signs”: zero, where prevalence in nymphs and adults was equal, positive (prevalence in nymphs is higher than in adults), or negative. The observations coded zero are then ignored, and the count of positive and negative observations compared (using a G-test with one degree of freedom) to the expectation that equal numbers of observations will fall into each category. Because prevalence in adults was consistently much higher than in nymphs, I analyzed prevalence patterns in nymphs and adults separately. I also used sign tests in the unplanned comparisons of mating status and overwintering adults.
My analysis of the 1999 data included location (as a factor nested within month and stream) and sex (nested within stream, month and location) as factors in the log linear model.
Neither was a significant factor in either this model or in sign tests. Location and sex are analyzed here as the paired factor in a non-parametric sign test. In 2000 and 2001, I continued
23 to include location in our experimental design, but in 2002 I dropped the up/down collections in all but one stream.
I analyzed density using two methods. First, I used a graphical approach to explore whether density and prevalence were correlated and tested for correlations between the two variables. I included the 1999 data in this analysis. Second, I used ANOVA to test whether variation in density could be explained by stream, month (within stream), stage (within stream and month) and location (within stream and month). As is standard for analyses of counts, I first log-transformed the data. In 1999, I used this same approach and concluded that density varied with month and stage, but not stream or location.
I used JMP (Ver. 4.0.2, SAS® Institute, Inc., 2002) for all statistical analyses and considered p-values ≤ 0.05 significant.
RESULTS
General observations
Between 1999 and 2002 I collected 7633 gerrids on 43 dates (Table 3). The 2000 field season was characterized by average rainfall (Figure 3), and streams were flowing (as opposed to drying up) throughout the field season. Above-normal precipitation fell in the 2001 field season, and streams were flowing for collections in both September and October. The first half of the 2002 season was characterized by above-average precipitation. Below-normal rainfall fell from June through September until increasing to above-normal levels again in October.
Spring and early summer stream levels were high for this season, but the streams dried up into pools by September. At this time in Darr’s Run, I collected estivating gerrids from a dry portion
24 of the streambed. I found gerrids hibernating from the hot and dry conditions in moist areas under streambed rocks.
For comparison, during the 1999 season, precipitation was below normal for most months during the field season and conditions were very dry (Figures 3, 4). As a result of the dry conditions, by June Harker’s lower and, and Collin’s upper site had dried into pools connected only by a trickle of water. By July, both Harker’s sites, Coulter’s lower, and Collin’s upper sites were isolated pools.
Population age structure, density, and life history patterns
To ensure an adequate data set, my goal was to collect 50 adults and 30 nymphs at each collection site. In 2000 I met this goal for adults in the summer and fall, but in the spring adult density was low. For nymphs, I met this goal in all months nymphs were present except July and October. In 2001, I collected fewer adult gerrids in October and hardly any nymphs in this shortened field season. In 2002 I met my goal for adults in all months but May and June. For nymphs, I met the collection goal two of the three months they were present in the populations
(Table 4).
The age structure of the gerrid population changed throughout each collection period in each collection year (Table 4). In all years, the post-diapause adults reproduced in early spring.
These adults were in low abundance and sometimes difficult to locate (see Tables 4 and 5 for average sample size and density estimates). After reproducing, these adults (the parental, or P1 generation) died by late spring or early summer. The time that nymphs began to hatch varied by year. In 1999 (Gurski and Ebbert, 2003) and 2000, nymphs were present in the streams by May, but in 2002 nymphs were not seen until June. By July, these first generation (G1) nymphs had molted into adults or died. In 1999 (Gurski and Ebbert, 2003) and 2002, nymph density
25 remained low for the remainder of the year. Adult density in 1999 remained high, but in the summer of 2002 adult density declined, presumably due to a severe drought. In contrast, in
2000 a second generation of nymphs (G2) peaked in density in September. Adult density at this period in time was also high.
Gerrid density was estimated by catch per unit effort for each collection. Although these estimates reflect relative gerrid density, they also reflect the stream contraction and expansion influenced by precipitation and temperature. For example, when conditions were dry, streams tended to contract into pools thus decreasing stream surface area and crowding the gerrids. As a result, measuring gerrid density as catch per unit effort may confound any relationship between density and other variables. These estimates (Table 3, 5) suggest that gerrid density varied by both month and gerrid age but not by stream or location within stream for each year. I separated density values for adult and nymph gerrids and tested for the effect of year, stream, month nested within stream and location nested within stream and month on each age class. Among nymphs, results suggest that density differed between year and month, but not between stream and stream location (Table 6, Figure 5). Adult gerrid density varied significantly among months but not between any other factors (Table 6, Figure 6).
I plotted trypanosomatid prevalence in adults against four density estimates: adult and nymph density for the same month and adult and nymph density for the previous month (Figure
7). I detected no relationship between prevalence in adults and density.
The stage structure also differed among years. All years were characterized with a peak in mating activity in the overwintered adults in the spring (Figure 8). In 1999 (Gurski and
Ebbert, 2003) and 2002 this was the only significant mating period while in 2000 a second peak in mating activity was detected in July. I used a log linear model to test for the effect of stream
26 and month on gerrid mating status. The model was significant (d.f.=27, X2=490.3, p<0.0001) with a significant effect of month (d.f.=24, X2=455.8, p<0.0001) but not stream (d.f.=3,
X2=0.0016, p=1.00). I tested the average percent gerrids mating across streams, and mating was significantly different among months March through June (d.f.=3, X2=138.7, p<0.0001). The model was no longer significant when June was removed from the analysis (d.f.=2, X2=1.22, p=0.5432) indicating that spring mating activity drop by June. The 2000 July peak in mating activity was significantly higher than mating activity in June (d.f.=1, X2=25.68, p<0.0001) and
September (d.f.=1, X2=67.54, p<0.0001).
First generation gerrids were mating during the July peak, and they produced a second generation. Evidence of a second generation was seen in the increase in nymphs in the following month and subsequently an increase in adult density (Figures 5, 6). These trends in mating activity and nymph density indicate that two life history patterns are present at my sites: in 1999 and 2002, the populations were univoltine, whereas in 2000 they showed a bivoltine pattern (Figure 8).
Spatial analysis of prevalence
I analyzed 55 pairs of upstream and downstream observations with respect to prevalence data from four field seasons. These were all the collections in which (a) adults were present in both upstream and downstream locations, and (b) infected and uninfected adults were present.
For each field season I compared prevalence between upstream and downstream locations with a paired sign test (Table 7). Results indicate that for all four years studied, location is not a significant factor in explaining prevalence of trypanosomatids in gerrids. Previously, Gurski
(2000) showed that neither land nor stream distance among streams is correlated with gerrid prevalence.
27 Mating status
I analyzed 46 pairs of mating and non-mating observations with respect to prevalence data from three field seasons. These were all the collections in which (a) mating and non- mating adults were present, and (b) infected and uninfected adults were present. I analyzed each year separately and combined for an effect of trypanosomatid prevalence on mating status. In all three years in which mating gerrids were collected, mating gerrids were no more likely to be infected than non-mating gerrids (Table 8).
Host sex
I analyzed 115 pairs of male and female observations with respect to prevalence data from four field seasons. These were all the collections in which (a) male and female adults were present, and (b) infected and uninfected adults were present. I analyzed each year separately and combined for an effect of trypanosomatid prevalence on host sex. Host sex was not a significant factor in explaining trypanosomatid prevalence in all years except 2002 (Table 9) when males were more likely to be infected than females (male: 16.7 ± 1.7%; female: 7.6 ±
1.3%).
Overwintering effect on prevalence
I analyzed eight pairs of fall and spring observations with respect to prevalence data from the 1999-2000 overwintering period. I pooled the collections from September and October
(fall) and from March and April (spring) by stream and compared the difference in prevalence among these seasons for each sex. I analyzed six pairs of prevalence data from 2001-2002 overwintering period. I pooled the collections from September and October by stream and compared the difference in prevalence to March for each sex. I did not include April 2002 collections because mating in this year was high compared to the same month in 2000. All of
28 these collections contained (a) male and female gerrids, and (b) infected and uninfected adults.
I analyzed the difference in trypanosomatid prevalence of adults before and after winter diapause for two overwintering periods (Table 10). Results suggest that prevalence significantly increased from fall 1999 to spring 2000 but significantly decreased between the 2001-2002 field seasons.
Host stage
I analyzed 65 pairs of adult and nymph observations with respect to prevalence data from four field collections. These were all the collections in which (a) adults and nymph gerrids were present, and (b) infected and uninfected gerrids were present. I analyzed each season separately and combined for the effect of trypanosomatid infection on gerrid stage. In all field seasons except 2002, adults were more likely to be infected than nymphs (Table 11). In 2002, results suggest that there was no difference in trypanosomatid prevalence between gerrid stages. In this year only five collections contained both adults and nymphs, and three of these collections were made in July when trypanosomatid prevalence in adult gerrids was very low (Tables 12, 13, 14).
Because overall, adult gerrids are more likely to be infected than nymphs, I chose to analyze trypanosomatid prevalence patterns for each stage separately.
Prevalence in nymphs
I collected 1373 nymphs from 2000 through 2002 and assayed 980 for trypanosomatid prevalence. 120 nymphs were infected for a total prevalence of 12.2 ± 1.0% across streams and these three years. Gurski and Ebbert (2003) collected 558 nymphs in 1999 and assayed 325 for trypanosomatid infection. Only three were infected for a total prevalence across streams and months of 0.9 ± 0.5%. For the entire collection, 1999 through 2002, I collected a total of 1931
29 nymphs and assayed 1305 for trypanosomatid infection; of these, 123 were infected for a total prevalence across all streams and years of 9.4 ± 0.8% (Table 13).
As in 1999 (Gurski and Ebbert, 2003), prevalence among nymphs was very low in 2001 and 2002. In 2002, I assayed only seven nymphs (all in September) and only one (from
Coulter’s Run) was infected (Table 13) for a total prevalence of 14.3 ± 13.2%. These data were similar to 1999, when only one infected nymph was collected in the fall. The similarity of 2001 to both 1999 and 2002 with respect to prevalence in nynphs could indicate that the population was primarily univoltine in 2001. I assayed a total of 244 nymphs in 2002, but very few were infected (n=6). All of these infected nymphs were collected in July and in Darr’s and Collin’s
Run for a total prevalence of 2.5 ± 1.0%. Because trypanosomatid prevalence was low in 2001 and 2002 and infected nymphs were only detected in a single season for each year, I did not analyze these data further.
Trypanosomatid prevalence in nymphs increased significantly throughout the 2000 field season (Table 14, Figure 9). Of the 729 nymphs assayed in 2000, 113 were infected with an overall of 15.5 ± 1.3%. The data set was not sufficient to test for stream, so I pooled all streams and used a log linear model to test for the effect of month on trypanosomatid prevalence: month had a significant effect (d.f.=4, X2=29.84, p<0.0001)(Table 15). Trypanosomatid prevalence was not different within summer months (comparison between June and July, d.f.=1, X2=0.09, p=0.77) or within the fall months (comparison between September and October, d.f.=1, X2=0.09, p=0.76). Prevalence did significantly differ among the spring (May), summer and fall seasons
(d.f.=2, X2=29.67, p<0.0001). Prevalence increased significantly from one season to the next within the field season. Similarly, Gurski and Ebbert (2003) found that trypanosomatid
30 prevalence in nymphs in 1999 was higher in the fall than in the spring and summer months, but these results were based on a small sample size (n=3) in the fall.
Prevalence in adults
I collected 3870 adults from 2000 through 2002 and assayed 3576 for trypanosomatid prevalence. 914 adults were infected for a total prevalence of 25.62 ± 0.7% across streams and these three years. Gurski and Ebbert (2003) collected 1832 adults in 1999 and assayed 1755 for trypanosomatid infection. 288 were infected for a total prevalence across streams and months of
16.1 ± 0.9%. For the entire collection, 1999 through 2002, I collected a total of 5702 adults and assayed 5531 for trypanosomatid infection. 1002 adults were infected for a total prevalence across all streams and years of 21.7 ± 0.6% (Table 12).
Prevalence in 2000 and 2001 were similar: 29.4 ± 1.0% (623 of 2120 assayed) in 2000, and 31.8 ± 2.0% (176 of 553 assayed) in 2001. 2002 prevalence was lower at 12.7 ± 1.1% (115 of 903 assayed) (Table 12).
I analyzed the prevalence data for adult gerrids for each year separately using a nested log linear model of stream and month nested within stream. I chose to exclude the factors of stream location and gerrid sex from all models because the paired sign tests indicated that neither of these factors was important in explaining prevalence either per year or pooled.
Adults 2000
The model provided a good fit to the 2000 data (d.f.=27, X2=206.6, p<0.0001) and both stream and month had a significant effect (Table 15). I examined the pattern of each stream over time in 2000 (Figure 10). I concluded that, although stream was a significant factor in explaining trypanosomatid prevalence, no one stream exhibited consistently higher or lower prevalence than another.
31 I pooled all streams in 2000 and tested for the effect of month on trypanosomatid prevalence. Month was no longer a significant factor when May and July were removed from the analysis (d.f.=4, X2=4.81, p=0.3072). The peaks in prevalence in May and June were significantly different from each other (d.f.=1, X2=17.79, p<0.0001). These results suggest that overall trypanosomatid prevalence peaked twice in 2000 (Figure 10).
Within each stream, month had a significant effect on prevalence with a sharp peak in
May and a smaller peak in July being the clearest patterns (Figure 10). In Harker’s Run, month was no longer a significant factor once July was removed from the analysis (d.f.=5, X2=8.46, p=0.1329) suggesting that there was a significant increase in prevalence in July. In Darr’s Run, prevalence significantly increased in May but prevalence remained high through July. Month was no longer a significant factor when the months of May, June and July were removed from the analysis (d.f.=3, X2=2.57, p=0.46) and these same months were not significantly different from each other (d.f.=2, X2=5.71, p=0.0575). For Coulter’s Run, prevalence increased in May and remained high for the remainder of the field season. Trypanosomatid prevalence was similar between March and April (d.f.=1, X2=1.19, p=0.27) and among all remaining months
(d.f.=4, X2=5.85, p=0.21), but was significantly different between early spring (March and
April) and all later months (d.f.=1, X2=24.03, p<0.0001).
The effect of month in Collin’s Run was more complex. Trypanosomatid prevalence appeared to peak in both May and July, but the effect of month was remained significant when these two months were removed from the analysis (d.f.=4, X2=24.51, p<0.0001). I divided the field season into sections. Month was a significant factor when I grouped March, April, May and June (d.f.=4, X2=38.05, p<0.0001). There was a significant effect of month between May and June (d.f.=1, X2=35.91, p<0.0001) and among March, April and May (d.f.=2, X2=13.74,
32 p=0.0010). Month was no longer significant when May was removed from the latter group
(d.f.=1, X2=0.57, p=0.45) indicating that the May peak in prevalence was significant. Month was a significant factor among June, July, September and October (d.f.=3, X2=14.11, p=0.0028), but month was no longer significant when July was removed from the analysis (d.f.=2, X2=1.28, p=0.5271) indicating that the July peak in trypanosomatid prevalence was significant. Thus, prevalence peaks in May and July were significant.
Adults 2001
In 2001, Collins Run was excluded from the analysis of prevalence in adults because I lacked October data for this stream. The model did not provide a good fit to the data (d.f.=5,
X2=8.57, p=0.1276), suggesting that prevalence did not differ with either factor in these two months (Figure 11).
Adults 2002
In 2002 the factor of stream was excluded from the model because I lacked data from each stream in each month. Month was a significant factor in the model (d.f.=6, X2=80.31, p<0.0001) with a June peak in prevalence being the clearest pattern. Month was no longer a significant factor only when both June and July were removed from the analysis (d.f.=4,
X2=3.30, p=0.5086). Trypanosomatid prevalence was significantly different in June and July
(d.f.=1, X2=76.93, p<0.0001). Month was a significant factor in July, September and October
(d.f.=2, X2=11.56, p=0.0031), but was no longer significant when July was removed from the analysis (d.f.=1, X2=0.13, p=0.72. These results suggest the prevalence in July is significantly lower than in all other months. Month was also a significant factor among March, April, May and June (d.f.=3, X2=37.12, p<0.0001), but was not longer significant when June was removed
33 from the analysis (d.f.=2, X2=76.93, p=0.56). These results suggest that prevalence in June was significantly higher than in all other months (Figure 12).
Re-analysis of 1999 data
In the 1999 field season I collected gerrids on two dates in April for a pilot study on gerrid rearing. I did not include these data in the prevalence analyses in 1999 because data came from two separate collections dates, from one location in one stream, and samples were small. I decided to include the April data in a re-analysis of 1999 data to obtain a more complete pattern for 1999 spring prevalence, and more easily compare this year to other field seasons. I compared prevalence and sex ratios between two collections made in April 1999.
Results suggest that neither prevalence nor numbers of males and females varied between the two collections (prevalence, d.f.=1, X2=1.849, p>0.17; sex ratio, d.f.=1, X2=0.541, p>0.46). As a result, I pooled the two collections and considered them as one collection in Harker’s Run for all subsequent analyses.
34 For the year 1999 Gurski and Ebbert (2003) report that month, but not stream, had a significant effect on trypanosomatid prevalence. I reanalyzed the 1999 prevalence data with a log-linear model of stream and month nested within stream only, and results suggests that both stream and month were significant factors (d.f.=20, X2=208.2, p<0.0001) (Table 15). As in the
2000 field season, although stream was a significant factor in explaining prevalence, no one stream exhibited consistently higher or lower prevalence than another throughout the season
(Figure 13). Within each stream, month was a significant factor with a significant peak in trypanosomatid prevalence in May (Gurski and Ebbert, 2003).
DISCUSSION
My prevalence patterns results show clear and consistent trends across four years and four streams: a spring peak and, typically, a decline through the rest of the collecting season.
Furthermore, the interaction persisted within all my sites. This consistency in the presence and pattern of infection allows me to consider explanations for trypanosomatid prevalence that can be valid over many gerrid populations.
Prevalence is the product of several forces within a population: host-parasite contact rates, establishment rates (how frequently a host becomes infected upon contact with a parasite), and duration of infection (the time an infection persists) (summarized in Fuxa and Tanada,
1987). Based on available literature, I make the following assumptions in my dissertation about these forces. First, I assume gerrids are infected for life, and therefore duration is not relevant in this study. Tieszen and Molyneux (1989) report that infected adult gerrids maintained at 4°C for 150 days retained their infections. Second, I assume that establishment rates are high (or at least similar between gerrid stages and seasons), and therefore changes in prevalence are likely
35 due to changes in host-parasite contact rates (Figure 14). Third, I assume there is no vertical transmission (Tieszen and Molyneux, 1989). Therefore, nymphs must acquire the infection from contact with infected individuals.
My results suggest I can assume three additional factors are of minimal importance in explaining prevalence. Gerrid migration among and within streams was minimal (Gurski,
2000), and I therefore assume it is not a major factor in explaining prevalence patterns within a year. Gerrids (winged or wingless) rarely move distances greater than 100 meters along the water surface (Fairbairn, 1986). Additionally, even though winged gerrids were found in this study, I assume that dispersal by flight was minimal given that the expected frequency of dispersal by flight per Aquarius remigis per generation is low (3 in 10,000 individuals;
Fairbairn, 1986). The possibility that any one individual is infected, winged, and disperses by flight is therefore low.
Prevalence patterns were similar across sites. Prevalence was not correlated with either distance among streams (across land or water) (Gurski and Ebbert, 2003) or location within streams (up- or downstream) (Gurski and Ebbert, 2003; this study). Thus, there is no evidence of a geographical pattern in the prevalence data. Sites close together were not more similar than sites further apart. This suggests that processes determining trypanosomatid infection rates are similar across all streams in my study. Finally, I ignore host sex because prevalence was not correlated with gerrid sex (Gurski and Ebbert, 2003; this study)
Prevalence and gerrid life history
I argue here that the similarities in prevalence patterns detected across streams and years from spring to fall are largely the result of patterns in gerrid life history.
Spring prevalence
36 The low density of adult gerrids in the spring (March through May) of 2000 was similar to that observed by Gurski and Ebbert (2003) and was probably due to high mortality during the winter. Winter mortality was estimated for two separate populations of Aquarius remigis at 60-
90% in Pennsylvania (Firko, 1986) and at 60-95% in Quebec (Fairbairn, 1985, 1986). Only unmated adults survive diapause; once these adults return to the streams, they mate and oviposit for two to three months. Mating entails an increase in predation risk for gerrids, especially females (Arnqvist, 1989; Rowe, 1992; Fairbairn, 1993,) and may contribute to the low spring adult density.
Average adult density in the spring of 2002 was more than twice as high as that observed in 1999 and 2000. It is possible that the above average winter temperatures in the winter of
2001-2002 decreased the overwintering mortality of gerrids. I observed changes in the structure of the streams both during and among field seasons that may account for these differences. For example, among months, stream volume fluctuated with precipitation and temperature altering stream flow and surface area. Over the winter, the collapse of trees and movement of sediment interrupted water flow and created new pools within the streams. Such changes in stream structure could alter the amount of available habitat for gerrid populations resulting in more or less space for gerrids in a given point in time. Aquarius remigis prefer shallow stream areas, and it is possible that gerrids emerging from diapause in 2002 had less suitable habitat available to them, therefore crowding them.
Gerrid nymphs appear late in the spring, and this generation is the result of the spring mating activities of the overwintered adults. Development time varies inversely with temperature and ranges from 40-65 days with the longest stages being at the egg and fourth and
37 fifth instar (Spence et al, 1980). Thus, the population size rapidly increases during the spring reproductive period with nymphs dominating the population age structure.
In the spring the density of adult gerrids was low, yet the prevalence of trypanosomatids was at a peak. In 2002 adult density was high for most of the spring, but when trypanosomatid prevalence peaked in June, adult density had decreased. Post-diapause gerrids can harbor heavy infections of trypanosomatids (Tiezsen and Molyneux, 1989; personal observation). Heavy infections can increase trypanosomatid contact in two ways. First, increased shedding of infective cells with fecal material can increase the chance of another gerrid ingesting trypanosomatids, and second, heavy infections can increase opportunities for contact by cannibalism of dead or dying overwintered adults (Tiezsen and Molyneux, 1989).
Contact rates among reproductive gerrids in the spring are high; they mate up to 12 hours at a time (although most matings are brief, lasting 15-60 seconds) and males encounter an average of 12 females an hour (Blanckenhorn et al., 1998). Mating encounters are very active and provide frequent opportunities for the transmission of trypanosomatids (Tieszen and
Molyneux, 1989). Males actively search for females and upon finding a mate will chase, leap, and grasp for her. Females in return resist attempted matings and flee. If the male is successful a struggle between the two ensues and the female will roll, somersault, and flip in an attempt to detach the male from her back (Rowe et al., 1994).
Mating individuals were not more likely to be infected than non-mating individuals. I conclude from this that trypanosomatid infection does not interfere with gerrid mating ability.
In contrast, Arnqvist and Maki (1990) report that male skating endurance, and therefore ability to pursue females, is negatively affected by heavier trypanosomatid infections. The lack of correlation between infection and mating status in this study does not exclude mating behavior
38 as a possible facilitator of transmission since infection status before and after mating encounters was not examined.
Unlike the adult gerrids, nymph density peaked in the spring, but trypanosomatid prevalence was very low, or absent in nymphs. Since vertical transmission does not occur in gerrids, first generation nymphs must initially obtain the infection from contact with adults.
Contact between nymphs and adults in the spring, and thus opportunities for trypanosomatid transmission, are low for several reasons. First, nymphs outnumber adults, and are therefore more likely to encounter each other than other adults. Second, first through third instars segregate themselves from adults by remaining close to shore in the vegetated areas (Nummelin et al., 1984). Third, adult gerrids invest most of their time in mating activities, thus reducing adult-nymph competition during foraging. In addition, because the post-diapause adults are older, they have had more time in which to encounter trypanosomatids and become infected.
Summer and fall prevalence in univoltine populations
Summer and fall prevalence patterns in adult and nymphs gerrids differed among the three full field seasons studied. The patterns in 1999 and 2002 (univoltine years) were similar.
Prevalence in adults peaked in early spring and was followed by a significant decline.
Prevalence remained low throughout the summer into fall. In contrast, 2000 was a bivoltine year, and prevalence peaked twice throughout the field season. I argue that variation in environmental conditions among the field seasons caused the life history variation in these gerrid populations. Conditions were dry during the summers of 1999 and 2002 and not favorable (low food and stream volume) to sustain a second generation of gerrids, thus the majority of the G1 generation delayed reproduction. Rainfall averages were close to normal in
2000 and food and available habitat were plentiful enough to sustain a G2 generation. Both
39 univoltine and bivoltine life cycles can exist in a single population of Aquarius remigis
(Blanckenhorn, 1994). Populations, such as those in this study, that are located in transitions areas between distinct uni- and bivoltine populations (Blanckenhorn, 1994) can demonstrate flexible life history patterns.
As spring progressed into summer (June and July), the population age structure became increasingly adult biased in all years. Although post-diapause adults died by June, they were replaced by large numbers of first generation nymphs, most of which had molted into adults. In
1999 and 2002 the first generation adults delayed reproduction and did not produce a second gerrid generation that same year. This life history pattern is characteristic of a univoltine population.
Although gerrid density was high during the summer (nymphs are molting into adults), trypanosomatid prevalence significantly decreased in June (July in year 2002). Much less contact among gerrids occurs in the summer as compared to spring (Arnqvist and Maki, 1990).
Gerrids are mating far less frequently and food is still plentiful enough that prey sharing and aggression due to prey defense are not frequent. New trypanosomatid infections may be lighter than in the post-diapause gerrids. The combination of lighter infections and decreased contact rates among adults could account for the decline in prevalence even when gerrid density was significantly higher.
Despite the low summer prevalence in these years trypanosomatids persisted within all streams. As the summer progressed into fall, prevalence was steady among adults, but rose significantly among nymphs in 1999. During this period in 1999, density and population age structure changed little compared to that in summer. In contrast, after July 2002, drought
40 caused a significant decrease in both the adult and nymph populations and trypanosomatid prevalence could only be detected in one gerrid population in one stream.
Although adult-to-adult contact via mating remains low as compared to spring (I observed 128 mating pairs in the spring and only eight mating pairs in the fall for 1999 and
2002 combined), contact during foraging may increase in the fall. Since gerrids are more food limited later in the summer (Preziosi and Fairbairn, 1992), interactions around food occur more frequently. First, multiple gerrids are more likely to attack a single prey item, and second, gerrids become more aggressive in defending productive territories (Blanckenhorn and Perner,
1996; Blanckenhorn et al. 1998). These behaviors may facilitate transmission during late summer and fall and allow the infection to persist when contact via mating activity is low or absent.
Towards the end of the summer there is also a tendency for those gerrids that will be entering diapause to increase foraging activity, presumably to increase energy reserves for winter (Blanckenhorn and Perner, 1996). An increase in foraging activity could lead to more chances to interact around food resources and increase trypanosomatid transmission. While the opportunities for contact among adults may increase in the fall, the data do not support a corresponding increase in prevalence.
Contact rates between nymphs and adults might also increase later in the summer and fall. Food is more limited and competition for prey may increase. This increased contact may facilitate trypanosomatid transmission both from adult to nymph and among nymphs. In addition, contact among gerrids may have been enhanced by decreasing stream volume, as little rain fell during either of these summer and fall the collection periods.
41 Summer and fall prevalence pattern in a bivoltine population
Mating activity patterns differed among years 1999, 2002 and 2000. Mating activity peaked in the spring for all years as overwintered adults reproduced at this time. The difference occurred later in the summer in when a second peak in mating activity was detected in 2000 but not in 1999 and 2002. The presence of two distinct mating periods in gerrid populations indicates that the population is bivoltine. First generation adults are directly reproducing rather than delaying reproduction until the following spring. Evidence of the presence of a second mating period in 2000 was also seen in the rise in nymph density in the fall.
Trypanosomatid prevalence in adult gerrids in 2000 differed from 1999 and 2002 in two aspects: pattern and degree. The peak in prevalence in late spring was present and declined in the summer, but in July prevalence rose a second time. Additionally, prevalence was higher throughout the field season than in either of the other two years.
Mating brings adult gerrids into close and frequent contact beginning with pre-mating struggles and ending with post-copulatory mate guarding. Although mating gerrids in this study were not more likely to be infected than single individuals, this increase in interactive behavior within the population increases host-to-host contact and opportunities for host-parasite contact and parasite transmission. While gerrid mating behavior can explain trypanosomatid prevalence in the spring, it may also account for the prevalence patterns during the summer and fall.
Evidence of this association was seen in the 2000 field season. High gerrid mating activity in
July corresponded to an increase in the trypanosomatid prevalence in adults in the same month.
In 1999 and 2002, a second peak in mating activity was not detected and summer and fall prevalence remained low these years. Increasing prevalence of trypanosomatids in adults indirectly increased the prevalence in nymphs as a result of increased nymph density.
42 For nymphs, prevalence in 2000 increased throughout the field season, and, just as for the adult gerrids, prevalence was higher than in 1999 or 2002. Increased nymph density may account for these differences in parasite prevalence. An increase in host density would cause an increase in host-to-host contact and increase prevalence. While adult density in 2000 was essentially the same as in 1999, nymph density was higher in May and September than in the same months in other field seasons. Adult density was always high in the summer and the fall, while prevalence in nymphs was low in 1999 but not in 2000. These trends suggest that while adult density does not appear to affect the prevalence in nymphs, nymph density may play in role in high trypanosomatid prevalence years. Nymphs and adults segregate themselves from each other, especially the younger instars, which remain nearer to protected areas along the stream edges. As a result, nymphs have more contact with each other than with other adults. I conclude that while nymph trypanosomatid prevalence may be explained by nymph density, adult density does not explain the observed patterns of trypanosomatid prevalence in adults. As noted previously, the catch per effort method used to estimate gerrid density was influenced by stream condition (surface area), and may not be an accurate measure of density. Thus, use of this method may have confounded the relationship between gerrid density and trypanosomatid prevalence.
Summary: gerrid life history patterns affect trypanosomatid prevalence
The differences in mating patterns among the years could be explained by environmental conditions. Gerrids in temperate regions have flexible life histories that change according to environmental conditions (Blanckenhorn, 1994; this study). I argue that the dry conditions in
1999 and 2002 provided a cue for the G1 gerrid generation to suppress reproduction in that summer and delay reproduction until the following year, thus inducing a univoltine life history
43 pattern. In contrast, conditions were favorable for the survival of a second gerrid generation in
2000, and several G1 adults reproduced that summer resulting in a bivoltine life history pattern.
Changes in life history patterns caused changes in gerrid interactive behavior with respect to mating activity. In a univoltine population, only one mating period occurs while two can be detected in a biovoltine population. Increased mating activity causes an increase in trypanosomatid transmission among adults. As a result, nymph density and trypanosomatid prevalence in nymphs increases. Thus, environmental conditions can change voltinism patterns in gerrid populations that can indirectly influence host-parasite dynamics and affect trypanosomatid prevalence patterns in gerrid populations.
44 CHAPTER 3
SPATIAL AND TEMPORAL VARIATION IN AQUARIUS REMIGIS (SAY) (HEMIPTERA:
GERRIDAE) MORPHOLOGY
45 Fitness is the average potential of individuals with a particular genotype to survive and reproduce relative to others in the population. This potential is affected by many aspects of individual behavior, life history and morphology. Which of these traits are “fitness components” varies with the population and species under study. Fitness in gerrids has long been of interest to evolutionary ecologists, particularly with respect to morphological variation shaped by sexual selection and selection on dispersal ability. Thus, there is a wealth of information on how morphology affects gerrid fitness and how those traits vary under “stress,” including food limitation, crowding and temperature variation. These previous studies, summarized below, provide direction in choosing fitness components for my study. Parasitic infection is one stressor that could affect morphology and fitness of gerrids. I chose to examine the effects of parasitic infection on gerrid morphology (Chapter 4), but first I had to describe the variation in uninfected gerrid morphology in my populations.
The relationship between gerrid adult body size and fitness
Body size is closely correlated to various estimates of Aquarius remigis fitness. Gerrids grow throughout the nymphal stages molting five times over the course of 45-60 days. Larger male and female gerrids eclose (molt from N5 nymph to adult) later than smaller individuals
(Blanckenhorn, 1994; Ferguson and Fairbairn, 2000), yet both large males and females have greater lifetime fecundity. While large females produce more mature eggs (Fairbairn, 1988b), the mates of large males also produce more eggs (Arnqvist and Danielsson, 1999). In addition, a greater proportion of eggs from large Gerris incognitus females hatch (Arnqvist et al., 1997).
Arnqvist and Danielsson (1999) suggest this increased fecundity is female-controlled: a female will produce few eggs after mating with a small male.
46 Large males have greater overall mating success (Fairbairn, 1988b; Sih and Krupa,
1992; Krupa and Sih, 1993; Fairbairn and Preziosi, 1994; Preziosi and Fairbairn, 1996). Several of these studies have shown that females choose males with relatively long genital segments.
Genital length is highly correlated with total male length (Fairbairn, 1988b; Sih and Krupa,
1992; Krupa and Sih, 1993; Fairbairn and Preziosi, 1994; Preziosi and Fairbairn, 2000).
Body size is also correlated with many behaviors of Aquarius remigis that are related to fitness. Larger females are more territorial, that is, they are more site-specific than smaller females (Rubenstein, 1984; Kaitala and Dingle, 1993) and are chosen more often by larger males as mates (Fairbairn, 1988b; Rowe and Arnqvist, 1996). Large females mate more frequently than smaller females and are found in tandem with males for a longer duration (Rowe and Arnqvist, 1996). While in tandem, larger females copulate longer and are guarded by males longer (Rowe and Arnqvist, 1996). Larger males engage in shorter premating struggles
(Weigensberg and Fairbairn, 1996), mate more frequently (Rowe and Arnqvist, 1996; Arnqvist et al., 1997), and remain in tandem longer (Rowe and Arnqvist, 1996). Larger males copulate for a shorter duration (because they transfer sperm faster than smaller males) (Rowe and
Arnqvist, 1996; Arnqvist et al., 1997), and mate-guard longer (Rowe and Arnqvist, 1996).
The relationship among components of adult gerrid body size and fitness
While the above studies consider total body size, other studies have also measured the components of body size and correlated them to fitness. Much work has been done on the length of the abdominal, thorax and genital segments, as well as various measures of leg length in Aquarius remigis.
Aquarius remigis female abdomen length is positively correlated with reproductive success and is a significant predictor of fecundity (Preziosi et al., 1996), but is negatively
47 correlated with reproductive longevity (Preziosi and Fairbairn, 2000). Female thorax length is also positively correlated with fecundity (Preziosi and Fairbairn, 2000) and survival when coupled with late eclosion date (Ferguson and Fairbairn, 2000). Male Aquarius remigis abdomen length is negatively correlated with eclosion date (Ferguson and Fairbairn, 2000) and is positively correlated to fitness, but only in individuals with an early eclosion date (Ferguson and Fairbairn, 2000).
Aquarius remigis female genital length is not correlated to fecundity (Preziosi et al.,
1996) or net fitness (Ferguson and Fairbairn, 2000) measured as the sum of pre-reproductive survival, daily reproductive success (mating frequency or fecundity), and reproductive lifespan.
Results vary with respect to the relationship between female genital length and pre-reproductive survival: while Preziosi and Fairbairn (2000) found no correlation, Ferguson and Fairbairn
(2000) suggest the two are positively related. Long male genital length is positively correlated to mating success in A. remigis (Preziosi and Fairbairn, 2000).
For Aquarius remigis females, Preziosi et al. (1996) suggest that both mid- and hind- femur length was positively correlated with fecundity while mid-femur length was not correlated with pre-reproductive survival (Preziosi and Fairbairn, 2000). In Gerris incognitus males, fore-femur length was positively correlated to the hatching success of eggs in the mated female (Arnqvist et al, 1997). Fore-femur width in A. remigis males was positively correlated to number of matings (Weigensberg and Fairbairn, 1996) while Preziosi and Fairbairn (2000) found the same for mid- and hind-femur length.
“Stress” in development: temperature, food, and crowding
Stress, defined by Bayne (1975) as,
48 a measurable alteration of physiological (or behavioural, biochemical, or cytological) steady state which is induced by an environmental change, and which renders the individual (or population, or the community) more vulnerable to further environmental change, can affect gerrid juvenile development, and thus survival and fecundity of adults. These effects of stress on juveniles have been documented in several studies. High temperatures, for example, affect juvenile morphology by decreasing development time (Vepsalainen, 1973; Spence et. al.,
1980; Blanckenhorn, 1991, 1994). A decrease in development time is associated with a decrease in adult body length (Blanckenhorn and Fairbairn (1995), a trait demonstrated to have significant implications for adult behavior and fitness.
Like adults, nymphs are scavengers and predators that typically feed on dead or dying insects trapped on the water surface. The amount of food nymphs consume affects development, mortality, morphology and adult fitness. High food consumption decreases development time and juvenile mortality, but increases adult body size (Arnqvist and Thornhill,
1998). An increase in developmental time is otherwise associated with increase in adult body length (Blanckenhorn, 1994; Blanckenhorn and Fairbairn, 1995). High food consumption also decreases adult female pre-oviposition period while increasing oviposition rate (equal to total fecundity/oviposition period) (Blanckenhorn, 1994). Additionally, limited food during juvenile stages has significant implications for adult reproductive diapause (Vepsalainen, 1971, 1974,
1978). Vepsalainen’s (1978) photoperiod threshold model predicts that individuals that do not reach the fourth instar by the summer solstice (June 21) will delay reproduction to the following year. Blanckenhorn (1994) showed that juveniles with limited food develop slower and therefore have less of a chance of reaching the fourth instar by the photoperiod threshold and reproducing that summer.
Finally, high densities of nymphs have several life-history effects on the gerrid Aquarius
49 paludum. Crowding of nymphs leads to faster nymph growth rates and may induce the population of nymphs to produce a higher proportion of winged adults (Harada et al., 1997). A longer pre-oviposition period in females was also observed as a result of nymph crowding
(Harada et al., 1997).
One morphological indicator that an organism has experienced stress is its developmental departure from that produced without stress, sometimes called the “ideal form”
(Zakharov, 1992). Assuming that there is a relationship between stress and a departure from the ideal form, and that the departure can be measured, the departure can provide a measure of stress experienced during development. The more stress an organism experiences, the greater the departures from the ideal form. In bilaterally symmetrical organisms, the degree of developmental stability can be measured as departures from perfect symmetry only if symmetry in the organism does not have a heritable basis. Non-directional departures from symmetry, termed fluctuating asymmetry, are easily observed in frequency distributions of left minus right sides of the organism (Palmer and Strobeck, 1986). Arnqvist et al. (1997) demonstrated that fluctuating asymmetry in all leg lengths of Gerris incognitus was negatively correlated with offspring survival.
My goal is to investigate how Aquarius remigis morphology varies spatially and temporally with respect to mating status in several streams within a watershed. This study will elucidate the factors important in explaining morphological variation in gerrids and provide direction for the investigation of the effect of trypanosomatid infection on gerrid morphology
(Chapter 4).
Hypotheses
50 Hypothesis 1: Gerrid morphology will differ between upstream and downstream collection locations and among streams. Gerrid habitat varies along a stream. Locations may differ, for example with respect to food availability, predation pressure, or abiotic factors. Gerrids experiencing these variable environments may therefore differ in adult size. Because of the existing variation in stream location, I predict that gerrid morphological traits will vary between stream locations.
Hypothesis 2: Gerrid morphology will differ between parental (P1) and first generation (G1)
Aquarius remigis. Parental and first generation gerrids in the current study hatched and developed in subsequent calendar years, and therefore, it is likely that each generation developed under different environmental conditions. Biotic and abiotic conditions, such as food availability and precipitation, have been demonstrated to have significant effects on gerrid development and adult morphology. Because these conditions can vary over time and space, I predict that gerrid morphological traits will vary among successive gerrid generations and among stream habitats.
Hypothesis 3: Gerrid morphology will differ between mating and non-mating individuals.
Studies demonstrate that larger adult male and female gerrids mate more often with each other than with smaller individuals. When compared to non-mating individuals in the same population (stream), I predict that mating gerrids will have larger morphological traits.
51 METHODS
General
Collection description
I used the adult and nymph gerrids caught in the 2002 monthly prevalence collections to investigate the effects of spatial and temporal variables on gerrid morphology (see methods
Chapter 2). I measured gerrids collected in the May, June, July, September and October field surveys , and I analyzed them for trypanosomatid infection using the methods described in
Chapter 2. All gerrids that were measured and confirmed uninfected were included in this analysis of gerrid morphology. Morphology of infected gerrids is considered in Chapter 4.
Measurement methods
Prior to dissecting gerrids (see methods Chapter 2), I measured all adult and nymph gerrids that were alive just prior to taking measurements according to the methods in Preziosi and Fairbairn (1996). I euthenized gerrids in their holding vials by soaking the cotton ball plug with 70% ethyl alcohol. I took measurements using a dissecting microscope fitted with an ocular micrometer in which 1cm = 7.20mm at 7X magnification. I measured all gerrid characters to the 0.01 mm. For increased clarity, smaller gerrid body components were measured at a higher magnification. Total magnification is indicated for each body component below. I recorded the following measures for each gerrid and will use the respective abbreviations for the remainder of the chapter:
1. right middle-femur length (MFR) (Mag.= 7X)
2. left middle-femur length (MFL) (Mag.= 7X)
3. right hind-femur length (HFR) (Mag.= 7X)
52 4. left hind-femur length (HFL) (Mag.= 7X)
5. total length (TOT) (Mag.= 7X)
6. thorax length (TH) (Mag.= 15X)
7. abdomen length (AB) (Mag.= 15X)
8. genital length (G) (Mag.= 30X)
9. head length (H) (Mag.= 30X)
Body segments were more accurately distinguished on the ventral side of gerrids while leg segment measurements were more accurately taken from the dorsal side. Some gerrids were photographed for archiving purposes on a dissecting microscope using a Nikon digital camera. I used a 1X objective and 10X ocular lens and adjusted a zoom lens to increase magnification. I made all measures working on an Olympus SZH10 dissecting microscope that allowed me to lock in the zoom magnification. This microscope feature ensured consistently accurate use as I switched among magnifications. I took all gerrid measurements on the same microscope and assumed that this microscope measured consistently.
Preliminary statistical analyses: methods and results
Prior to analyzing the effects of space and time on gerrid morphology, I performed a preliminary analysis of the data to make sure the data met the assumptions for a multivariate analysis of variance (MANOVA)(Tabachnick and Fidell, 2001; Grimm and
Yarnold, 1995). I used JMP statistical software (JMP, Ver. 4.0.2, SAS® Institute, Inc., 2002) for all statistical analyses. I considered p-values ≤ 0.05 as significant for all statistical tests, and I used Pillai’s Trace lambda values for MANOVA analyses.
Data correction and outlier analysis
53 I measured nine characters on 297 male, 213 female and 178 nymph gerrids for a total of
6642 measurements. I created distributions of each measured character for each gerrid sex and nymph instar. I ran an outlier analysis for each distribution and identified extreme data points using jackknife distances. I rechecked these individuals against the data sheets, and I compared extreme traits to others measured for the same individual. Given the high correlations among the traits (see below), I assumed that extreme values not consistent with other traits in the same individual were errors. I removed these entries and treated them as missing data. After these preliminary analyses, I excluded 15 male, 10 female and four nymphs for a total of 5931 measures.
Measurement error
I used a subset of gerrids to assess my accuracy by making repeated measurements on different bugs. I collected 54 adult gerrids in October, and I used these gerrids for the analysis of measurement error. I made three measurements of both the left hind femur and the abdomen lengths of 40 males and 14 females. I measured each sex on separate days, but all individuals of each sex within the same day. Each day I measured all individuals before making replicate measurements. I randomly selected the next individual to be measured within each replicate and recorded values for each replicate on separate notebook pages. These methods allowed me to make each set of measurements without reference to the previous set.
I analyzed the three replicates of abdomen and left hind femur of all individuals with an
ANOVA and found that replicate was not a significant factor in explaining the variation in the data (HFL: d.f.=2, F=0.0225, p=0.98; AB: d.f.=2, F=0.0012, p=1.00). These results suggest that variation in morphometrics due to my measurement error was low.
Choosing morphological variables for analysis
54 I checked for multicollinearity of the nine variables using multivariate correlations and variance inflation factors (VIF). While the multivariate correlations describe the relationship among pairs of variables, VIF describe overall relationships among all the variables. I used these values to identify measures that would give me different perspectives on gerrid size without being redundant. Highly correlated measures indicates that measures are redundant, so
I considered pairs of variables with a correlation factor greater that |0.80| (highly correlated) and
VIF values greater than 10 (highly correlated with one or more of the other variables) for possible exclusion.
Both right and left leg lengths and front and back legs were highly correlated, so I chose one leg length for analysis. MFL and HFR data were missing for two individuals, while MFR and HFL had only missing values for one individual. I chose to include MFR in the analysis.
Total body length was highly correlated with most other measures so I focused on individual body sections instead. I then reanalyzed the remaining five variables (MFR, TH, AB, G, and H) for multicollinearity. AB and G were highly negatively correlated and had a high VIF. Four individuals had missing values for AB and only two were missing values for G, therefore I chose to include G in the analysis. I reanalyzed MFR, TH, G, and H for multicollinearity and all correlations were less than |0.80| and had VIF values less than 10. These were the four variables I used in all subsequent analyses of morphology.
I analyzed the variables of MFR, TH, G and H for homogeneity of covariance, an important assumption of MANOVA, between male and female gerrids. I generated a covariance matrix for each gerrid group separately, and all covariances of variables were nearly equal when compared across groups and among gerrid groups.
55 Analysis of variance assumes variables are normally distributed. As long as distributions are roughly bell-shaped, however, ANOVA is still a reliable statistical tool (i.e., the procedure is “robust” to violations of the normality assumption). I analyzed the distributions of each of these four dependent variables for both gerrid sexes. Five of the eight distribution curves significantly departed from normal (Shapiro-Wilk goodness-of-fit, w<0.05). I transformed the data using a Box-Cox function in JMP and rechecked the distributions for normality. Four of the eight distribution curves remained significantly different from normal.
Because transforming the data only minimally improved the normality of the data and because the distributions were approximately normal, I used the non-transformed variables in the primary analysis.
Primary statistical analysis
As in Chapter 2, all individuals were assigned to a category under sex, stream, mating status, location, stage and month. In addition, I coded adults according to generation. I used
MANOVA to test effects of these factors on the dependent variables MFR, TH, G and H.
The parental generation (P1) contained the overwintered adults that mated and died by early summer. Adults from May and June were coded as P1. Adults from September and
October included the offspring of the P1 generation and were coded as G1. Both generations were present in July, and I therefore coded July gerrids as G1.5.
I evaluated and analyzed gerrids for the presence of fluctuating asymmetry using the methods outlined in Palmer (1994). I estimated the symmetry of gerrid leg lengths as the difference of left minus right values for mid-femur and hind-femur leg segment lengths
(abbreviated SMF and SHF). Individual gerrids with damaged leg segments were excluded
56 from the analyses. I used an ANOVA model to test the effect of generation on the dependent variables of SMF and SHF.
RESULTS
Effect of upstream and downstream location
I investigated the effect of stream location using data from Harker’s Run, the only stream with upstream and downstream locations in the 2002 collections. I included male and female gerrids from collections in May, June and July, and I used a MANOVA to test for the effect of stream location and gerrid sex and the interaction of location and sex on MFR, TH, G, and H.
Both location and sex, as well as their interaction, had an effect on the morphology of uninfected gerrids (d.f.=12, l=1.3207, p<0.0001)(Table 16). I tested male and female gerrids separately for the effect of location and results indicate that for both sexes location has a significant effect on morphology (Male: d.f.=4, l=0.5938, p<0.0001; Female: d.f.=4, l=0.4786, p=0.0002)(Table 16). Gerrids in the downstream location tended to have larger body size components than those in the upstream location (Table 17).
Location had a significant effect on gerrid morphology, and therefore, I could not pool these two sites analyzed them as one stream. Neither could I analyze the two collections as separate sites because they are not independent samples with respect to stream. Therefore, I chose to exclude Harker’s Run downstream location from all subsequent analyses because fewer collections were made at this site than from the upstream site.
Effect of stream, sex and generation
57 I collected 114 P1 and 151 G1 adults (male and female combined), from three streams. I tested for the effect of gerrid generation, sex, and stream on morphology, and results indicate that all three main effects were significant as well as the interaction of stream and generation
(d.f.=44, l=1.5764, p<0.0001)(Table 18). I separated the data and tested each sex separately for the effect of generation and stream and the interaction on morphology. Results suggest that for both sexes generation, stream and the interaction were significant (Male: d.f.=20, l=0.6279, p<0.0001; Female: d.f.=20, l=0.6479, p<0.0001) (Table 18). Generation one gerrids tended to be larger than the parental generation for both sexes (Table 19). I separated the data further by sex and by stream and tested for the effect of generation. Results indicate that generation had a significant effect on morphology in each stream for each sex except for female gerrids in
Harker’s Run (Table 18). With respect to body size components, G1 generation gerrids were larger than the P1 generation in Darr’s Run (males and females) and Harker’s Run (males only).
In Collin’s Run, the relative size among body components varied with generation (Table 20).
Symmetry
I looked for a correlation of body size and symmetry. A correlation between these two values could result from sexual dimorphism or simply because larger individuals might have more or less symmetry. I plotted total body length against the absolute value of SMF and SHF in uninfected adult gerrids. I observed no correlation between the degree of asymmetry between right and left leg segments and total body length (Figure 15). As a result, I did not include a correction factor for body length in the analysis of fluctuating asymmetry, and I analyzed male and female gerrids as one group.
I used distributions of SMF and SHF to determine if uninfected gerrid femur lengths exhibited anti- or directional asymmetry. I detected neither condition in these populations of
58 gerrids. If one side of an organism was always larger, but there was no trend to either side
(antisymmetry), then these distributions would be bimodal. Directional symmetry (one side larger than the other and always the same side) would have been indicated if the mean difference of left minus right was not equal to zero. Neither condition occurred in my data: both SMF and SHF showed a normal distribution (Shapiro-Wilk goodness-of-fit, w<0.05) and a mean of zero. I then analyzed the data for the presence of fluctuating asymmetry. I tested for the effect of generation and stream on the symmetry of gerrid femur leg lengths. Results suggest that neither generation or stream had an effect on SMF or SHF (SMF: d.f.=5, F=0.7582, p=0.58; SHF: d.f.=5, F=1.0364, p=0.40)(Table 21).
Mating adults
I tested for the effect of mating status on gerrid morphology to determine if a size difference existed between mating and non-mating individuals within a population. The majority of gerrid mating pairs were caught in May; therefore, I chose to include only May in the analysis of the effect of gerrid mating status on morphology. I included stream in the model because each stream contains reproductively distinct populations of gerrids, that is, gerrids from one stream in my study are unlikely to encounter and mate with gerrids from another stream. I generated a multivariate covariance matrix for the separate sexes of mating and non-mating status of the non-transformed variables of MFR, TH, G, and H to check for homogeneity of variance. I used MANOVA model to test the effect of mating status, stream, sex and the interaction of these factors on the dependent variables of MFR, TH, G, and H. Results indicate that while stream and gerrid sex were significant, mating status was not an important factor in explaining morphology (d.f.=33, l=1.9320, p<0.0001) (Table 22).
59 I investigated the effect of gerrid mating status on femur length symmetry to determine if symmetrical individuals were chosen as mates more frequently than asymmetrical individuals. I used an ANOVA model with mating status, stream and the interaction of mating status and stream as the main effect and SMF and HMF as dependent variables. Results suggest that in all streams mating individuals are as likely as non-mating individuals to have symmetrical middle- and hind-femur leg lengths (SMF: d.f.=5, F=0.2932, p=0.92; SHF: d.f.=5, F=1.423, p=0.23)(Table 23).
Nymphs
I collected 168 uninfected gerrid nymphs from three age classes (N3-N5) in June and
July. Of these, I was unable to sex 4 nymphs, and I excluded them from the analysis of nymph morphology. Because all nymphs represented one generation I chose to analyze the two months as one collection. First I used the collections in Harker’s Run upstream and downstream locations and MANOVA model to test for the effect of sex, age and location and all interactions on the variables of MFR, TH, G and H. All factors had a significant (Table 24) effect on morphology so I analyzed male and female nymphs separately for the effect of age, location and the interaction on the variables. While male nymph morphology was only affected by age, female nymph morphology varied with age and stream location (Table 24). For both sexes, body size was positively associated with nymph age, and females in Harker’s Run upstream location tended to be larger than in the downstream location (Table 25). Because location was a significant factor in females, I excluded the effect of location from further analyses of nymph morphology.
I collected only 23 nymphs from Collin’s Run, and because all ages of both sexes were not represented in the collection I chose to exclude this stream from the analysis of the effect of
60 stream on nymph morphology. I pooled the Harker’s and Darr’s Runs collections and used a
MANOVA to test for the effect of sex, age and stream and all interactions on the variables of
MFR, TH, G and H. All of the factors were significant (Table 24) so I separated the data by nymph sex and tested for the effect of age and stream and the interaction on nymph morphology. Nymph age was the only significant factor for both sexes (Table 24) with body size positively correlating to nymph age (Table 26).
DISCUSSION
The size of gerrid morphological traits varied (2-5%) with time (between generations), and space (among streams and location within stream), but not between mating and non-mating gerrids in this study. Environmental variation in weather, food availability or stream structure over time and space could account for the observed differences in gerrid morphology.
Body size in gerrids can be affected by abiotic and biotic factors. As noted earlier, high temperatures decrease nymph development time (Vepsalainen, 1973; Spence et al., 1980;
Blanckenhorn, 1991a, 1994), and decreased nymph development time results in shorter adult body length (Blanckenhorn, 1991a, 1994; Blanckenhorn and Fairbairn, 1995).
Precipitation and temperature can affect stream volume and surface area, and thus available habitat for gerrids. The effect of reduced stream volume and surface area initially increases the density of gerrids. Under crowded conditions Harada et al. (1997) and
Klingenberg and Spence (1996) have demonstrated that Aquarius paludum and Gerris buenoi nymphs respectively develop faster. Thus, precipitation and temperature, by increasing gerrid density, may also indirectly decrease adult body length.
61 Food availability, like temperature, has direct effects on nymph development time. High food consumption by nymphs decreases their development time and leads to larger adult body size (Arnqvist and Thornhill, 1998). High food consumption in nymphs also decreases juvenile mortality (Arnqvist and Thornhill, 1998).
I observed a significant difference in the size of adult morphological traits between the two generations in this study. P1 generation gerrids were significantly smaller than the G1 generation, and thus, it is likely that the P1 generation developed under relatively more stressful conditions. The G1 gerrids developed during the spring of 2002. P1 gerrids developed in 2001, possibly in the fall. I argue in Chapter 2 that the 2001 season might have been bivoltine. If this was the case, the P1 generation in the present study most likely would have hatched and developed late in the season in 2001, a time of when gerrids are food limited (Firko, 1986;
Blanckenhorn, 1994). At this time adult gerrid density is typically high (Chapter 2), and the P1 generation nymphs may have developed under crowded conditions. Finally, late summer temperatures are typically the highest in the season. These three conditions, low food, crowding and high temperatures can induce faster nymph development and result in relatively shorter adult body size.
In contrast, the G1 generation in the present study not only hatched and developed in a different year but it did so at a different time of year than the P1 generation. The G1 generation nymphs developed early in the season when food is not as limited as it is later in the season, adult gerrid density is low, and temperatures are cooler. These conditions are correlated with slower nymph development and result in relatively longer adult gerrid body size.
Although the overall the trend of the data was for increased size in the G1 generation, I also detected variation among streams in the effect of generation. In Darr’s Run all
62 morphological traits analyzed were on average larger in the G1 generation for both gerrid sexes.
In Harker’s Run, the same trend was true but for males only. I detected no effect of generation in female gerrids in Harker’s Run. The variation in gerrid morphology with respect to generation was even greater in Collin’s Run. While P1 males had longer mid and hind femurs and thoraces, G1 males had longer genital and head segments. Results were similar for females in Collin’s Run. P1 females had longer mid and hind femurs, thoraces, and genital segments, but a shorter head segment. These results suggest that despite the overall trend for larger size in the G1 generation, the effect of generation on gerrid size is inconsistent when individual streams are considered.
With respect to stream location, male and female adult and female nymph gerrids were larger in the downstream location than in the upstream location. The landscape along Harker’s
Run varies between these locations. The downstream location is adjacent to two manicured turf fields and a parking lot, whereas the upstream location is surrounded by a forest reserve.
Abiotic and biotic factors may have varied among the streams in this study area, and therefore, could account for the observed variation in gerrid size.
Adult gerrid morphology, especially body size, has a wide array of effects on fitness components including survival to reproductive maturity, mating success, food acquisition, and predator avoidance.
Survival to reproductive maturity
Despite producing smaller body size, faster development of nymphs can be adaptive. G1 generation nymphs hatching early in the season and in conditions favorable for a subsequent generation (bivoltine life cycle) will benefit from conditions favoring faster development.
Vepsalainen’s (1978) photoperiod threshold model predicts that nymphs that do not reach the
63 fourth instar by the summer solstice will delay reproduction until the following year. Delaying reproduction can significantly affect gerrid fitness since overwintering gerrid mortality can be as high as 80%. Thus, faster development in the G1 generation may actually increase gerrid fitness in some years.
Fast development may be of increased importance for the P1 nymphs in this study, assuming they were a G2 generation in 2001 (that is, the offspring resulting from a second reproductive period in a bivoltine life cycle; P1 adults in 2002 are the same bugs as G2 nymphs in 2001). None of the individuals in a G2 generation directly reproduce the same year in these populations of gerrids, and therefore, they are destined to overwinter. Since only adult gerrids can successfully enter diapause and overwinter, nymphs of a G2 generation have relatively little time to complete development and accumulate lipid reserves before the onset of winter. The environmental conditions present late in the season, low food, crowded conditions and high temperatures, may actually indirectly increase G2 generation gerrids fitness by decreasing development time. Those nymphs that do not eclose to the adult stage and accumulate enough fat stores by the onset of winter will not successfully overwinter (Wilcox and Ruckdeschel,
1982) and thus not reproduce. Faster development of the G2 generation may also be adaptive for male gerrids. Ferguson and Fairbairn (2000) demonstrated that males with shorter body lengths survive to reproductive maturity more often than larger males. These results could suggest that smaller size confers increased survivorship during diapause actually benefiting male gerrids.
Mating success
The gerrid mating system is characterized as a weak positive assortative mating by size
(Rowe and Arnqvist, 1996; Arnqvist et al., 1996). In other words, larger individuals mate more
64 often with each other than with smaller individuals, and those individuals of each sex that are mating have a larger body size than those that are not mating. Although my study did not find a correlation between body size and mating status, many studies have demonstrated the importance of body size and reproductive activities of gerrids.
Mating is initiated by males that compete for females in a scramble-like fashion attempting to mount with each female encountered (Rowe, 1992). Mating is typically terminated by the female. When the male attempts to mate with the female she actively resists about 85% of mating attempts by trying to dislodge him from her back with rolls and somersaults (Weigensberg and Fairbairn, 1994). She is typically successful at repelling more than half of male mating attempts (Weigensberg and Fairbairn, 1994). If a male is successful,
64% of copulations are terminated by the female by similar efforts described for the resistance of mating attempts (Weigensberg and Fairbairn, 1994). Females can store sperm and maintain maximum fertility for at least 15 days (Preziosi and Fairbairn, 1997), and they therefore gain little (if anything) by mating frequently. As a result, an intersexual conflict of interest in mating frequency exists in gerrid populations (Hammerstein and Parker, 1987; Rowe et al., 1994.
Fairbairn and Preziosi (1996) conclude that sexual selection in Aquarius remigis occurs through female reluctance to mate with certain males rather than by active female choice.
In this assortative mating system characterized by female reluctance to mate, small males are at a significant reproductive disadvantage. In previous studies of Aquarius remigis, mating females were larger that single females in field populations (Faribairn, 1988b; Arnqvist et al., 1996), and large females mated more frequently (Krupa and Sih, 1993). Other studies have made similar conclusions for males, but the effects of male size on mating success were weaker and more variable (Fairbairn, 1988b; Sih and Krupa, 1992; Krupa and Sih, 1993;
65 Arnqvist et al., 1996; Fairbairn and Preziosi, 1994). Rowe and Arnqvist (1996) suggest that the weaker effects found for male body size might be explained by the separate activities that occur during the mating season. Although large males mate more frequently, engage in shorter premating stuggles (Weigensberg and Faribairn, 1996) and mate guard longer than small males, small males copulate longer than large males. The authors explain that small males require more time for sperm transfer and displacement in the female, and therefore must remain in copula longer than that required for larger males. Once copulation is complete, males guard their mates while in tandem to increase the chances for fertilization with their sperm and not that of the next male to mate with the same female. During the mate-guarding phase, females are able to dislodge small males more easily than larger males thereby decreasing the chances for small males to successfully fertilize eggs.
Regardless of the difference in copulation duration that exists between large and small male gerrids, large males are still at a reproductive advantage. Large males have less to gain from extending the duration of copulation because they mate more frequently, transfer sperm faster (Rowe and Arnqvist, 1996; Arnqvist et al., 1997), and are able to mate guard longer
(Rowe and Arnqvist, 1996) ensuring the fertilization of more eggs.
Female body size is positively correlated with the number of mature eggs she contains in her abdomen (Fairbairn, 1988b), and the number of mature eggs in females is positively correlated with the total duration of successful mating encounters (Weigensberg and Fairbairn,
1996). One would assume that large female body size results in longer mating durations that results in more mature eggs, yet Weigensberg and Fairbairn (1996) demonstrated that a direct relationship exists between mating duration and egg number rather than mating duration and female body size (it is unclear whether mating duration in this study included mate guarding
66 duration in addition to actual copulation time). Additionally, the number of mature eggs that a female carries is not only positively correlated with the duration of successful matings, but also positively correlated with the duration of premating struggles with males (Fairbairn, 1988).
These studies clearly demonstrate that body size has a significant impact on the reproductive activities of both male and female gerrids.
Food acquisition
Although food consumption by nymphs is positively correlated with adult body size, body size can also have a significant effect on ability of Aquarius remigis to acquire food. A. remigis are scavengers and predators and feed primarily on other live insects trapped on water surface. The upper size range of prey items that A. remigis can successfully capture and consume is positively correlated with body size (McLean, 1990). In other words, larger gerrids have available to them more types of food because they can subdue larger prey items than smaller gerrids can.
Territoriality is food-based in stream-dwelling gerrids and has been demonstrated to have consequences for gerrid fitness (Blanckenhorn, 1991b,c). Aquarius remigis of both sexes defend territories in habitats of food resources (Wilcox and Ruckdeschel, 1982; Rubenstein,
1984). Rubenstein (1984) has suggested that by maintaining foraging positions in the faster flowing areas in a stream, A. remigis increase their prey encounter rate. The author demonstrated that only the larger of A. remigis females were successful in defending these foraging positions against males. Blanckenhorn (1991b) reported that competitively dominant individuals have significantly higher foraging success and mass gain prior to diapause. The author suggests that foraging success prior to diapause can affect overwintering survivorship.
Predation risk
67 For populations of Gerris buenoi, predation by other arthropods has been reported to be an important determinant of survival (Spence, 1986). Although the effect of adult gerrid body size on predation risk has not been reported, it may have an indirect effect with regard to mating activities. Movement in gerrids attracts backswimmers (Notonecta undulata) an insect predator
(Fairbairn, 1993; Lowe, 1994), and gerrid mating activities result in vigorous movement. Lowe
(1994) demonstrated a positive correlation between backswimmer predation risk and the length of Aquarius remigis pre- and postmating struggles. I predict that because larger gerrids mate more frequently, they may be at a high risk for predation. Conversely, because small males mate less frequently, they spend more time in search of mates. Searching for mates requires high movement rates which also attracts predators (Lowe, 1994).
Summary
In my study I have demonstrated the variability of gerrid body size across space and time. Body size in gerrids is clearly important in terms of gerrid fitness in natural populations.
Gerrid mating interactions and ability to forage are positively affected by large body size.
Because of the temporal and spatial variation in relative gerrid body size, I suggest that studies involving components of gerrid fitness with respect to body size consider more than a
“snapshot-in-time” approach. My study demonstrates a need for studies to assess parameters related to body size within several gerrid populations not only over time, but on a stream by stream basis. Understanding the causes for variation in body size among populations could also be enhanced with supporting data on environmental conditions, especially temperature and rainfall.
68 CHAPTER 4
TRYPANOSOMATID (KINETOPLASTIDA: TRYPANOSOMATIDAE) INFECTION
REDUCES ADULT AQUARIUS REMIGIS (SAY) (HEMIPTERA: GERRIDAE)
BODY SIZE
69 The effects of parasites on host fitness are central to their epidemiology. Most insect trypanosomatids are considered non-pathogenic (Schaub, 1994). But in most cases host species have only been investigated under optimum laboratory conditions. Once examined in the context of a natural, potentially more stressful environment, pathogenic effects of these ‘non- pathogenic’ trypanosomatids were detected (e.g., Schmid-Hempel, 2001). Schaub (1992) reviews the negative effects associated with infection in laboratory studies subjecting hosts to various stressors, such as temperature and starvation. His review shows that trypanosomatids can have a detrimental effect on hosts, yet only two trypanosomatid species have been thoroughly investigated with respect to their potential effects on host fitness: Blastocrithidia triatomae in the terrestrial hemipteran Triatoma infestans, (Schaub, 1992) and Crithidia bombi in bumblebees (review in Schmid-Hempel, 2001). In an aquatic environment, trypanosomatid effects been investigated in gerrid hosts in relatively few studies, and it is the primary goal of my project to document the effect of infection on gerrid fitness components.
Gerris odontogaster infected with trypanosomatids showed reduced host vigor and increased adult mortality when hosts are starved. Thus, these parasites may play an important role in population regulation of gerrids during periods of stress (Arnqvist and Maki, 1990). The authors also suggest that trypanosomatid infections may decrease overwintering survival of gerrids, reduce fecundity of females during times of food stress, and have significant negative effects on the survival of nymphs, but they do not provide evidence for these effects.
Klingenberg et al. (1997) showed that trypanosomatid infections increase development time (by 5%) and decrease adult body length (by 2-3%) in laboratory populations of Gerris buenoi. These effects, in turn, influence the population dynamics of gerrids. Because mortality is high in gerrid nymphal stages, increased development time may decrease chances of survival
70 (Spence, 1986). Because only adults enter diapause, increased development time may also cost second-generation gerrids their chance of overwintering and reproducing the following spring.
As discussed in detail in Chapter 3, decreased adult size may affect a variety of gerrid fitness parameters including increased predation risk, lowered male mating success (review in Rowe and Arnqvist, 1996) or fecundity of females (Fairbairn, 1988b).
Arnqvist (1992) found that trypanosomatid load (number of parasites per host) in male
Gerris odontogaster had a significant effect on mating success, but was not correlated with several morphological measures. Parasite load was very low in each of the three populations.
The author suggests that while low parasite loads appear to have little impact on gerrid fitness, higher trypanosomatid parasite loads may have a relatively large effect.
In the last two decades much attention has focused on using fluctuating asymmetry as an indication of developmental stability in an organism under given environmental conditions
(reviews in Palmer, 1996, 2000; Moller, 1997). Of those environmental factors investigated, parasitism is the most common factor positively correlated with high degrees of fluctuating asymmetry (review in Moller, 1996). These studies indicate that parasitism is associated with developmental instability and this relationship could be explained by two mechanisms: developmentally unstable hosts may be more susceptible to parasites due to their poor condition, or parasite infections cause developmental instability. Few studies of fluctuating asymmetry with regard to parasitism in insects exist (review in Moller, 1996; Ward et al., 1998), and all of those studies consider only terrestrial insects. My study intends to determine if a relationship exists between parasitism in an aquatic insect and fluctuating asymmetry.
As demonstrated above, little is known about the capability of trypanosomatids to adversely affect gerrid fitness. This aspect of the trypanosomatid-gerrid relationship is the focus
71 of this chapter. Before pathogenicity can be documented however, an understanding of what fitness is to the host organism must first be established. In Chapter 3 I established that Aquarius remigis morphology varies with spatial and temporal factors. Variation in body size has several implications for gerrid fitness including effects on mating success, foraging efficiency and predation risk.
Hypothesis
Several studies have investigated effects of the environment on gerrid body size and the impact that body size has on gerrid fitness, but only a few studies have documented the effects of trypanosomatid parasites on gerrids. I predict that trypanosomatid infections in gerrids will stress the organisms and exert negative effects on gerrids similar to those described in Chapter
3.
Hypothesis: Trypanosomatid infection during development reduces adult size and is therefore a significant cost to gerrid fitness. The stress of a parasitic infection in the developmental period could channel energy to the immune system for fighting the infection. As a result, less energy is left to support the functions of normal growth and development. I predict that morphometrics of adult and nymph gerrids infected with trypanosomatids will be smaller when compared to uninfected gerrids. I also predict that adult and nymph gerrids infected with trypanosomatids will exhibit greater asymmetry in mid- and hind-femur leg length than that of uninfected gerrids.
METHODS
General
Data collection
72 I used the adult and nymph gerrids caught in the 2002 monthly prevalence collections to investigate the effect of trypanosomatids on gerrid morphology (see methods Chapter 2). I measured gerrids collected in the May, June, July, September and October field surveys according to methods described in Chapter 3. I dissected and assayed gerrids for trypanosomatid infection within two hours of taking measurements according to methods described in Chapter 2.
Preliminary statistical analysis
I compared infected gerrids over space and time to the uninfected gerrids analyzed in
Chapter 3. Based on the preliminary statistical analyses in Chapter 3, I chose to use the same morphological variables (MFR, TH, G and H) in the analysis of the effect of infection on gerrid size. I analyzed the variables of MFR, TH, G and H for homogeneity of covariance across and among groups of gerrids of interest (uninfected female, infected female, uninfected male, and infected male). I generated a covariance matrix for each gerrid group separately. All covariances of variables were nearly equal when compared across groups and among gerrid groups.
I analyzed the distributions of each of these four dependent variables by the four groups described above. Six of the 16 distribution curves significantly departed from normal. I transformed the data with a Box-Cox function in JMP and rechecked the distributions for normality. Five of the 16 distribution curves remained significant. As in Chapter 3, I used the non-transformed variables in all subsequent analyses. I used JMP statistical software (JMP,
Ver. 4.0.2, SAS® Institute, Inc., 2002) for all statistical analyses. I considered p-values ≤ 0.05 as significant for all statistical tests, and I used Pillai’s Trace lambda values for MANOVA analyses.
73 Primary statistical analyses
I separated adult and nymph gerrids for which both morphometric and prevalence data were available. Only six of the 174 nymphs that I measured were infected, thus I could not analyze the effect of infection on nymph morphology. I therefore focused on adult gerrids for the analysis of trypanosomatid infection.
Previous analyses of the effect of location on gerrid morphology revealed a significant difference in gerrid morphology between upstream and downstream locations (see Chapter 3).
Because this test result was significant and the factor of location is nested within stream in statistical analyses, I chose to exclude the Harker’s downstream site from all analyses of infection and morphology within a stream.
I used MANOVA to test for the effects of infection, sex, generation and stream on gerrid morphology as in Chapter 3. I excluded the July collections from this analysis because both gerrid generations were present in this month and were indistinguishable.
I investigated the effect of infection and location on fluctuating asymmetry in adult gerrids using the methods outlined in Chapter 3.
RESULTS
In May, I collected 12 male gerrids (10 uninfected, 2 infected) from Harker’s downstream site and 18 male gerrids (15 uninfected, 3 infected) from Harker’s upstream site. In this small sample, morphology was significantly affected by stream location, but not by gerrid infection status or the interaction of location and infection (Table 27).
I included a total of 307 male and female gerrids from the May, June, September and
October collections (excluding Harker’s Run downstream site) in the analysis of gerrid sex,
74 infection status, generation and stream on morphology. Results of this analysis suggest several significant interactions among the factors, including infection status (Table 28). I modified the model by removing the 4-way interaction. The model remained significant (d.f.=84, l=1.6432, p<0.0001) and the interaction of gerrid infection status, generation and stream was just above the 0.05 cut off for significance. I further modified the model by removing the other, clearly insignificant, three-way interactions. Because the three-way interaction of infection, generation and stream remained significant, I separated the data into subgroups for further analysis.
Samples sizes of infected gerrids were small, and I could not test for the effect of infection for each sex in each generation in each stream. I did have enough data to analyze one generation of males (G1). I tested a total of 93 males (32 infected and 61 uninfected) in
Harker’s, Darr’s and Collin’s Runs for the effect of infection, stream and their interaction on gerrid morphology. While infection was not significant, stream and the interaction of infection and stream were (Table 29). I separated these data by stream, and infection was significant in
Harker’s and Darr’s Runs, but not in Collin’s (Table 29). The effect of infection was variable with respect to morphological trait in both streams (Table 30).
I chose to exclude generation from the primary analyses for two reasons: first, generation appeared to have an inconsistent effect on gerrid morphology (Chapter 3). Analyses of each gerrid sex within each stream suggest that although gerrid generation can affect morphology, its effect within each stream varied. Second, I did not have a large enough sample of uninfected gerrids from each stream in each generation for each sex. Because I chose to exclude generation from the analyses, I included gerrids from the July collections.
A total of 485 male and female gerrids from collections in May, June, July, September and October were included in the primary analysis of infection and gerrid morphology. Sixty
75 three were infected. I tested for the effect of infection, stream and sex, and all three factors significantly affected gerrid morphology (d.f.=44, l=1.1556, p<0.0001)(Table 31).
Because sex had a strong effect on morphology, I chose to analyze each sex separately to clarify effects of stream and infection. Neither factor had a significant effect on female morphology, but male morphology was significantly affected by infection, stream and their interaction (Table 31). I tested for the effect of infection on male gerrid morphology in each of the three streams separately. Infection was significant in Harker’s Run and Darr’s Run while infection had no effect on male morphology in Collin’s Run (Table 31). Reference to the mean values for these streams suggest that infected male gerrids in Harker’s and Darr’s streams had significantly shorter body components than do uninfected male gerrids in these streams (Table
32).
Because stream location has a significant effect on morphology (Chapter 3) and abiotic and biotic factors may differ between these locations (see Chapter 3), I tested stream location for differences in fluctuating asymmetry in relation to infection status. I tested a total of 30 male gerrids in the May Harker’s Run collection for the effect of infection on body symmetry in up- and downstream locations. A total of three and two infected gerrids were tested from upstream and downstream locations respectively. Neither infection, location nor their interaction had an effect on SMF or SHF (SMF: d.f.=3, F=0.1213, p=0.95; SHF: d.f.=3,
F=0.6469, p=0.59) (Table 33).
In the analysis of gerrid infection status and fluctuating asymmetry, I included a total of
419 male and female gerrids from collections in May, June, July, September, and October in the analysis. Neither infection or stream or their interaction had an effect on the SMF and SHF
(SMF: d.f.=5, F=0.6193, p=0.69; SHF: d.f.=5, F=0.5948, p=0.70) (Table 34).
76 DISCUSSION
Trypanosomatid infection had significant effects on male Aquarius remigis morphology but did not affect the body size of female gerrids. Infected male gerrids had significantly smaller body size components (middle femur, thorax, genital, and head lengths) than those that were uninfected. Trypanosomatid infection did not have an effect on gerrid femur symmetry within or among streams in this study.
This study showed that body length was shorter (by 2-3%) in Aquarius remigis males that harbored trypanosomatid infections than in those that were uninfected. This figure is similar to the variation (2-5%) found between gerrid generations (Chapter 3). My results agree with Klingenberg et al. (1997) who showed that trypanosomatid infections increase development time (by 5%) but decrease adult body length (by 2-3%) in laboratory populations of Gerris buenoi.
Arnqvist (1992) found that trypanosomatid load (intensity of parasitic infection) in male
Gerris odontogaster had a significant effect on mating success, but was not correlated with several morphological measures. These results contrast to the findings of my study. I found that while infection status was correlated with morphological measures, it was not correlated with mating status (Chapter 2). Yet, Arnqvist (1992) reported very light trypanosomatid infections in Gerris odontogaster and suggests that heavier infections may account for the lack of correlation with morphology.
Although I detected a significant effect of trypanosomatid infection in male gerrids, I interpret these results cautiously. Generation had a significant effect on morphology (Chapter
3), and I detected an overall trend for second generation (G1) gerrid to be larger. But upon
77 closer examination I found that the effect of generation on body size was inconsistent among gerrid populations. As body size varies among seasons and populations, so might the effect of parasitic infection on body size. I tested G1 males in three streams for the effect of infection on body size. In two of the three streams infection had a significant effect on gerrid size but was variable with respect to morphological trait.
I could not include a temporal factor (generation) in my infection analyses because samples of infected gerrids of each sex in each stream were too small and inconsistent among streams. I therefore discuss the results of trypanosomatid infection on male body size as significant, but I consider these findings preliminary until I can investigate this effect further.
Trypanosomatids can infect gerrids at both life stages: the adult and nymphs stage.
Infection experiments demonstrate that adult Gerris lacustris gerrids can acquire and sustain a trypanosomatid infection (Tieszen and Molyneux (1989) via host water intake, and all ages of nymphs (N1-N5) have been found to naturally harbor trypanosomatids (Tieszen and Molyneux,
1989).
The relationship between male size and infection could result from several mechanisms.
First, trypanosomatid infections may disrupt developmental function in gerrids resulting in small adult male size. I focus on this mechanism below. Second, adult and nymph male gerrids that are small may be more likely to become infected. Small size can be the result of stress an individual experiences during the developmental period (see Chapter 3 and below). Stressed individuals may be more susceptible to infections or pathogens perhaps because they are less resistant. I do not know that environmental stress increases the likelihood of gerrids acquiring a trypanosomatid infection or that the effects of stress during development can affect the susceptibility of adult gerrids to infection. Another possibility is that small gerrids may be more
78 likely to come into contact with trypanosomatids. I can find no evidence nor offer any biological reason for this mechanism, but it remains a plausible explanation for the observations in this study. A third mechanism for the relationship between male size and infection may be that a third factor both decreases size and increases infection, for example, stress. Crowding within gerrid populations increases both host contact rates and stress. Increased contact rates facilitate parasites transmission, and stress can decrease gerrid size. For the purpose of this discussion I will ignore the latter two mechanisms for explaining trypanosomatid infection in small gerrids and interpret my results under the assumption that trypanosomatid infections negatively affect nymph development in a way that leads to small adult body size.
Several mechanisms have been proposed to explain how an established trypanosomatid infection in gerrids may disrupt normal body function. Once in the host hindgut, trypanosomatids may reduce host viability by 1) competing with the host for nutrients or blocking the surface of the gut which reduces nutrient uptake (Schaub and Losch, 1989), 2) interfering with host excretion (Schaub and Schnitker, 1988), and/or 3) a loss of microvilli in the gastric caecum (Tieszen et al., 1983). Scanning microscopy images of trypanosomatid infections depict these protozoans completely carpeting the gerrid intestinal lining (Tieszen et al., 1983) suggesting that the effects of trypanosomatids on digestive processes described above are plausible. Reduced nutrient absorption during developmental stages in gerrids can affect growth performance and adult size. Arnqvist and Thornhill (1998) demonstrated that low food consumption by nymphs increases development time and leads to a smaller body size.
Just as low food consumption in gerrid nymphs causes an increase in development time and affects adult size, so might trypanosomatid infection. I suggest trypanosomatid infection affects nymph development in a similar fashion as has been demonstrated for food consumption.
79 Trypanosomatid infections interfere with digestive processes and result in slow nymph development and smaller adult body size possibly via the three mechanisms previously suggested.
Considering studies listed describing the effect of stress and nymph development, I suggest that nymph developmental time appears to 1) vary with the form of stress the organism experiences, and 2) result in variation with respect to body length or size. While high temperatures and crowding conditions induce fast nymph development that results in shorter adult body length, low food consumption and trypanosomatid infection slows nymph development and results in small adult gerrids.
Arnqvist and Maki (1990) studied the effect of trypanosomatid infection in Gerris odontogaster, and they found that the presence of infection reduces male gerrid vigor. Their study compared the skating ability of infected and uninfected gerrids and found that infected male gerrids could not swim against water currents as well as those that were uninfected. I suggest that the mechanism underlying this result could lie in a difference in body size between the two groups of gerrids. First, the authors did not find the effect in female gerrids, and I did not find an effect of infection in the body size of female gerrids. Second, my study included an assessment of femur leg length as a component of body size. I found that infected male gerrids had shorter femurs than uninfected individuals, and I suggest that male skating ability is hampered by the direct effect of trypanosomatid infection on male gerrid leg length.
Arnqvist and Maki (1990) also report that trypanosomatid infections increase adult mortality when the hosts are starved. These results suggest that in times of food shortage, smaller gerrids (those with trypanosomatid infections) are less likely to survive. During the
Aquarius remigis life cycle, food shortage occurs in the summer, when adults are either
80 preparing for diapause (in a univoltine population) or entering a mating period (in a bivoltine population). In either life cycle, the smaller infected male gerrids will suffer increased mortality over the uninfected larger gerrids. This scenario could increase the average size of males in a mating pool of gerrids both in univoltine (in the spring mating period) and bivoltine populations.
Male size has been shown to have significant effects on the mating dynamics of A. remigis males, as well as foraging and predation risk (summary in Chapter 3).
Of the relatively few studies that have examined the effects of trypanosomatids on gerrid fitness components, two have demonstrated a differential effect of trypanosomatids on gerrid male and females (Arnqvist and Maki, 1990; this study). Yet, males and females are equally likely to be infected with the parasites (Tieszen and Molyneux, 1989; Gurski and Ebbert, 2003; chaper 2). I can only speculate that the physiological difference between male and female gerrids is the reason for this differential effect. Further studies of the effects of trypanosomatids are needed to determine if these parasites have an effect on females.
I did not find evidence to support the hypothesis that trypanosomatid infection correlates to fluctuating asymmetry of gerrid leg lengths. This result is similar to those found for other insects. Of the studies of insect parasites and fluctuating asymmetry, most have reported no correlation between parasitism and fluctuating asymmetry (review in Moller, 1996; Ward et al.,
1998; exception in Thomas et al., 1998). Arnqvist et al. (1997) demonstrated that fluctuating asymmetry in all leg lengths of Gerris incognitos was negatively correlated with offspring survival, but he did not assess parasitic infection.
I conclude that in the Four Mile Creek watershed, trypanosomatid infections in Aquarius remigis may negatively affect male nymph development by decreasing adult male body size, probably via disruption of digestive processes. Small size, especially when coupled with
81 stressful environmental conditions, has significant effects on components of gerrid fitness.
Because gerrid trypanosomatids have the potential to significantly affect many aspects of gerrid life history, I suggest that future studies regarding gerrid fitness consider assessing the effects of parasitic infection.
82 CHAPTER 5
DISSERTATION CONCLUSION
83 This dissertation had three main goals. First, I documented the prevalence of trypanosomatid parasites in natural population of gerrids within and among annual field seasons.
Second, I addressed the question of how uninfected gerrid morphology varies spatially, temporally and with mating status. And third, I examined the effect of trypanosomatid parasites on field-caught gerrid morphological development. I will briefly summarize the findings from my research, discuss possible relationships among these findings and provide suggestions for future studies of gerrid-trypanosomatid interactions.
Effects of space and time on trypanosomatid prevalence in Aquarius remigis populations
Mating activity in gerrids was highly associated with trypanosomatid prevalence in adults. The patterns of gerrid mating activity varied within and among the three full field seasons studied: in two years the populations were univoltine whereas the third was bivoltine. I assumed these patterns were the result of varying environmental conditions among the years, primarily with respect to precipitation and temperature. In all years, trypanosomatid prevalence patterns were associated with gerrid mating patterns over time, and not gerrid density. Contrary to a common epidemiological assumption (Fuxa and Tanada, 1987), density only appeared to be important in explaining prevalence in nymphs, but not adults. Mating activities facilitate trypanosomatid transmission and allow the parasite to persist despite low host density (e.g., in the spring).
Trypanosomatid prevalence varied with host age, but did not differ between males and females, among streams or between locations within streams. These observations also indicate that the prevalence varies temporally, but not spatially in gerrid populations. Adult gerrids were more likely to be infected than nymphs: adults have more time to acquire infection and are more
84 likely to transmit it to other adults than to nymphs. Temporal variation in trypanosomatid infection has significant implications for gerrid fitness (Klingenberg et al, 1997; Arnqvist and
Maki, 1990; this study) because infection can negatively affect body size.
The effect of space and time on uninfected and infected Aquarius remigis morphology
In uninfected gerrids, morphological traits varied spatially (within and among streams) and temporally (between subsequent gerrid generations). I assume that differences in adult morphology are the result of the environmental conditions during nymph development rather than genetic differences. The adults of the two generations developed in different field seasons and most likely at different times within their respective field season. Yet, within each stream the size of morphological traits varied with generation for both male and female gerrids. Since many components of fitness are correlated with gerrid body size, I suggest that gerrid fitness will also vary with space and time.
Small body size was associated with trypanosomatid infections in male gerrids. I assume that infections interfere with nymph development and decrease adult body size probably via disruption of digestive processes. Similar to uninfected gerrid morphology, the effect of infection on gerrid male morphology was variable with respect to spatial factors. Within a generation, the relative size of morphological traits with respect to infection varied with gerrid population. I concluded that although small body size caused by parasitic infection can have significant effects on gerrid fitness, further investigation is needed to confirm this effect.
85 Direction of future studies
Although this dissertation addressed the relationship among many potential factors and trypanosomatid prevalence, several more are likely to be import in explaining observed patterns.
Little is known about gerrid-trypanosomatid winter ecology. This dissertation documented the fall and spring prevalence for two overwintering episodes and found inconsistent prevalence patterns between the two periods. Field studies are needed to document infection rates over the winter to determine what factors are important in explaining prevalence during gerrid diapause. Although studies suggest that trypanosomatids survive the winter inside their hosts (Teizsen and Molyneux, 1989; this study), empirical evidence remains to be obtained.
The intensity of parasitic infection may also be an important factor in gerrid- trypanosomatid interactions. Not only may parasite intensity be an important factor in explaining prevalence patterns, but it may also have significant affects on gerrid life history. For example, Arnqvist (1992) reported trypanosomatid intensity in gerrids had a significant effect on mating success. Although he did not find an effect on gerrid morphology, only light intensity infections were present in this system. I observed a range of parasite loads in gerrids considered in this study, with many infections being very heavy. I suggest that in studies infection intensity be assessed and evaluated for its role in gerrid-trypanosomatid interactions.
This dissertation only characterized trypanosomatid species to a recognizable taxonomic unit. I was able to recognize at least two morphotypes in trypanosomatid samples, but these morphotypes were neither distinguished in this study nor identified further taxonomically. Just as parasite intensity may differentially affect gerrid life history traits, so might the presence of multiple parasite species within one host or infections of different species among hosts. I suggest that until adequate molecular techniques for taxonomic identification of insect
86 trypanosomatids are available, qualifying infections by morphotypes would provide insight into the effects that different species may exert on gerrid hosts.
Additional field studies of gerrid-trypanosomatid interactions would foster a better understanding of the gerrid-trypanosomatid relationship. These studies would include assessing the effect of parasites on gerrid reproductive success and survival. Samples of infected and uninfected field-caught nymphs could be monitored in the lab for molting, eclosing, mating and reproductive success as well as survival. Or, these same factors could be monitored in a
“natural” setting but in enclosures similar to those used by Klingenberg et al. (1997). Controlled experiments with laboratory reared and infected gerrids would complement observations made under field conditions.
Much work remains before the relationship between gerrid hosts and their trypanosomatids parasites is fully understood. Once the interactions between these organisms are identified, then work on the role they play in the community may commence. The research in this dissertation has established some of the basic interactions between gerrids and trypanosomatids and provides a springboard for further investigation of the role of insect parasites in animal communities.
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102 Table 1: Breakdown of data set from the number of gerrids caught to the number actually used in prevalence analyses. Year 1999 2000 Gerrid stage Adult Nymph Adult Nymph # Caught 1832 558 2289 991
male 928 N3 23 male 334 N3 135 Adult sex or nymph female 904 N4 235 female 236 N4 392 age N5 300 N5 464
# Gerrids for which a 1794 529 2268 987 slide was made # Gerrids removed due to 39 204 148 258 unreliable slide # Gerrids included in 1755 325 2120 729 prevalence analyses Year 2001 2002 Gerrid stage Adult Nymph Adult Nymph # Caught 570 13 1011 369 N2 5 male 334 N3 1 male 576 N3 58 Adult sex or nymph female 236 N4 10 female 435 N4 110 age N5 2 N5 122 N4/N5 74 # Gerrids for which a 561 8 945 244 slide was made # Gerrids removed due to 8 1 42 0 unreliable slide # Gerrids included in 553 7 903 244 prevalence analyses
103 Table 2: Experimental factors potentially related to (A) trypanosomatid prevalence in gerrids and (B) gerrid density. Category Factor Data type Nested? Expectation A Planned Spatial Stream nominal no equal Temporal Month ordinal w/in stream equal Spatial Location nominal w/in stream and month up>down Population Sex nominal w/in stream, month and site equal Population Stage nominal w/in stream, month and site equal Population Density continuous w/in stream, month and site positive correlation Unplanned Temporal Winter ordinal w/in stream, month and site equal Population Mating nominal w/in stream, month, site and sex equal
B Planned Spatial Stream nominal no equal Temporal Month ordinal w/in stream equal Spatial Location nominal w/in stream and month equal Population Stage nominal w/in stream and month equal
104 Table 3: Number, sex, age, and relative density (catch per unit effort, CPUE*) estimates of gerrids collected in four Oxford, Ohio area streams. Location in Adult CPUE Nymph CPUE Stream Date stream** Male Female (#/hr) Nymph (#/hr) Harker's 4/3/99 down 10 9 - 0 - 4/21/99 down 11 6 - 0 - 5/23/99 down 10 1 15 50 200 up 17 11 41 49 213 6/22/99 down 26 24 111 30 67 up 27 23 119 30 71 7/22/99 down 33 17 119 7 17 up 21 29 152 0 0 9/2/99 down 30 20 79 14 22 up 20 30 86 1 2 10/10/99 down 29 18 94 1 2 up 21 31 90 0 0 Darr's 5/20/99 down 27 12 19 31 45 up 26 11 20 29 42 6/22/99 down 21 28 175 31 111 up 12 37 272 30 167 7/21/99 down 26 24 86 4 7 up 24 26 152 1 3 9/2/99 down 27 23 132 0 0 up 28 22 91 0 0 10/10/99 down 14 14 67 0 0 up 20 30 100 1 2 Coulter's 5/21/99 down 16 20 27 30 68 up 17 11 25 30 79 6/21/99 down 25 25 59 30 35 up 23 27 106 30 64 7/21/99 down 21 29 200 0 0 up 20 30 152 0 0 9/3/99 down 26 24 200 1 4 up 16 19 117 2 7 10/11/99 down 29 21 75 0 0 up 26 24 179 0 0 Collin's 5/21/99 down 23 22 73 30 143 up 31 19 56 30 100 105 6/21/99 down 21 28 61 30 38 up 24 25 94 31 60 7/22/99 down 24 38 189 0 0 up 25 28 126 0 0 9/2/99 down 27 23 167 1 3 up 28 11 93 0 0 10/12/99 down 26 24 79 1 2 up 22 25 65 3 4 Harker's 3/23/00 up 30 23 60 0 0 down 6 4 27 0 0 4/20/00 up 25 24 55 0 0 down 6 2 23 0 0 5/23/00 up 0 0 0 30 257 down 1 0 5 30 150 6/23/00 up 23 29 208 52 92 down 19 38 86 48 72 7/20/00 up 29 26 254 10 50 down 27 24 87 37 63 9/8/00 down 25 23 206 50 91 9/9/00 up 26 26 156 8 24 10/15/00 up 28 23 99 4 8 down 23 27 176 2 7 Darr's 3/23/00 up 25 17 65 0 0 down 26 26 65 0 0 4/24/00 up 18 13 56 0 0 down 20 18 46 0 0 5/22/00 up 7 2 27 29 249 down 3 6 25 30 257 6/22/00 up 26 28 108 50 79 down 25 29 108 50 100 7/20/00 up 31 23 125 4 9 down 21 33 125 27 62 9/9/00 up 31 19 231 20 55 down 27 22 245 50 100 10/13/00 up 25 27 95 15 27 down 34 21 206 6 23 Coulter's 3/23/00 up 20 11 65 0 0 down 14 9 45 0 0 4/24/00 up 2 7 28 0 0 down 1 1 9 0 0
106 5/22/00 up 6 3 68 30 143 down 5 8 27 30 360 6/22/00 up 32 21 59 50 150 down 32 22 324 50 88 7/20/00 up 32 18 86 0 0 down 34 20 130 0 0 9/9/00 up 26 28 101 38 71 down 26 29 330 12 72 10/13/00 up 22 26 169 3 11 down 32 18 130 4 10 Collin's 3/22/00 up 32 18 53 0 0 down 24 26 57 0 0 4/19/00 up 30 17 71 0 0 down 27 23 73 0 0 5/22/00 down 17 3 50 30 225 5/23/00 up 11 6 39 30 360 6/23/00 up 22 30 95 13 23 down 33 23 216 50 73 7/21/00 up 31 21 284 0 0 down 25 27 390 0 0 9/8/00 up 30 20 214 34 85 down 33 15 262 51 85 10/15/00 up 20 28 93 3 6 down 24 28 173 11 37 Harker's 9/29/01 up 27 29 193 0 0 down 31 26 204 7 28 10/31/01 up 31 22 106 0 0 down 29 21 115 0 0 Darr's 9/27/01 up 35 15 125 0 0 down 24 29 133 0 0 10/30/01 up 6 3 32 0 0 down 17 9 87 0 0 Coulter's 9/28/01 up 26 25 109 1 3 down 33 18 93 3 9 10/31/01 up 9 3 45 0 0 Collin's 9/26/01 up 29 20 98 1 2 down 37 16 127 1 2 Harker's 3/8/02 up 27 21 290 0 0 down 29 21 100 0 0 4/11/02 up 33 19 284 0 0
107 down 32 22 295 0 0 5/10/02 up 29 22 146 0 0 down 12 2 49 0 0 6/9/02 up 29 5 50 26 38 down 4 1 14 16 46 7/11/02 up 22 27 155 52 41 down 22 28 130 50 94 7/26/02 middle 19 23 - 74 - 10/11/02 up 40 14 99 0 0 Darr's 3/8/02 down 36 20 67 0 0 4/11/02 down 28 22 136 0 0 5/10/02 down 35 15 91 0 0 6/9/02 down 13 2 19 55 70 7/11/02 down 24 27 250 45 95 9/5/02 down 37 33 - 0 0 Collin's 3/8/02 down 24 26 104 0 0 4/11/02 down 12 10 78 0 0 5/10/02 down 18 14 66 0 0 7/11/02 down 25 25 97 46 66 9/12/02 down 26 26 51 5 12 * CPUE is the number of gerrids caught per hour. ** up=upstream location within the stream, down=downstream location
108 Table 4: Monthly averages (number of collections, standard deviation) for number of gerrids collected and analyzed in Oxford, Ohio area streams. Year Month Adult Nymph Collected Analyzed Collected Analyzed 1999 April 36.0 (1, 0.0) 36.0 (1, 0.0) 0.0 (0, 0.0) 0.0 (0, 0.0) May 34.3 (8, 11.3) 31.5 (8, 10.2) 34.9 (8, 8.5) 26.5 (8, 7.6) June 49.5 (8, 0.5) 46.0 (8, 4.9) 30.3 (8, 0.4) 11.8 (8, 5.9) July 51.9 (8, 4.0) 51.9 (8, 4.0) 4.0 (3, 2.4) 4.0 (3, 2.4) September 46.8 (8, 5.7) 43.3 (8, 5.3) 3.8 (6, 5.1) 0 (0, 0.0) October 46.8 (8, 7.3) 46.8 (8, 7.3) 1.5 (4, 0.9) 1.5 (4, 0.9) 2000 March 38.9 (8, 15.9) 35.5 (8, 14.9) 0.0 (0, 0.0) 0.0 (0, 0.0) April 29.3 (8, 20.1) 26.8 (8, 18.7) 0.0 (0, 0.0) 0.0 (0, 0.0) May 11.1 (7, 6.2) 7.0 (7, 6.4) 29.9 (8, 0.4) 22.6 (8, 3.2) June 54.0 (8, 1.4) 48.8 (8, 4.1) 45.4 (8, 13.1) 37.8 (8, 13.4) July 52.8 (8, 1.8) 50.3 (8, 2.4) 19.5 (4, 15.2) 13.4 (4, 9.8) September 50.8 (8, 2.7) 49.4 (8, 2.4) 32.9 (8, 17.6) 24.1 (7, 14.1) October 50.8 (8, 2.3) 48.3 (8, 2.2) 6.0 (8, 4.6) 4.8 (8, 3.5) 2001 September 52.5 (8, 2.8) 50.8 (8, 1.8) 2.6 (5, 2.6) 1.4 (5, 0.5) October 30.0 (5, 20.7) 29.4 (5, 19.9) 0.0 (0, 0.0) 0.0 (0, 0.0) 2002 March 53.5 (4, 4.1) 52.5 (4, 4.8) 0.0 (0, 0.0) 0.0 (0, 0.0) April 44.5 (4, 15.1) 40.3 (4, 14.8) 0.0 (0, 0.0) 0.0 (0, 0.0) May 36.8 (4, 17.5) 26.8 (4, 9.4) 0.0 (0, 0.0) 0.0 (0, 0.0) June 18.0 (3, 14.7) 17.3 (3, 15.3) 32.3 (3, 20.3) 19.3 (3, 7.6) July 48.4 (5, 3.6) 40.2 (5, 6.6) 53.4 (5, 11.9) 37.2 (5, 16.7) September 61.0 (2, 12.7) 59.0 (2, 14.1) 5.0 (1, 0.0) 0.0 (0, 0.0) October 54.0 (1, 0.0) 54.0 (1, 0.0) 0.0 (0, 0.0) 0.0 (0, 0.0)
109 Table 5: Average monthly and seasonal gerrid density estimated by catch per unit effort (# gerrids caught/hour ± standard deviation)
1999 Month Adult Nymph May 35 ± 20.6 111 ± 67.0 June 125 ± 69.8 76 ± 43.3 July 147 ± 37.0 3 ± 6.0 September 121 ± 43.4 5 ± 7.4 October 94 ± 36.7 1 ± 1.5 (n=8)
Season Adult Nymph Spring 35 ± 19.2 111 ± 62.7 Summer 136 ± 53.4 40 ± 46.7 Fall 107 ± 39.9 3 ± 5.3 (spring n=8, summer and fall n=16)
2000 Month Adult Nymph March 55 ± 13.2 0 ± 0.0 April 45 ± 23.1 0 ± 0.0 May 30 ± 22.3 250 ± 81.4 June 151 ± 90.2 85 ± 35.3 July 185 ± 111.0 23 ± 29.7 September 218 ± 68.7 73 ± 24.1 October 143 ± 44.0 16 ± 11.4 (n=8)
Season Adult Nymph Spring 43 ± 21.8 250 ± 81.4 Summer 168 ± 99.3 54 ± 44.8 Fall 180 ± 68.0 45 ± 34.5 (spring: adult n=24, nymph n=8; summer and fall n=16)
2001 Month Adult Nymph September 135 ± 41.6 6 ± 9.6 October 48 ± 48.6 0 ± 0.0 (n=8)
110 Season Adult Nymph Fall 92 ± 62.7 3 ± 7.1 (n=8)
2002 Month Adult Nymph March 140 ± 101.2 0 ± 0.0 April 198 ± 108.1 0 ± 0.0 May 88 ± 42.3 0 ± 0.0 June 21 ± 21.1 39 ± 29.0 July* 158 ± 65.8 74 ± 25.8 September 13 ± 25.5 3 ± 6.0 October** 25 ± 49.5 0 ± 0.0 (n=4)
Season Adult Nymph Spring 142 ± 93.2 0 ± 0.0 Summer* 89 ± 86.2 56 ± 31.7 Fall** 19 ± 37.0 2 ± 4.2 (spring n=12, summer and fall n=8) * does not include Middle Harker's collection ** does not include Darr's collection
111 Table 6: ANOVA results for the effect of independent variables on adult and nymph gerrid density. Mean Source* d.f. Square F ratio** p>F Adult year 3 18.94 1.767 0.1613 stream 3 4.20 0.3918 0.7592 month[stream] 24 31.34 2.923 0.0003 location[stream, month] 28 5.40 0.5033 0.9773 error 71 10.72 total 129
Nymph year 3 103.5 10.36 <0.0001 stream 3 5.91 0.5915 0.6226 month[stream] 24 68.54 6.859 <0.0001 location[stream, month] 28 4.90 0.4903 0.9811 error 72 9.99 total 130 * Nested factors are indicated as B[A] where B is nested within A. ** Error and Mean Square used to calculate F ratio.
112 Table 7: Years 1999 - 2002 contingency tables for non- parametric paired sign tests of prevalence vs. stream location.
1999 observed expected* up > downstream 10 9.5 downstream > up 9 9.5 (n=19, d.f. =1, X 2 =0.05, p =0.82)
2000 observed expected up > downstream 18 13.5 downstream > up 9 13.5 (n=27, d.f. =1, X 2 =3.06, p =0.0803)
2001 observed expected up > downstream 5 3 downstream > up 1 3 (n=6, d.f. =1, X 2 =2.91, p =0.088)
2002 observed expected up > downstream 2 1.5 downstream > up 1 1.5 (n=3, d.f. =1, X 2 =0.34, p =0.56)
All observed expected Years up > downstream 35 27.5 downstream > up 20 27.5 (n=55, d.f. =1, X 2 =4.13, p =0.0418) *Observed results were compared against the expectation that prevalence did not differ between up- and downstream locations.
113 Table 8: Years 1999 - 2002 contingency tables for non-parametric paired sign tests of prevalence vs. gerrid mating status.
1999 observed expected* mating > non-mating 7 5 non-mating > mating 3 5 (n=10, d.f. =1, X 2 =1.65, p =0.20)
2000 observed expected mating > non-mating 16 11.5 non-mating > mating 7 11.5 (n=23, d.f. =1, X 2 =3.62, p =0.0572)
2002 observed expected mating > non-mating 5 6.5 non-mating > mating 8 6.5 (n=13, d.f. =1, X 2 =0.70, p =0.40)
All observed expected Years mating > non-mating 28 23 non-mating > mating 18 23 (n=46, d.f. =1, X 2 =2.19, p =0.1388) *Observed results were compared against the expectation that prevalence did not differ between mating and non-mating gerrids.
114 Table 9: Years 1999 - 2002 contingency tables for non- parametric paired sign tests of prevalence vs. gerrid sex.
1999 observed expected* female>male 22 18 male>female 14 18 (n=36, d.f. =1, X 2 =1.79, p =0.18)
2000 observed expected female>male 27 24 male>female 21 24 (n=48, d.f. =1, X 2 =0.75, p =0.39)
2001 observed expected female>male 5 6 male>female 7 6 (n=12, d.f. =1, X 2 =0.33, p =0.56)
2002 observed expected female>male 5 9.5 male>female 14 9.5 (n=19, d.f. =1, X 2 =4.44, p =0.0351)
All observed expected Years female>male 59 57.5 male>female 56 57.5 (n=115, d.f. =1, X 2 =0.078, p =0.7797) *Observed results were compared against the expectation that prevalence did not differ between the two sexes.
115 Table 10: Years 1999 - 2002 contingency tables for non- parametric paired sign tests of prevalence vs. overwintering seasons.
1999- observed expected 2000 fall>spring 1 4 spring>fall 7 4 (n=8, d.f. =1, X 2 =5.06, p =0.02446)
2001- observed expected 2002 fall>spring 6 3 spring>fall 0 3 (n=6) *Observed results were compared against the expectation that prevalence did not differ between fall and spring.
116 Table 11: Years 1999 - 2002 contingency tables for non- parametric paired sign tests of prevalence vs. gerrid stage.
1999 observed expected* adult>nymph 19 10.5 nymph>adult 2 10.5 (n=21, d.f. =1, X 2 =15.90, p <0.0001)
2000 observed expected adult>nymph 27 17 nymph>adult 7 17 (n=34, d.f. =1, X 2 =12.56, p =0.0004)
2001 observed expected adult>nymph 4 2.5 nymph>adult 1 2.5 (n=5, d.f. =1, X 2 =1.93, p =0.17)
2002 observed expected adult>nymph 3 2.5 nymph>adult 2 2.5 (n=5, d.f. =1, X 2 =0.20, p =0.65)
All observed expected Years adult>nymph 53 32.5 nymph>adult 12 32.5 (n=65, d.f. =1, X 2 =27.9, p <0.0001) *Observed results were compared against the expectation that prevalence did not differ between nymphs and adults.
117 Table 12: Monthly trypanosomatid prevalence in adult gerrids per stream.
% Infected ± binomial Year Stream Month #Infected error n 1999 Harker's April 5 13.9 ± 5.8 36 May 17 44.7 ± 8.2 38 June 6 6.1 ± 2.4 98 July 6 6.0 ± 2.4 100 September 12 12.8 ± 3.4 94 October 10 10.1 ± 3.0 99 total 50 12.0 ± 1.5 465 Darr's May 25 37.3 ± 5.9 67 June 13 14.3 ± 3.7 91 July 12 12.0 ± 3.2 100 September 11 11.8 ± 3.3 93 October 15 19.2 ± 4.5 78 total 76 17.7 ± 1.8 429 Coulter's May 21 36.2 ± 6.3 58 June 11 13.1 ± 3.7 84 July 4 4.0 ± 2.0 100 September 1 1.4 ± 1.4 74 October 10 10.0 ± 3.0 100 total 47 11.3 ± 1.6 416 Collin's May 55 61.8 ± 5.2 89 June 13 13.7 ± 3.5 95 July 14 12.2 ± 3.1 115 September 12 14.1 ± 3.8 85 October 15 15.5 ± 3.7 97 total 109 22.7 ± 1.9 481 Total 288 16.1 ± 0.9 1791
2000 Harker's March 8 13.6 ± 4.5 59 April 13 24.5 ± 5.9 53 May 1 100.0 ± 0.0 1 June 16 16.2 ± 3.7 99 July 34 35.1 ± 4.8 97 September 17 17.5 ± 3.9 97 October 25 25.3 ± 4.4 99
118 total 114 22.6 ± 1.9 505 Darr's March 25 29.1 ± 4.9 86 April 18 30.0 ± 5.9 60 May 9 90.0 ± 9.5 10 June 58 56.3 ± 4.9 103 July 55 53.9 ± 4.9 102 September 30 31.3 ± 4.7 96 October 39 39.0 ± 4.9 100 total 234 42.0 ± 2.1 557 Coulter's March 3 6.4 ± 3.6 47 April 0 0.0 ± 0.0 10 May 4 57.1 ± 18.7 7 June 26 26.0 ± 4.4 100 July 34 34.3 ± 4.8 99 September 33 31.4 ± 4.5 105 October 38 39.2 ± 5.0 97 total 138 29.7 ± 2.1 138 Collin's March 30 32.6 ± 4.9 92 April 25 27.5 ± 4.7 91 May 20 64.5 ± 8.6 31 June 8 9.1 ± 3.1 88 July 29 27.9 ± 4.4 104 September 14 14.4 ± 3.6 97 October 11 12.2 ± 3.4 90 total 137 23.1 ± 1.7 593 Total 623 29.4 ± 1.0 2120
2001 Harker's September 31 29.5 ± 4.5 105 October 24 24.0 ± 4.3 100 total 55 26.8 ± 3.1 205 Darr's September 37 37.8 ± 4.9 98 October 12 34.3 ± 8.0 35 total 49 36.8 ± 4.2 133 Coulter's September 34 33.3 ± 4.7 102 October 1 8.3 ± 8.0 12 total 35 30.7 ± 4.3 114 Collin's September 37 36.6 ± 4.8 101 October 0 0.0 ± 0.0 0 total 37 36.6 ± 4.8 101 Total 176 31.8 ± 2.0 553
119 2002 Harker's March 10 9.4 ± 2.8 107 April 14 14.0 ± 3.5 100 May 7 14.9 ± 5.2 47 June 19 50.0 ± 8.1 38 July 0 0.0 ± 0.0 117 September 0 0.0 ± 0.0 0 October 6 11.1 ± 4.3 54 total 56 12.1 ± 1.5 463 Darr's March 9 16.4 ± 5.0 55 April 8 19.1 ± 6.1 42 May 4 13.8 ± 6.4 29 June 8 57.1 ± 13.2 14 July 1 2.5 ± 2.5 40 September 5 7.3 ± 3.1 69 October 0 0.0 ± 0.0 0 total 35 14.1 ± 2.2 249 Collin's March 7 14.6 ± 5.1 48 April 1 5.3 ± 5.1 19 May 7 22.6 ± 7.5 31 June 0 0.0 ± 0.0 0 July 3 6.8 ± 3.8 44 September 6 12.2 ± 4.7 49 October 0 0.0 ± 0.0 0 total 24 12.6 ± 2.4 191 Total 115 12.7 ± 1.1 903
120 Table 13: Monthly trypanosomatid prevalence in nymph gerrids per stream. % Infected ± binomial Year Stream Month #Infected error n 1999 Harker's April 0 0.0 ± 0.0 0 May 0 0.0 ± 0.0 73 June 0 0.0 ± 0.0 30 July 0 0.0 ± 0.0 7 September 0 0.0 ± 0.0 0 October 0 0.0 ± 0.0 1 total 0 0.0 ± 0.0 111 Darr's May 0 0.0 ± 0.0 56 June 0 0.0 ± 0.0 20 July 1 20.0 ± 17.9 5 September 0 0.0 ± 0.0 0 October 0 0.0 ± 0.0 1 total 1 1.2 ± 1.2 82 Coulter's May 0 0.0 ± 0.0 50 June 0 0.0 ± 0.0 29 July 0 0.0 ± 0.0 0 September 0 0.0 ± 0.0 0 October 0 0.0 ± 0.0 0 total 0 0.0 ± 0.0 79 Collin's May 0 0.0 ± 0.0 34 June 0 0.0 ± 0.0 15 July 0 0.0 ± 0.0 0 September 0 0.0 ± 0.0 0 October 2 50.0 ± 25.0 4 total 2 3.8 ± 2.6 53 Total 3 0.9 ± 0.5 325
2000 Harker's March 0 0.0 ± 0.0 0 April 0 0.0 ± 0.0 0 May 5 10.2 ± 4.3 49 June 9 14.1 ± 4.4 64 July 0 0.0 ± 0.0 27 September 15 40.5 ± 8.1 37 October 0 0.0 ± 0.0 6
121 total 29 15.9 ± 2.7 183 Darr's March 0 0.0 ± 0.0 0 April 0 0.0 ± 0.0 0 May 2 4.4 ± 3.0 46 June 28 30.8 ± 4.8 91 July 5 41.7 ± 14.2 12 September 11 42.3 ± 9.7 26 October 4 25.0 ± 10.8 16 total 50 26.2 ± 3.2 191 Coulter's March 0 0.0 ± 0.0 0 April 0 0.0 ± 0.0 0 May 1 2.7 ± 2.7 37 June 2 2.1 ± 1.5 96 July 0 0.0 ± 0.0 0 September 12 37.5 ± 8.6 32 October 2 33.3 ± 19.2 6 total 17 9.9 ± 2.3 171 Collin's March 0 0.0 ± 0.0 0 April 0 0.0 ± 0.0 0 May 3 6.1 ± 3.4 49 June 5 9.8 ± 4.2 51 July 0 0.0 ± 0.0 0 September 6 8.1 ± 3.2 74 October 3 30.0 ± 14.5 10 total 17 9.2 ± 2.1 184 Total 113 15.5 ± 1.3 729
2001 Harker's September 0 0.0 ± 0.0 2 October 0 0.0 ± 0.0 0 total 0 0.0 ± 0.0 2 Darr's September 0 0.0 ± 0.0 0 October 0 0.0 ± 0.0 0 total 0 0.0 ± 0.0 0 Coulter's September 1 33.3 ± 27.2 3 October 0 0.0 ± 0.0 0 total 1 33.3 ± 27.2 3 Collin's September 0 0.0 ± 0.0 2 October 0 0.0 ± 0.0 0 total 0 0.0 ± 0.0 2 Total 1 14.3 ± 13.2 7
122 2002 Harker's March 0 0.0 ± 0.0 0 April 0 0.0 ± 0.0 0 May 0 0.0 ± 0.0 0 June 0 0.0 ± 0.0 32 July 0 0.0 ± 0.0 134 September 0 0.0 ± 0.0 0 October 0 0.0 ± 0.0 0 total 0 0.0 ± 0.0 166 Darr's March 0 0.0 ± 0.0 0 April 0 0.0 ± 0.0 0 May 0 0.0 ± 0.0 0 June 0 0.0 ± 0.0 26 July 2 8.0 ± 5.4 25 September 0 0.0 ± 0.0 0 October 0 0.0 ± 0.0 0 total 2 3.9 ± 2.7 51 Collin's March 0 0.0 ± 0.0 0 April 0 0.0 ± 0.0 0 May 0 0.0 ± 0.0 0 June 0 0.0 ± 0.0 0 July 4 14.8 ± 6.8 27 September 0 0.0 ± 0.0 0 October 0 0.0 ± 0.0 0 total 4 14.8 ± 6.8 27 Total 6 2.5 ± 1.0 244
123 Table 14: Average monthly and seasonal trypanosomatid prevalence across all sites (percent ± binomial error). Includes Mid Harker’s July collection. 1999 Month Adult n Nymph n April 13.9 ± 3.0 36 0.0 ± 0.0 0 May 46.8 ± 3.1 252 0.0 ± 0.0 212 June 11.7 ± 1.7 368 0.0 ± 0.0 94 July 8.7 ± 1.4 415 8.3 ± 8.0 12 September 10.4 ± 1.6 346 0.0 ± 0.0 0 October 13.4 ± 1.8 374 33.3 ± 19.2 6
Season Adult n Nymph n Spring 42.7 ± 2.9 252 0.0 ± 0.0 212 Summer 10.1 ± 1.1 783 0.9 ± 0.9 106 Fall 11.9 ± 1.2 720 33.3 ± 19.2 6
2000 Month Adult n Nymph n March 23.2 ± 2.5 284 0.0 ± 0.0 0 April 26.2 ± 3.0 214 0.0 ± 0.0 0 May 69.4 ± 6.6 49 6.1 ± 1.8 181 June 27.7 ± 2.3 390 14.6 ± 2.0 302 July 37.8 ± 2.4 402 12.8 ± 5.3 39 September 23.8 ± 2.1 395 26.0 ± 3.4 169 October 29.3 ± 2.3 386 23.7 ± 6.9 38
Season Adult n Nymph n Spring 28.5 ± 1.9 547 6.1 ± 1.8 181 Summer 32.8 ± 1.7 792 14.4 ± 1.9 341 Fall 26.5 ± 1.6 781 25.6 ± 3.0 207
2001 Month Adult n Nymph n September 34.2 ± 2.4 406 14.3 ± 13.2 7 October 25.2 ± 3.6 147 0.0 ± 0.0 0
Season Adult n Nymph n Fall 31.8 ± 2.0 553 14.3 ± 13.2 7
2002 Month Adult n Nymph n 124 March 12.4 ± 2.3 210 0.0 ± 0.0 0 April 14.3 ± 2.8 161 0.0 ± 0.0 0 May 16.8 ± 3.6 107 0.0 ± 0.0 0 June 51.9 ± 6.9 52 0.0 ± 0.0 58 July 2.0 ± 1.0 201 3.2 ± 1.3 186 September 9.3 ± 2.7 118 0.0 ± 0.0 0 October 11.1 ± 4.3 54 0.0 ± 0.0 6
Season Adult n Nymph n Spring 14.0 ± 1.6 478 0.0 ± 0.0 0 Summer 12.3 ± 2.1 253 2.5 ± 1.0 244 Fall 9.9 ± 2.3 172 0.0 ± 0.0 0
125 Table 15: Effect of independent variables on trypanosomatid prevalence in adults and nymphs (Logistic model/Liklihood ratio Chi-square analysis). Source* d.f. X 2 p>F Adult 1999 model 20 208.2 <0.0001 stream 3 23.17 <0.0001 month[stream] 17 179.6 <0.0001
2000 model 27 206.6 <0.0001 stream 3 46.5 <0.0001 month[stream] 24 142.8 <0.0001
2001 model 5 8.567 0.1276 stream 2 4.875 0.0874 month[stream] 3 4.805 0.1866
2002 stream 6 80.31 <0.0001
Nymph 2000 model 9 52.91 <0.0001 stream 1 9.882 0.0017 month[stream] 8 46.85 <0.0001 * Nested factors are indicated as B[A] where B is nested within A.
126 Table 16: MANOVA results for the effect of independent variables on (A) adult, (B) male and (C) female morphology. Source d.f. l p>F A All model 12 1.3207 <0.0001 Adults location 4 0.4711 <0.0001 sex 4 143.9 <0.0001 sex*location 4 0.0823 0.0384
B Male model 4 0.5938 <0.0001 location 4 0.5938 <0.0001
C Female model 4 0.4756 0.0002 location 4 0.4786 0.0002
127 Table 17: Mean and standard deviation of body component length (mm) between Harker's Run upstream and downstream locations for male and female gerrids.
Male Female Body Component Harker Up Harker Down Harker Up Harker Down Middle femur x 10.08 10.38 10.08 10.43 (rt) S.D. 0.50 0.49 0.47 0.31 range 9.11-10.94 9.32-11.32 9.17-10.88 9.71-10.93 N 43 31 32 28 Thorax x 5.49 5.70 5.49 6.07 S.D. 0.25 0.19 0.25 0.13 range 5.08-6.06 5.30-5.95 5.38-6.30 5.71-6.27 N 43 31 32 28 Genitalia x 2.12 2.21 0.61 0.67 S.D. 0.10 0.08 0.08 0.07 range 1.94-2.28 2.05-2.35 0.47-0.82 0.50-0.76 N 43 31 32 28 Head x 1.44 1.43 1.48 1.51 S.D. 0.05 0.06 0.05 0.04 range 1.35-1.55 1.29-1.52 1.34-1.55 1.39-1.58 N 43 31 32 28
128 Table 18: MANOVA results for the effect of independent variables on (A) adult, (B) male, (C) female, (D) Collin's Run male and female, (E) Darr's Run male and female and (F) Harker's Run male and female morphology. Source l d.f. p>F A All model 1.5764 44 <0.0001 Adults generation 0.1892 4 <0.0001 sex 109.8 4 <0.0001 stream 0.1908 8 <0.0001 generation*sex 0.0078 4 0.7622 generation*stream 0.2432 8 <0.0001 sex*stream 0.0528 8 0.1180 generation*sex*stream 0.0288 8 0.5418
B Male model 0.6279 20 <0.0001 generation 0.2918 4 <0.0001 stream 0.2057 8 0.0001 generation*stream 0.2377 8 <0.0001
C Female Whole model 0.6479 20 <0.0001 generation 0.1315 4 0.0194 stream 0.2813 8 0.0003 generation*stream 0.2990 8 0.0001
D Collin's Male generation 1.1865 4 0.0003
Female generation 1.1450 4 <0.0001
E Darr's Male generation 0.9619 4 <0.0001
Female generation 0.5822 4 0.0024
F Harker's Male generation 0.1975 4 0.0247
Female generation 0.2291 4 0.2931
129 Table 19: Mean and standard deviation of body component length (mm) between subsequent generations of male and female gerrids Male Female Body Component P1 G1 P1 G1 Middle femur x 9.82 10.10 9.76 10.01 (rt) S.D. 0.42 0.54 0.39 0.54 range 8.88-10.79 9.01-11.29 9.15-11.18 8.61-11.07 N 61 89 40 64 Thorax x 5.38 5.56 5.70 5.84 S.D. 0.19 0.27 0.22 0.25 range 4.93-5.81 4.96-6.13 5.38-6.18 5.04-6.32 N 61 89 40 64 Genitalia x 2.07 2.11 0.58 0.61 S.D. 0.08 0.10 0.08 0.07 range 1.85-2.27 1.83-2.41 0.46-0.85 0.46-0.84 N 61 88 40 63 Head x 1.41 1.46 1.44 1.48 S.D. 0.05 0.06 0.05 0.07 range 1.30-1.54 1.31-1.65 1.34-1.52 1.26-1.65 N 61 88 40 63
130 Table 20: Mean and standard deviation of body component length (mm) of uninfected (A) male and (B) female gerrids. A Body Male P1 G1 Component Harker's Darr's Collin's Harker's Darr's Collin's
Middle x 9.90 9.68 9.81 9.56 10.43 10.08 femur (rt) S.D. 0.46 0.38 0.35 0.26 0.46 0.50 range 9.11-10.79 8.88-10.42 9.25-10.39 9.04-9.90 9.42-11.29 9.01-10.93 N 31 18 12 20 34 35 Thorax x 5.38 5.31 5.44 5.33 5.71 5.53 S.D. 0.19 0.18 0.19 0.14 0.23 0.26 range 5.08-5.81 4.93-5.64 5.16-5.69 5.03-5.65 4.96-6.13 4.96-5.93 N 31 18 12 20 34 35 Genitalia x 2.09 2.07 2.05 2.06 2.16 2.10 S.D. 0.08 0.06 0.09 0.11 0.05 0.09 range 1.94-2.27 1.95-2.18 1.85-2.19 1.83-2.41 1.94-2.31 1.91-2.33 N 31 18 12 20 33 35 Head x 1.43 1.42 1.36 1.42 1.49 1.46 S.D. 0.05 0.05 0.04 0.04 0.05 0.07 range 1.35-1.54 1.33-1.50 1.30-1.43 1.35-1.49 1.41-1.65 1.32-1.61 N 31 18 12 20 33 35
B Female P1 G1 Harker's Darr's Collin's Harker's Darr's Collin's
Middle x 9.69 9.22 9.99 10.00 10.24 9.74 femur (rt) S.D. 0.36 0.31 0.44 0.57 0.44 0.51 range 9.17-10.43 9.15-10.19 9.60-11.18 8.75-10.85 8.61-10.83 8.88-11.07 N 15 13 12 13 28 23 Thorax x 5.62 5.62 5.89 5.85 5.90 5.75 S.D. 0.18 0.21 0.05 0.33 0.24 0.19 range 5.38-5.92 5.38-6.04 5.60-6.18 5.20-6.32 5.04-6.30 5.40-6.24 N 15 13 12 13 28 23 Genitalia x 0.55 0.59 0.61 0.56 0.65 0.60 S.D. 0.05 0.05 0.12 0.04 0.07 0.06 range 0.47-0.64 0.52-0.70 0.46-0.85 0.49-0.63 0.52-0.84 0.46-0.67 N 15 13 12 13 27 23 Head x 1.45 1.47 1.40 1.48 1.49 1.47 S.D. 0.05 0.04 0.02 0.06 0.09 0.05 range 1.34-1.52 1.39-1.52 1.36-1.44 1.37-1.58 1.26-1.65 1.37-1.64 N 15 13 12 13 27 23
131 Table 21: ANOVA results for the effect of independent variables on (A) middle femur and (B) hind femur fluctuating asymmetry. Sum of Source d.f. squares F ratio p>F A Middle generation 1 0.0040 1.5587 0.2130 Femur stream 2 0.0013 0.2427 0.7847 generation*stream 2 0.0039 0.7524 0.4723 error 246 0.6385 total 251 0.6483
B Hind generation 1 0.0009 0.3808 0.5377 Femur stream 2 0.0053 1.1455 0.3197 generation*stream 2 0.0069 1.1455 0.3197 error 246 0.5677 total 251 0.5797
132 Table 22: MANOVA results for the effect of independent variables on adult mating morphology. Source l d.f. p>F model 1.932 44 <0.0001 mating 0.0169 4 0.8983 sex 121.6 4 <0.0001 stream 0.621 8 <0.0001 mating*sex 0.1028 4 0.1805 mating*stream 0.1806 8 0.1344 sex*stream 0.1616 8 0.1998 mating*sex*stream 0.0827 8 0.6996
133 Table 23: ANOVA results for the effect of independent variables on (A) middle femur and (B) hind femur fluctuating asymmetry. Sum of Source d.f. squares F ratio p>F A Middle mating 1 0.0005 0.2322 0.6314 Femur stream 2 0.0016 0.3925 0.6768 mating*stream 2 0.0005 0.1273 0.8807 error 71 0.1405 total 76 0.1434
B Hind mating 1 0.0107 3.3368 0.0720 Femur stream 2 0.0124 1.9221 0.1538 mating*stream 2 0.0022 0.3396 0.7132 error 71 0.2284 total 76 0.2513
134 Table 24: MANOVA results for the effect of independent variables on nymph morphology with regard to (A) stream location and (B) stream. Source l d.f. p>F A model 2.551 44 <0.0001 All Nymphs sex 5.386 4 <0.0001 age 1.459 8 <0.0001 location 0.2366 4 0.0018 sex*age 0.5027 8 <0.0001 sex*location 0.1399 4 0.0314 age*location 0.2449 8 0.0058 sex*age*location 0.0706 8 0.6548
Male model 1.787 20 <0.0001 age 1.413 8 <0.0001 location 0.2019 4 0.1184 age*location 0.2656 8 0.1596
Female model 2.178 20 <0.0001 age 1.564 8 <0.0001 location 0.5899 4 0.0012 age*location 0.4634 8 0.0063
B model 2.436 44 <0.0001 All Nymphs sex 4.500 4 <0.0001 age 1.418 8 <0.0001 stream 0.1699 4 0.0112 sex*age 0.5319 8 <0.0001 sex*stream 0.0656 4 0.2607 age*stream 0.0465 8 0.8595 sex*age*stream 0.1563 8 0.0885
Male model 1.63 20 <0.0001 age 1.424 8 <0.0001 stream 0.1928 4 0.1429 age*stream 0.1815 8 0.4631
Female model 1.813 20 <0.0001 age 1.443 8 <0.0001 stream 0.2494 4 0.0531 age*stream 0.2317 8 0.2189
135 Table 25: Mean and standard deviation of body component length (mm) of (A) male and (B) female nymphs in upstream and downstream Harker's Run locations. A Body Male Harker's Upstream Harker's Downstream Component N3 N4 N5 N3 N4 N5
Middle x 5.46 7.93 8.22 5.22 8.08 8.11 femur (rt) S.D. 0.28 0.18 0.25 0.08 0.18 0.26 range 5.21-5.89 7.58-8.18 7.90-8.61 5.13-5.28 7.83-8.33 7.76-8.64 N 5 9 8 3 7 16 Thorax x 3.18 4.30 4.70 3.18 4.40 4.76 S.D. 0.24 0.21 0.17 0.02 0.12 0.13 range 2.88-3.43 3.96-4.55 4.52-5.00 3.16-3.21 4.18-4.52 4.56-4.97 N 5 9 8 3 7 16 Genitalia x 0.52 1.21 1.25 0.70 1.19 1.25 S.D. 0.15 0.12 0.07 0.02 0.20 0.05 range 0.34-0.70 0.90-1.31 1.14-1.33 0.68-0.72 0.76-1.33 1.13-1.33 N 5 9 8 3 7 16 Head x 0.84 1.13 1.18 0.80 1.15 1.17 S.D. 0.02 0.03 0.06 0.07 0.02 0.05 range 0.81-0.86 1.09-1.18 1.12-1.30 0.72-0.85 1.12-1.17 1.08-1.28 N 5 9 8 3 7 35
B Female Harker's Upstream Harker's Downstream N3 N4 N5 N3 N4 N5
Middle x 5.43 7.83 7.81 5.25 7.94 8.07 femur (rt) S.D. 0.08 0.29 0.29 0.04 0.28 0.17 range 5.31-5.56 7.26-8.29 7.35-8.18 5.22-5.28 7.60-8.40 7.69-8.22 N 5 17 8 2 6 9 Thorax x 3.22 4.32 4.58 3.04 4.47 4.84 S.D. 0.24 0.16 0.09 0.02 0.21 0.07 range 2.93-3.49 3.94-4.62 4.47-4.71 3.03-3.05 4.21-4.79 4.72-4.95 N 5 17 8 2 6 9 Genitalia x 0.44 0.62 0.46 0.46 0.61 0.63 S.D. 0.04 0.09 0.04 0.003 0.12 0.04 range 0.38-0.49 0.44-0.79 0.40-0.52 0.45-0.46 0.49-0.75 0.58-0.68 N 5 17 8 2 6 9 Head x 0.86 1.14 1.16 0.79 1.14 1.18 S.D. 0.02 0.04 0.04 0.003 0.03 0.03 range 0.85-0.88 1.06-1.20 1.12-1.23 0.79-0.79 1.11-1.18 1.12-1.21 N 5 17 8 2 6 9
136 Table 26: Mean and standard deviation of body component length (mm) of male and female nymphs. Body Male Female Component N3 N4 N5 N3 N4 N5
Middle x 5.51 7.90 8.18 5.44 7.86 7.93 femur (rt) S.D. 0.19 0.18 0.25 0.08 0.28 0.26 range 5.21-5.89 7.47-8.18 7.76-8.61 5.31-5.57 7.26-8.29 7.35-8.33 N 14 19 14 8 26 17 Thorax x 3.22 4.23 4.68 3.21 4.33 4.64 S.D. 0.17 0.22 0.15 0.18 0.19 0.12 range 2.88-3.53 3.79-4.55 4.49-5.00 2.93-3.49 3.82-4.73 4.47-4.82 N 14 19 14 8 25 17 Genitalia x 0.62 1.23 1.25 0.45 0.63 0.57 S.D. 0.12 0.10 0.07 0.04 0.09 0.20 range 0.34-0.80 0.90-1.36 1.14-1.33 0.38-0.49 0.44-0.79 0.40-1.27 N 14 19 14 8 26 17 Head x 0.85 1.13 1.17 0.85 1.13 1.16 S.D. 0.02 0.03 0.05 0.02 0.05 0.03 range 0.81-0.88 1.08-1.18 1.09-1.30 0.82-0.88 1.00-1.20 1.16-1.23 N 14 19 14 8 26 17
137 Table 27: MANOVA results for the effect of independent variables on Harker's Run male morphology. Source l d.f. p>F Whole model 1.1143 12 0.0002 Intercept 1643.9 4 <0.0001 infection 0.1419 4 0.5300 location 2.0639 4 <0.0001 infection*location 0.4026 4 0.0878
138 Table 28: MANOVA results for the effect of independent variables on adult morphology for the (A) whole model, (B) four-way interaction removed and (C) four- way and non-significant three-way interactions removced. Source l d.f. p>F A All model 1.6526 92 <0.0001 Adults infection 0.0066 4 0.763 sex 37.64 4 <0.0001 stream 0.1197 8 <0.0001 generation 0.0888 8 <0.0001 infection*sex 0.0113 4 0.5330 infection*stream 0.0359 8 0.2486 infection*generation 0.0079 4 0.6975 sex*stream 0.0360 8 0.2476 sex*generation 0.0123 4 0.4887 generation*stream 0.1167 8 <0.0001 infection*sex*stream 0.0209 8 0.6559 infection*sex*generation 0.0047 4 0.8602 infection*stream*generation 0.0523 8 0.0596 sex*stream*generation 0.0330 8 0.3092 infection*sex*stream*generation 0.0131 8 0.8813
B All model 1.643 84 <0.0001 Adults infection 0.0064 4 0.7716 sex 39.03 4 <0.0001 stream 0.1220 8 <0.0001 generation 0.0916 4 <0.0001 infection*sex 0.0103 4 0.5726 infection*stream 0.0361 8 0.2409 infection*generation 0.0087 4 0.6551 sex*stream 0.0341 8 0.2795 sex*generation 0.0099 4 0.5955 generation*stream 0.1240 8 <0.0001 infection*sex*stream 0.0211 8 0.6444 infection*sex*generation 0.0037 4 0.9033 infection*stream*generation 0.0519 8 0.0600 sex*stream*generation 0.0350 8 0.2627
C All model 1.6073 64 <0.0001 Adults infection 0.0080 4 0.6796 sex 54.07 4 <0.0001 stream 0.1164 8 <0.0001 generation 0.1069 4 <0.0001 infection*sex 0.0080 4 0.6797 infection*stream 0.0388 8 0.1831 infection*generation 0.0086 4 0.6532 sex*stream 0.0498 8 0.0675 sex*generation 0.0106 4 0.5498 generation*stream 0.1371 8 <0.0001 infection*stream*generation 0.0580 8 0.0297 139 Table 29: MANOVA results for the effect of independent variables on (A) G1 male, (B) Collin's Run G1 male, (C) Darr's Run G1 male and (D) Harker's Run G1 male morphology. Source l d.f. p>F A G1 Male model 0.7006 20 <0.0001 infection 0.0422 4 0.4760 stream 0.4584 8 <0.0001 infection*stream 0.1967 8 0.0219
B Collin's infection 0.1312 4 0.8094 G1 Male
C Darr's infection 0.4788 4 0.0447 G1 Male
D Harker's infection 0.3679 4 0.0092 G1 Male
140 Table 30: Mean and standard deviation of body component length (mm) of infected and uninfected male G1 gerrids. Harker's Darr's Collin's Body Component Infected Uninfected Infected Uninfected Infected Uninfected
Middle femur x 9.75 9.90 9.72 9.68 9.61 9.81 (rt) S.D. 0.47 0.46 0.44 0.38 0.44 0.35 range 8.92-10.57 9.11-10.79 9.00-10.42 8.88-10.42 9.03-10.07 9.25-10.39 N 16 31 11 18 5 12 Thorax x 5.43 5.38 5.40 5.31 5.30 5.44 S.D. 0.24 0.19 0.24 0.18 0.16 0.19 range 5.09-5.79 5.08-5.81 5.05-5.90 4.93-5.64 5.16-5.48 5.16-5.69 N 16 31 11 18 5 12 Genitalia x 2.11 2.09 2.03 2.07 2.00 2.05 S.D. 0.09 0.08 0.10 0.06 0.07 0.09 range 1.97-2.27 1.94-2.27 1.85-2.19 1.95-2.18 1.91-2.08 1.85-2.19 N 16 31 11 18 5 12 Head x 1.45 1.43 1.41 1.42 1.34 1.36 S.D. 0.05 0.05 0.07 0.05 0.05 0.04 range 1.37-1.53 1.35-1.54 1.27-1.48 1.33-1.50 1.29-1.42 1.30-1.43 N 16 31 11 18 5 12
141 Table 31 - MANOVA results for the effect of independent variables on (A) adult, (B) male and (C) female morphology. Source l d.f. p>F A model 1.1556 44 <0.0001 All Adults infection 0.0308 4 0.0153 sex 41.01 4 <0.0001 stream 0.0434 8 0.0225 inf*sex 0.0076 4 0.5443 inf*stream 0.0334 8 0.0901 sex*stream 0.0174 8 0.5233 inf*sex*stream 0.0141 8 0.6765
B Male model 0.2502 20 <0.0001 infection 0.0843 4 0.0007 stream 0.0767 8 0.0168 infection*stream 0.0764 8 0.0174
C Female model 0.1988 20 0.0217 infection 0.0145 4 0.6634 stream 0.0639 8 0.2085 infection*stream 0.0405 8 0.5526
142 Table 32: Mean and standard deviation of body component length (mm) of infected and uninfected male gerrids. Harker's Darr's Collin's Body Component Infected Uninfected Infected Uninfected Infected Uninfected
Middle femur x 9.81 10.08 9.79 10.32 9.78 9.97 (rt) S.D. 0.43 0.50 0.49 0.54 0.40 0.50 range 8.92-10.57 9.01-10.94 9.00-10.56 8.88-11.29 9.03-10.25 9.04-10.94 N 21 78 12 73 10 52 Thorax x 5.46 5.51 5.42 5.64 5.41 5.49 S.D. 0.24 0.25 0.24 0.27 0.24 0.22 range 5.09-5.85 5.40-6.06 5.05-5.90 4.93-6.13 5.16-5.93 5.04-6.13 N 21 78 12 74 10 52 Genitalia x 2.11 2.11 2.04 2.16 2.03 2.12 S.D. 0.08 0.09 0.11 0.11 0.08 0.12 range 1.97-2.27 1.91-2.33 1.85-2.19 1.94-2.39 1.91-2.19 1.83-2.41 N 21 78 12 73 10 52 Head x 1.45 1.45 1.42 1.48 1.39 1.43 S.D. 0.05 0.06 0.08 0.06 0.07 0.07 range 1.37-1.53 1.32-1.61 1.27-1.58 1.33-1.65 1.29-1.47 1.30-1.64 N 21 78 12 73 10 52
143 Table 33: ANOVA results for the effect of independent variables on (A) middle femur and (B) hind femur fluctuating asymmetry. Sum of Source d.f. squares F ratio p>F A Middle infection 1 0.0001 0.0208 0.8865 Femur location 1 0.0021 0.3522 0.558 infection*location 1 0.0013 0.2174 0.6449 error 26 0.1608 total 29 0.163
B Hind infection 1 0.0008 0.2593 0.6149 Femur location 1 0.0000 0.0147 0.9044 infection*location 1 0.0036 1.1908 0.2852 error 26 0.0786 total 29 0.0845
144 Table 34: ANOVA results for the effect of independent variables on (A) middle femur and (B) hind femur fluctuating asymmetry. Sum of Source d.f. squares F ratio p>F A Middle infection 1 0.0002 0.0824 0.7743 Femur stream 2 0.0052 1.0073 0.3661 infection*stream 2 0.0049 0.9361 0.3930 error 412 1.0698 total 417 1.0779
B Hind infection 1 0.0002 0.0887 0.7660 Femur stream 2 0.0034 0.6950 0.4996 infection*stream 2 0.0033 0.6744 0.5100 error 412 1.0031 total 417 1.0103
145 Figure 1: Host-parasite interactions as they affect prevalence in a host population.
146 PREVALENCE OF PARASITIC INFECTION
INCIDENCE OF PARASITIC INFECTION
DURATION OF PARASITIC HOST- ESTABLISHMENT INFECTION PARASITE OF PARASITE IN CONTACT HOST
HOST HOST HOST DENSITY BEHAVIOR SUSCEPTABILITY
HOST TOLERANCE HOST STATUS OF PARASITIC AS AFFECTED INFECTION BY…
HOST SEX BIOTIC ENVIRONMENT AGE VARIATION WITHIN… ABIOTIC PREY INDIVIDUAL ENVIRONMENT COMPETITORS SPECIES CONSPECIFICS POPULATION TEMPERATURE PREDATORS
147 Figure 2: Map of the study area in Butler County, OH showing paired upstream and downstream collection sites at each of four streams.
148 N
Harker’s Run 8 7
Oxford
6 Darr’s Run
5 Coulter’s Run 4 Darrtown 2 Collin’s Run
3 1 Road Stream Collection site Four Mile Creek
1 mile
149 Figure 3: Actual and normal monthly precipitation (cm) recorded at Dayton International
Airport, Dayton, OH.
150 Actual precipitation (cm) Normal precipitation (cm) 18 16 14 12 10 8 6 4 Precipitation (cm) 2 0 Jul-00 Jul-99 Jul-01 Jul-02 Jan-99 Sep-99 Oct-99 Jan-01 Jan-00 Sep-00 Oct-01 Jan-02 Oct-00 Sep-01 Sep-02 Oct-02 Dec-00 Feb-00 Feb-99 Dec-99 Jun-01 Jun-99 Feb-02 Jun-00 Dec-01 Feb-01 Jun-02 Dec-02 Nov-00 Apr-99 Nov-02 Apr-01 Apr-00 Nov-99 Apr-02 Nov-01 Aug-99 Aug-01 Aug-00 Aug-02 Mar-99 Mar-00 Mar-02 Mar-01 May-99 May-00 May-01 May-02 Month-Year
151 Figure 4: Actual and normal average monthly temperature (C) recorded at the Dayton
International Airport, Dayton, OH.
152 Average actual temperature (C) Average normal temperature (C) 30 25 20 15 10 5 0 -5
Temperature (C) -10 -15 -20 Jul-99 Jul-01 Jul-00 Jul-02 Jan-99 Sep-99 Oct-99 Sep-00 Jan-00 Oct-00 Oct-01 Sep-02 Sep-01 Oct-02 Jan-01 Jan-02 Feb-00 Dec-99 Feb-99 Jun-99 Feb-01 Dec-00 Jun-00 Feb-02 Dec-01 Jun-02 Jun-01 Dec-02 Apr-99 Apr-00 Apr-01 Nov-99 Apr-02 Nov-00 Nov-02 Nov-01 Aug-00 Aug-99 Aug-02 Aug-01 Mar-99 Mar-00 Mar-01 Mar-02 May-99 May-00 May-02 May-01 Month-Year
153 Figure 5: Average monthly nymph density (# individuals caught/hr) over four field seasons.
154 300 1999 2000 250 2001 2002 200
150
100 Relative nymph density (# individuals caught/hr) 50
0 March April May June July Sept Oct Month
155 Figure 6: Average monthly adult density (# individuals caught/hr) over four field seasons.
156 250 1999 2000 2001 200 2002
150
100 Relative adult density (# individuals caught/hr) 50
0 March April May June July Sept Oct Month
157 Figure 7: Correlations between trypanosomatid prevalence in adults (%) and A) adult density, B) nymph density, C) adult density the previous month and D) nymph density the previous month.
158 A B
0.7 0.7 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 Prevalence in adults (%) Prevalence in adults (%) 0.1 0.1 0 0 0 100 200 300 400 500 0 50 100 150 200 250 Adult density (CPUE) Nymph density (CPUE) C D
0.7 0.7 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 Prevalence in adults (%) 0.1 Prevalence in adults (%) 0.1 0 0 0 100 200 300 400 0 100 200 300 400 Adult density (CPUE) previous month Nymph density (CPUE) previous month
159 Figure 8: Average monthly gerrid mating activity (% mating ± binomial error) over four field seasons.
160 100 90 80 1999 70 2000 60 2002 50 40 Mating (%) 30 20 10 0 March April May June July Sept Oct Month
161 Figure 9: Average monthly trypanosomatid prevalence (± binomial error) in gerrid nymphs in all streams in 2000.
162 100
90 Collin's Coulter's 80 Darr's 70 Harker's Average 60
50
40
30
2000 Prevalence in Nymphs (%) 20
10
0 May June July Sept Oct Month
163 Figure 10: Average monthly trypanosomatid prevalence (± binomial error) in gerrid adults in all streams in 2000.
164 100
90 Collin's Coulter's
80 Darr's Harker's Average 70
60
50
40
2000 Prevalence in Adults (%) 30
20
10
0 March April May June July Sept Oct Month
165 Figure 11: Average monthly trypanosomatid prevalence (± binomial error) in gerrid adults in all streams in 2001.
166 60 Collin's Coulter's 50 Darr's Harker's 40 Average
30
20
2001 Prevalence in Adults (%) 10
0 Sept Oct Month
167 Figure 12: Average monthly trypanosomatid prevalence (± binomial error) in gerrid adults in all streams in 2002.
168 100
90 Collin's 80 ) Darr's 70 Harker's Average dults (% 60
50
40
30 2002 Prevalence in A 20
10
0 March April May June July Sept Oct Month
169 Figure 13: Average monthly trypanosomatid prevalence (± binomial error) in gerrid adults in all streams in 1999.
170 100
Collin's 90 Coulter's 80 Darr's Harker's 70 Average
60
50
40
30 1999 Prevalence in Adults (%)
20
10
0 March April May June July Sept Oct Month
171 Figure 14: Hypothetical host-parasite interactions as they affect prevalence of trypanosomatid parasites.
172 PREVALENCE OF PARASITIC INFECTION
INCIDENCE OF PARASITIC INFECTION
HOST- PARASITE CONTACT
HOST HOST DENSITY BEHAVIOR
HOST STATUS AS AFFECTED BY…
HOST SEX BIOTIC ENVIRONMENT AGE VARIATION WITHIN… ABIOTIC PREY INDIVIDUAL ENVIRONMENT COMPETITORS SPECIES CONSPECIFICS POPULATION TEMPERATURE PREDATORS
173 Figure 15: Correlation of adult gerrid body length (cm) to A) mid-femur symmetry
(SMF) and B) hind-femur symmetry (SHF).
174 A
18.00 16.00 14.00 12.00 10.00 8.00 6.00 dult body length (mm)
A 4.00 2.00 0.00 0.00 0.05 0.10 0.15 0.20 0.25 0.30 SHF
B
18.00 16.00 14.00 12.00 10.00 8.00 6.00 4.00
Adult body length (mm) 2.00 0.00 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35
SMF
175 Appendix A: Number and infection status of gerrids collected and analyzed in four streams in the Oxford, Ohio area.
Collection Infection Stream Date Location Sex status Count Harkers Apr-99 down M inf 4 ok 17 F inf 1 ok 14 N inf 0 ok 0 May-99 down M inf 2 ok 8 F inf 1 ok 0 N inf 0 ok 41 up M inf 6 ok 10 F inf 8 ok 3 N inf 0 ok 32 Jun-99 down M inf 1 ok 25 F inf 1 ok 21 N inf 0 ok 14 up M inf 3 ok 24 F inf 1 ok 22 N inf 0
176 ok 16 Jul-99 down M inf 0 ok 33 F inf 0 ok 17 N inf 0 ok 7 up M inf 2 ok 19 F inf 4 ok 25 N inf 0 ok 0 Sep-99 down M inf 2 ok 26 F inf 4 ok 16 N inf 0 ok 0 up M inf 3 ok 16 F inf 3 ok 24 N inf 0 ok 0 Oct-99 down M inf 1 ok 28 F inf 2 ok 16 N inf 0 ok 1 up M inf 2 ok 19 F inf 5 ok 26 N inf 0 ok 0 Mar-00 down M inf 0 ok 6 F inf 0
177 ok 4 N inf 0 ok 0 up M inf 7 ok 23 F inf 1 ok 18 N inf 0 ok 0 Apr-00 down M inf 0 ok 6 F inf 0 ok 1 N inf 0 ok 0 up M inf 6 ok 18 F inf 7 ok 15 N inf 0 ok 0 May-00 down M inf 1 ok 0 F inf 0 ok 0 N inf 1 ok 23 up M inf 0 ok 0 F inf 0 ok 0 N inf 4 ok 21 Jun-00 down M inf 3 ok 15 F inf 2 ok 32 N inf 2 ok 30 up M inf 6
178 ok 16 F inf 5 ok 20 N inf 7 ok 25 Jul-00 down M inf 12 ok 13 F inf 11 ok 9 N inf 0 ok 23 up M inf 7 ok 22 F inf 4 ok 19 N inf 0 ok 4 Sep-00 down M inf 2 ok 23 F inf 8 ok 15 N inf 15 ok 22 up M inf 4 ok 19 F inf 3 ok 23 N inf 0 ok 0 Oct-00 down M inf 5 ok 18 F inf 6 ok 20 N inf 0 ok 2 up M inf 7 ok 21 F inf 7 ok 15 N inf 0
179 ok 4 Sep-01 down M inf 6 ok 21 F inf 4 ok 20 N inf 0 ok 2 up M inf 13 ok 12 F inf 8 ok 21 N inf 0 ok 0 Oct-01 down M inf 5 ok 23 F inf 2 ok 19 N inf 0 ok 0 up M inf 10 ok 20 F inf 7 ok 14 N inf 0 ok 0 Mar-02 down M inf 4 ok 24 F inf 3 ok 18 N inf 0 ok 0 up M inf 1 ok 26 F inf 2 ok 29 N inf 0 ok 0 Apr-02 down M inf 5 ok 25 F inf 1
180 ok 21 N inf 0 ok 0 up M inf 8 ok 22 F inf 0 ok 18 N inf 0 ok 0 May-02 down M inf 2 ok 10 F inf 0 ok 1 N inf 0 ok 0 up M inf 3 ok 15 F inf 2 ok 14 N inf 0 ok 0 Jun-02 down M inf 2 ok 1 F inf 0 ok 1 N inf 0 ok 11 up M inf 13 ok 16 F inf 4 ok 1 N inf 0 ok 21 Jul-02 down M inf 0 ok 12 F inf 0 ok 17 N inf 0 ok 32 middle M inf 0
181 ok 19 F inf 0 ok 23 N inf 0 ok 66 up M inf 0 ok 20 F inf 0 ok 26 N inf 0 ok 36 Sep-02 down M inf 0 ok 0 F inf 0 ok 0 N inf 0 ok 0 up M inf 0 ok 0 F inf 0 ok 0 N inf 0 ok 0 Oct-02 down M inf 0 ok 0 F inf 0 ok 0 N inf 0 ok 0 up M inf 5 ok 35 F inf 1 ok 13 N inf 0 ok 0 Darrs May-99 down M inf 9 ok 16 F inf 3 ok 8 N inf 0
182 ok 27 up M inf 8 ok 13 F inf 5 ok 5 N inf 0 ok 29 Jun-99 down M inf 1 ok 17 F inf 4 ok 22 N inf 0 ok 5 up M inf 3 ok 8 F inf 5 ok 31 N inf 0 ok 15 Jul-99 down M inf 4 ok 22 F inf 6 ok 18 N inf 1 ok 3 up M inf 0 ok 24 F inf 2 ok 24 N inf 0 ok 1 Sep-99 down M inf 4 ok 23 F inf 5 ok 18 N inf 0 ok 0 up M inf 0 ok 23 F inf 2
183 ok 18 N inf 0 ok 0 Oct-99 down M inf 4 ok 10 F inf 1 ok 13 N inf 0 ok 0 up M inf 4 ok 16 F inf 6 ok 24 N inf 0 ok 1 Mar-00 down M inf 10 ok 15 F inf 8 ok 15 N inf 0 ok 0 up M inf 3 ok 21 F inf 4 ok 10 N inf 0 ok 0 Apr-00 down M inf 3 ok 14 F inf 5 ok 10 N inf 0 ok 0 up M inf 5 ok 12 F inf 5 ok 6 N inf 0 ok 0 May-00 down M inf 1
184 ok 0 F inf 1 ok 0 N inf 0 ok 25 up M inf 7 ok 0 F inf 0 ok 1 N inf 2 ok 19 Jun-00 down M inf 13 ok 11 F inf 14 ok 11 N inf 15 ok 34 up M inf 17 ok 9 F inf 14 ok 14 N inf 13 ok 29 Jul-00 down M inf 12 ok 8 F inf 13 ok 19 N inf 5 ok 6 up M inf 14 ok 14 F inf 16 ok 6 N inf 0 ok 1 Sep-00 down M inf 7 ok 20 F inf 10 ok 12 N inf 9
185 ok 11 up M inf 6 ok 23 F inf 7 ok 11 N inf 2 ok 4 Oct-00 down M inf 12 ok 21 F inf 7 ok 12 N inf 1 ok 3 up M inf 9 ok 12 F inf 11 ok 16 N inf 3 ok 9 Sep-01 down M inf 4 ok 18 F inf 6 ok 22 N inf 0 ok 0 up M inf 18 ok 16 F inf 9 ok 5 N inf 0 ok 0 Oct-01 down M inf 5 ok 12 F inf 3 ok 6 N inf 0 ok 0 up M inf 3 ok 3 F inf 1 186 ok 2 N inf 0 ok 0 Mar-02 down M inf 8 ok 28 F inf 1 ok 18 N inf 0 ok 0 Apr-02 down M inf 5 ok 20 F inf 3 ok 14 N inf 0 ok 0 May-02 down M inf 3 ok 14 F inf 1 ok 11 N inf 0 ok 0 Jun-02 down M inf 8 ok 4 F inf 0 ok 2 N inf 0 ok 26 Jul-02 down M inf 0 ok 22 F inf 1 ok 17 N inf 2 ok 23 Sep-02 down M inf 2 ok 34 F inf 3 ok 30 N inf 0 ok 0 Oct-02 down M inf 0
187 ok 0 F inf 0 ok 0 N inf 0 ok 0 Coulters May-99 down M inf 3 ok 11 F inf 9 ok 9 N inf 0 ok 24 up M inf 6 ok 9 F inf 3 ok 8 N inf 0 ok 26 Jun-99 down M inf 2 ok 15 F inf 0 ok 17 N inf 0 ok 6 up M inf 4 ok 19 F inf 5 ok 22 N inf 0 ok 23 Jul-99 down M inf 2 ok 19 F inf 2 ok 27 N inf 0 ok 0 up M inf 0 ok 20 F inf 0 ok 30 N inf 0
188 ok 0 Sep-99 down M inf 0 ok 18 F inf 0 ok 23 N inf 0 ok 0 up M inf 1 ok 15 F inf 0 ok 17 N inf 0 ok 0 Oct-99 down M inf 5 ok 24 F inf 2 ok 19 N inf 0 ok 0 up M inf 1 ok 25 F inf 2 ok 22 N inf 0 ok 0 Mar-00 down M inf 1 ok 11 F inf 1 ok 6 N inf 0 ok 0 up M inf 0 ok 18 F inf 1 ok 9 N inf 0 ok 0 Apr-00 down M inf 0 ok 1 F inf 0
189 ok 1 N inf 0 ok 0 up M inf 0 ok 2 F inf 0 ok 6 N inf 0 ok 0 May-00 down M inf 3 ok 1 F inf 1 ok 1 N inf 0 ok 21 up M inf 0 ok 1 F inf 0 ok 0 N inf 1 ok 15 Jun-00 down M inf 6 ok 25 F inf 10 ok 12 N inf 1 ok 47 up M inf 4 ok 24 F inf 6 ok 13 N inf 1 ok 47 Jul-00 down M inf 6 ok 26 F inf 5 ok 13 N inf 0 ok 0 up M inf 14
190 ok 17 F inf 9 ok 9 N inf 0 ok 0 Sep-00 down M inf 6 ok 19 F inf 8 ok 20 N inf 1 ok 5 up M inf 10 ok 15 F inf 9 ok 18 N inf 11 ok 15 Oct-00 down M inf 11 ok 20 F inf 6 ok 12 N inf 1 ok 2 up M inf 9 ok 13 F inf 12 ok 14 N inf 1 ok 2 Sep-01 down M inf 12 ok 21 F inf 4 ok 14 N inf 1 ok 1 up M inf 11 ok 15 F inf 7 ok 18 N inf 0
191 ok 1 Oct-01 down M inf 0 ok 0 F inf 0 ok 0 N inf 0 ok 0 up M inf 1 ok 8 F inf 0 ok 3 N inf 0 ok 0 Collins May-99 down M inf 13 ok 8 F inf 18 ok 4 N inf 0 ok 20 up M inf 15 ok 15 F inf 9 ok 7 N inf 0 ok 14 Jun-99 down M inf 3 ok 18 F inf 4 ok 24 N inf 0 ok 8 up M inf 3 ok 21 F inf 3 ok 19 N inf 0 ok 7 Jul-99 down M inf 4 ok 20 F inf 7
192 ok 31 N inf 0 ok 0 up M inf 1 ok 24 F inf 2 ok 26 N inf 0 ok 0 Sep-99 down M inf 2 ok 24 F inf 5 ok 16 N inf 0 ok 0 up M inf 4 ok 24 F inf 1 ok 9 N inf 0 ok 0 Oct-99 down M inf 5 ok 21 F inf 3 ok 21 N inf 0 ok 1 up M inf 6 ok 16 F inf 1 ok 24 N inf 2 ok 1 Mar-00 down M inf 5 ok 16 F inf 9 ok 16 N inf 0 ok 0 up M inf 10
193 ok 21 F inf 6 ok 9 N inf 0 ok 0 Apr-00 down M inf 8 ok 17 F inf 5 ok 16 N inf 0 ok 0 up M inf 9 ok 19 F inf 3 ok 14 N inf 0 ok 0 May-00 down M inf 9 ok 6 F inf 1 ok 0 N inf 0 ok 25 up M inf 9 ok 1 F inf 1 ok 4 N inf 3 ok 21 Jun-00 down M inf 0 ok 27 F inf 5 ok 15 N inf 4 ok 38 up M inf 1 ok 16 F inf 2 ok 22 N inf 1
194 ok 8 Jul-00 down M inf 6 ok 19 F inf 3 ok 24 N inf 0 ok 0 up M inf 13 ok 18 F inf 7 ok 14 N inf 0 ok 0 Sep-00 down M inf 3 ok 29 F inf 1 ok 14 N inf 2 ok 40 up M inf 7 ok 23 F inf 3 ok 17 N inf 4 ok 28 Oct-00 down M inf 1 ok 19 F inf 3 ok 22 N inf 3 ok 5 up M inf 4 ok 15 F inf 3 ok 23 N inf 0 ok 2 Sep-01 down M inf 17 ok 19 F inf 8
195 ok 8 N inf 0 ok 1 up M inf 6 ok 23 F inf 6 ok 14 N inf 0 ok 1 Oct-01 down M inf 0 ok 0 F inf 0 ok 0 N inf 0 ok 0 up M inf 0 ok 0 F inf 0 ok 0 N inf 0 ok 0 Mar-02 down M inf 5 ok 17 F inf 2 ok 24 N inf 0 ok 0 Apr-02 down M inf 1 ok 8 F inf 0 ok 10 N inf 0 ok 0 May-02 down M inf 5 ok 12 F inf 2 ok 12 N inf 0 ok 0 Jun-02 down M inf 0
196 ok 0 F inf 0 ok 0 N inf 0 ok 0 Jul-02 down M inf 1 ok 20 F inf 2 ok 21 N inf 4 ok 23 Sep-02 down M inf 4 ok 20 F inf 2 ok 23 N inf 0 ok 0 Oct-02 down M inf 0 ok 0 F inf 0 ok 0 N inf 0 ok 0
197 Appendix B1: Gerrid morphological trait measures in units. For mm: MFR, MFL, MHR, TOT * (1.389); TH and AB * (0.6061); G and H * (0.3030). Number MFR MRL HFR HFL TOT TH AB G H 10392 7.46 7.45 6.67 6.73 9.47 9.20 5.39 6.82 4.75 10393 7.11 7.20 6.25 6.20 10.00 9.33 8.80 1.98 4.88 10394 7.00 7.10 6.33 6.31 9.35 8.83 5.49 6.78 4.67 10395 6.72 6.77 5.99 5.90 9.89 9.40 8.83 1.87 4.92 10396 7.27 7.24 6.50 6.50 9.57 8.98 5.64 7.21 4.82 10397 6.70 6.74 6.00 6.03 9.74 9.05 8.69 1.55 4.70 10398 7.25 7.30 6.49 6.44 9.16 8.78 5.18 6.60 4.70 10399 7.15 7.12 6.20 6.18 10.18 9.65 8.95 1.78 4.75 10400 7.17 7.18 6.51 6.48 9.68 8.83 5.83 7.22 4.85 10401 7.16 7.16 6.41 6.38 9.95 9.27 8.87 1.78 4.90 10402 7.11 7.13 6.10 6.10 9.30 8.83 5.57 6.73 4.72 10403 6.94 6.91 6.20 6.20 9.72 9.05 9.65 1.70 4.59 10404 7.47 7.47 6.43 6.50 9.37 8.96 5.43 6.83 4.73 10405 7.39 7.41 6.60 6.49 10.46 9.62 9.25 1.80 4.75 10406 7.34 7.30 6.49 6.50 9.19 8.66 5.41 6.72 4.71 10407 6.80 6.81 5.88 5.90 9.78 9.00 8.72 1.90 5.00 10409 6.89 7.00 6.17 6.29 10.02 9.03 9.08 1.95 4.74 10413 7.51 7.53 6.58 6.57 10.53 9.77 9.12 2.01 4.90 10415 6.99 7.00 6.20 6.23 10.11 9.16 9.07 2.01 4.74 10416 7.12 7.18 6.23 6.29 9.19 8.58 5.35 6.83 4.65 10417 7.09 7.10 6.40 6.37 9.27 9.12 5.48 6.40 4.66 10421 7.23 7.25 6.43 6.41 9.39 9.19 5.47 6.90 4.93 10422 6.82 6.83 6.09 6.10 9.01 8.41 5.15 6.80 4.70 10424 6.71 6.77 5.97 6.00 9.21 8.83 5.40 6.51 4.62 10425 6.80 6.81 6.12 6.15 9.20 8.79 5.29 6.79 4.71 10426 6.68 6.61 5.89 5.90 8.90 8.46 5.11 6.68 4.48 10429 6.57 6.56 5.83 5.91 8.94 8.40 5.24 6.62 4.59 10430 7.20 7.27 6.49 6.50 9.53 9.02 5.49 7.00 4.77 10431 7.10 7.13 6.35 6.31 9.48 9.01 5.10 6.90 4.56 10433 6.73 6.76 5.80 5.90 9.83 9.25 8.90 1.63 4.63 10434 6.60 6.67 5.87 5.80 9.43 8.87 8.29 1.64 4.41 10436 6.85 6.85 5.93 5.91 9.64 8.90 8.56 1.89 4.71 10440 7.07 7.06 6.10 6.10 10.25 9.66 9.05 2.12 4.85 10441 7.40 7.43 6.43 6.42 10.61 9.87 9.55 1.83 5.00 10447 6.57 6.61 5.79 5.81 9.17 8.56 5.50 6.93 4.45
198 10448 6.68 6.60 5.85 5.80 9.24 8.87 8.10 1.73 4.71 10449 6.95 6.95 6.33 6.18 8.79 8.35 5.25 6.69 4.59 10450 7.19 7.20 6.28 6.20 10.29 9.60 9.12 1.91 4.93 10452 6.91 7.00 6.08 6.11 10.01 9.18 8.92 1.99 4.76 10453 7.31 7.38 6.69 6.60 9.55 9.23 5.50 6.83 4.82 10454 7.34 7.35 6.55 6.52 10.66 9.97 9.42 2.05 5.02 10455 6.39 6.34 5.63 n/a 8.55 8.13 4.89 6.45 4.39 10456 6.97 7.00 6.10 6.10 9.80 9.21 8.52 1.81 4.90 10458 6.81 6.80 5.99 6.00 9.95 9.21 8.58 2.13 5.01 10459 7.06 7.09 6.44 6.43 9.30 8.85 5.50 6.82 4.62 10464 7.11 7.10 6.40 6.32 9.52 9.04 5.59 7.02 4.85 10465 7.34 7.30 6.67 6.70 9.46 9.24 5.32 6.90 4.83 10466 7.20 7.11 6.35 6.41 9.25 8.66 5.49 6.89 4.62 10468 7.03 7.03 6.30 6.37 9.39 8.79 5.53 6.94 4.95 10469 7.27 7.26 6.48 6.49 9.39 8.77 5.49 6.91 4.79 10470 6.65 6.71 6.00 5.96 8.88 8.49 5.11 6.52 4.47 10472 6.98 7.00 6.20 6.19 8.99 8.65 5.19 6.52 4.60 10473 6.94 6.96 6.20 6.19 9.01 8.49 5.37 6.61 4.57 10474 6.84 6.88 6.12 6.10 9.26 8.63 5.51 6.84 4.62 10483 7.50 7.55 6.60 6.60 9.44 9.02 5.59 6.85 4.82 10484 7.13 7.13 6.30 6.41 9.59 9.31 5.45 6.96 4.93 10485 6.76 6.73 6.12 6.13 9.14 8.63 5.27 7.00 4.65 10486 6.83 6.85 6.06 6.00 10.07 9.10 8.77 1.95 4.97 10487 6.88 6.90 5.99 6.04 10.11 9.33 8.93 1.89 4.75 10488 6.94 6.98 6.01 5.99 9.67 8.93 8.63 1.88 4.58 10489 6.90 6.89 6.09 6.09 9.80 9.16 8.48 1.80 4.75 10491 7.09 7.20 6.10 6.10 10.15 9.45 8.90 2.15 4.83 10492 6.84 6.85 6.08 5.99 10.18 9.20 9.30 1.99 4.76 10493 6.81 6.79 6.09 6.10 9.40 8.52 n/a 6.12 4.33 10494 8.05 8.01 7.00 7.00 10.67 10.15 9.56 2.12 4.57 10495 7.22 7.25 6.45 6.45 9.87 9.34 5.51 6.74 4.60 10496 6.99 7.04 6.28 6.20 10.12 9.38 8.91 2.81 4.62 10497 6.89 6.90 5.99 6.00 9.49 8.85 5.52 7.00 4.64 10498 7.07 7.00 6.20 6.23 10.02 9.91 8.52 1.94 4.55 10499 6.66 6.65 5.97 6.00 9.11 8.80 n/a 6.71 4.28 10500 7.02 7.08 6.38 6.40 10.00 9.43 9.02 1.71 4.58 10502 7.05 7.15 6.13 6.20 10.32 9.89 9.34 1.51 4.65 10503 6.50 6.58 5.68 5.68 8.85 8.51 5.19 6.31 4.32 10504 7.10 7.08 6.40 6.35 10.29 9.70 9.17 2.00 4.71 10505 7.31 7.30 6.53 6.50 9.66 9.39 5.57 6.84 4.71
199 10506 7.00 7.01 6.34 6.38 9.40 9.03 5.72 6.85 4.51 10507 7.24 7.22 6.50 6.50 9.41 9.05 5.44 6.88 4.69 10508 7.48 7.51 6.77 6.75 9.85 9.31 5.95 7.22 4.56 10509 7.25 7.10 6.43 6.42 9.43 9.04 5.19 6.61 4.53 10510 6.78 6.72 5.60 5.65 8.89 8.58 5.21 6.42 4.25 10511 7.07 7.00 6.27 6.20 9.53 9.29 5.74 6.62 4.65 10512 6.80 6.80 6.07 6.10 9.02 8.67 n/a 6.69 4.42 10513 6.99 6.99 6.21 6.25 9.08 8.56 5.50 6.40 4.60 10514 7.33 7.30 6.18 6.30 9.37 9.18 5.21 7.10 4.37 10515 7.15 7.12 6.30 6.35 9.08 8.70 5.19 6.73 4.37 10516 6.83 6.83 6.10 6.10 9.10 8.56 5.50 6.75 4.32 10517 7.02 7.15 6.14 6.20 10.45 9.58 9.43 2.01 4.60 10518 7.50 7.50 6.58 6.55 10.60 9.73 9.28 2.69 4.60 10519 7.30 7.30 6.60 6.65 10.81 10.20 9.53 1.96 4.64 10520 7.00 7.02 6.18 6.12 9.94 9.24 9.09 2.12 4.48 10521 6.70 6.73 6.09 5.99 9.91 9.32 8.79 1.90 4.40 10522 6.59 6.61 5.73 5.80 9.38 8.80 8.25 1.20 4.29 10523 7.24 7.25 6.32 6.50 10.45 9.94 9.30 1.73 4.61 10524 6.91 6.98 6.08 6.07 9.81 9.33 8.81 1.52 4.75 10525 7.19 7.25 6.40 6.50 9.60 9.23 5.57 7.02 4.53 10526 6.99 6.92 6.10 6.06 9.93 9.42 8.67 1.76 4.58 10527 7.37 7.31 6.50 6.48 9.52 9.24 5.72 6.70 4.52 10528 7.20 7.27 6.23 6.42 9.33 9.07 5.51 6.77 4.44 10529 7.35 7.34 6.54 6.51 9.68 9.36 5.67 7.02 4.58 10530 7.10 7.14 6.19 6.30 9.59 9.38 5.58 6.93 4.43 10531 7.37 7.40 6.70 6.70 9.45 8.94 5.61 7.13 4.54 10532 6.84 6.91 6.27 6.20 9.15 8.77 5.19 7.75 4.58 10533 6.71 6.76 6.00 6.03 9.07 8.78 5.21 6.90 4.37 10534 6.98 6.95 6.30 6.30 9.33 8.75 5.42 7.20 4.40 10535 7.39 7.43 6.55 6.55 9.87 9.56 5.55 7.51 4.29 10536 7.30 7.41 6.54 6.55 9.66 9.30 5.51 7.21 4.59 10537 7.30 6.99 6.42 6.42 9.54 8.98 5.69 7.00 4.48 10539 6.76 6.76 6.07 6.10 9.08 8.69 5.23 6.72 4.19 10540 7.30 7.30 6.47 6.48 10.24 9.90 9.08 1.89 5.01 10541 6.84 6.90 6.10 6.20 9.40 8.94 5.48 7.20 4.60 10543 6.81 6.83 6.08 6.05 9.20 8.80 5.34 6.80 4.48 10544 7.04 7.04 6.24 6.22 9.24 8.84 5.55 6.43 4.73 10545 6.77 6.80 6.10 6.10 9.08 8.66 5.28 6.79 4.59 10546 7.50 7.42 6.77 6.71 10.10 9.74 5.88 7.22 4.90 10547 6.92 6.95 6.44 6.42 9.49 9.00 5.19 6.82 4.83
200 10548 7.30 7.32 6.52 6.52 9.41 8.95 5.57 6.22 4.87 10549 7.10 7.39 6.30 6.40 9.40 8.99 5.50 6.70 4.65 10550 6.48 6.50 5.80 5.78 8.72 8.33 5.19 6.09 4.47 10551 7.21 7.21 6.34 6.31 9.60 9.08 5.58 7.06 4.80 10552 6.76 6.75 6.10 6.08 9.21 8.80 5.45 6.53 4.45 10553 6.59 6.69 5.99 6.00 9.70 8.90 8.48 2.31 4.96 10554 6.10 6.10 5.38 5.40 6.47 7.40 2.73 3.88 3.80 10559 5.78 5.77 5.01 5.10 6.92 7.83 4.30 1.61 3.81 10560 5.82 5.82 5.18 5.21 6.62 7.74 n/a 2.00 3.90 10561 5.88 5.90 5.20 5.30 7.30 7.87 3.81 4.17 3.78 10563 5.79 5.80 5.09 5.12 7.69 7.95 5.50 1.78 3.81 10565 5.81 5.88 5.19 5.17 6.37 7.27 3.59 1.78 3.89 10566 3.94 3.95 3.51 3.50 4.89 5.82 1.99 2.63 2.80 10567 5.82 5.90 5.21 5.20 6.45 7.17 2.51 4.47 3.85 10568 5.60 5.60 5.19 5.10 5.27 n/a 1.85 1.90 3.79 10570 5.70 5.70 5.11 5.11 6.36 7.24 3.58 1.80 3.71 10574 5.97 5.99 5.33 5.32 6.88 7.80 4.10 1.65 3.89 10577 5.85 5.90 5.26 5.39 5.59 7.04 2.08 2.02 3.81 10588 4.10 4.10 3.55 3.60 4.51 5.27 2.02 2.00 2.71 10589 3.87 3.86 3.40 3.38 4.21 5.19 1.79 1.41 2.69 10590 3.87 3.89 3.41 3.44 4.60 5.21 2.08 2.16 2.77 10591 3.89 3.86 3.47 3.47 4.28 5.15 1.58 2.11 2.70 10592 4.01 4.01 3.49 3.50 4.21 5.39 1.50 1.53 2.72 10593 3.98 3.90 3.59 3.49 4.50 5.31 1.88 2.05 2.85 10596 4.20 4.20 3.69 3.70 4.30 5.28 1.52 2.00 2.87 10598 4.07 4.01 3.57 3.59 4.51 5.50 1.60 2.40 2.78 10599 3.90 3.94 3.57 3.54 4.02 5.07 1.08 2.10 2.78 10600 3.87 3.87 3.38 3.31 4.58 5.36 2.50 1.31 2.69 10601 3.80 3.80 3.39 3.31 4.01 4.92 1.54 1.50 2.67 10603 4.00 4.01 3.50 3.50 4.70 5.47 2.00 2.38 2.90 10604 3.90 3.91 3.48 3.49 4.25 5.29 1.73 1.56 2.72 10609 6.70 6.80 6.04 6.04 9.01 8.46 5.21 6.71 4.57 10610 7.00 7.00 6.07 6.08 10.12 9.42 9.12 1.88 4.87 10611 6.72 6.74 6.00 6.00 8.90 8.40 5.12 6.91 4.51 10612 6.90 6.88 6.08 6.20 10.02 9.45 8.76 2.30 4.87 10613 7.20 7.18 6.48 6.50 9.36 8.87 5.45 7.00 4.80 10614 7.07 7.10 6.22 6.30 10.04 9.42 9.89 1.90 4.85 10615 6.59 6.59 5.91 5.87 8.97 8.65 5.19 6.43 4.59 10616 6.94 6.90 6.08 6.07 9.98 9.10 8.91 1.89 4.83 10617 7.10 7.00 6.22 6.20 9.70 9.15 n/a 7.36 4.90
201 10618 7.60 7.60 7.00 7.07 10.00 9.47 5.93 7.33 5.07 10619 7.47 n/a n/a 6.61 9.57 9.01 5.52 6.99 4.82 10620 7.09 7.04 6.30 6.31 9.37 8.91 5.37 6.61 4.48 10621 7.21 7.21 6.48 6.50 9.62 9.05 5.21 7.21 4.80 10622 6.42 6.50 5.69 5.88 9.02 8.51 5.18 6.90 4.58 10623 7.29 7.29 6.78 6.68 9.88 9.56 5.69 7.32 5.04 10624 7.30 7.30 6.58 6.65 9.79 9.29 5.72 7.18 5.00 10625 6.56 6.58 5.90 5.84 9.09 8.61 5.24 6.73 4.58 10626 7.48 7.46 6.67 6.68 10.03 9.40 5.93 7.50 4.88 10627 6.76 6.70 6.01 6.00 9.02 8.43 5.29 6.70 4.60 10628 6.69 6.73 6.11 6.12 9.34 8.88 5.49 6.91 4.99 10629 7.00 7.00 6.30 6.23 9.36 8.93 5.40 6.94 4.97 10630 7.16 7.12 6.38 6.34 9.34 8.80 5.39 6.88 4.70 10631 7.77 7.81 6.98 6.97 10.19 9.59 5.90 7.50 5.06 10632 7.00 7.00 6.16 6.20 9.27 8.91 5.19 6.98 4.72 10633 6.64 6.63 5.96 5.95 8.80 8.38 5.28 6.47 4.45 10634 7.39 7.40 6.56 6.51 9.77 9.50 5.57 7.02 4.72 10635 6.91 6.93 6.20 6.20 9.23 8.57 5.67 6.71 4.68 10636 7.73 7.73 6.68 6.69 10.09 9.58 5.86 6.90 4.90 10637 7.39 7.39 6.71 6.70 9.02 8.99 5.59 7.00 4.74 10638 7.61 7.61 6.87 6.87 9.84 9.47 5.81 7.15 4.91 10639 7.22 7.29 6.50 6.49 9.71 9.12 5.74 7.21 4.92 10640 6.97 6.96 6.10 6.20 9.18 8.83 5.22 6.58 4.87 10641 6.82 6.81 6.01 6.04 8.99 8.67 5.17 6.81 4.55 10642 7.39 7.37 6.45 6.48 10.70 10.03 9.58 1.93 5.04 10643 6.20 6.19 5.68 5.67 7.73 8.05 4.36 4.32 3.98 10644 5.93 5.94 5.30 5.29 7.27 7.65 4.12 3.89 3.70 10645 5.84 5.76 5.18 5.20 7.68 7.90 4.51 4.21 3.97 10646 5.97 5.98 5.39 5.40 5.93 7.25 2.60 1.89 3.89 10650 5.89 5.94 5.28 5.30 6.79 7.51 3.20 3.88 3.84 10651 5.60 5.65 4.97 5.00 5.82 7.00 2.74 1.89 3.67 10652 5.78 5.82 5.32 5.21 6.49 7.36 2.51 4.20 3.91 10653 5.79 5.77 5.07 5.04 6.41 7.30 2.30 2.97 3.65 10654 5.89 5.57 4.92 4.95 6.23 7.45 3.09 2.00 3.69 10655 5.90 5.89 5.31 5.20 6.65 7.62 3.62 2.11 3.81 10658 3.76 3.79 3.37 3.40 3.73 4.75 1.16 1.40 2.68 10659 3.90 3.90 3.47 3.46 4.11 5.25 1.49 1.24 2.80 10660 3.93 3.81 3.48 3.50 4.94 5.75 2.80 1.43 2.79 10661 3.90 3.97 3.40 3.58 3.90 5.05 1.08 1.63 2.87 10662 4.00 4.00 3.65 3.57 3.79 4.83 1.08 1.51 2.91
202 10663 4.00 4.01 3.50 3.60 4.32 4.99 1.83 2.15 2.80 10664 3.91 3.95 3.57 3.55 5.19 5.60 3.10 1.11 2.80 10665 3.75 3.80 3.29 3.30 4.70 5.22 2.74 1.70 2.73 10666 3.82 3.87 3.20 3.20 4.87 5.66 2.65 1.49 2.79 10667 4.24 4.25 3.82 3.90 5.36 5.66 3.18 2.30 2.85 10668 3.80 3.80 3.46 3.42 4.77 5.61 2.54 1.58 2.74 10670 7.01 7.08 6.31 6.32 10.58 10.00 9.44 2.01 4.90 10671 6.83 6.82 6.14 6.16 9.14 8.60 5.33 6.91 4.68 10672 6.22 6.29 5.59 5.50 8.80 8.20 5.32 6.50 4.39 10673 6.80 6.78 6.10 6.10 9.26 8.83 5.29 6.90 4.70 10674 6.00 5.99 5.48 5.49 6.92 7.38 3.34 3.85 3.85 10675 5.59 5.60 4.87 4.90 6.63 7.37 3.86 1.71 3.67 10677 5.47 5.50 4.90 4.90 5.64 6.95 2.52 1.61 3.68 10679 3.80 3.87 3.36 3.39 4.61 5.22 2.40 2.25 2.70 10681 3.80 3.90 3.40 n/a 4.26 5.29 2.05 2.37 2.80 10682 3.80 3.80 3.38 3.39 4.02 5.00 1.63 1.51 2.62 10684 3.76 3.72 3.37 3.32 4.15 5.04 1.80 1.50 2.61 10685 3.69 3.70 3.30 3.29 4.37 5.23 1.64 2.30 2.38 10690 7.80 7.80 7.00 7.10 10.23 9.60 6.19 7.43 4.47 10691 7.47 7.51 6.79 6.78 10.22 9.63 5.93 7.24 5.01 10692 7.88 7.93 7.21 7.28 10.40 9.40 5.80 7.31 4.81 10693 7.22 7.15 6.44 6.38 9.55 8.94 5.57 7.30 4.52 10694 7.44 7.43 6.60 6.60 9.95 9.38 5.80 7.21 4.82 10695 7.51 7.54 6.69 6.65 10.00 9.39 5.89 7.32 4.90 10696 7.74 7.74 7.05 7.10 9.87 9.19 5.88 6.88 4.87 10698 7.48 7.44 6.67 6.66 9.94 9.30 5.75 7.50 4.82 10699 7.72 7.78 7.10 7.10 10.28 9.79 5.95 7.51 5.00 10705 7.59 7.57 7.00 6.96 10.18 9.58 5.99 7.40 5.00 10707 7.66 7.65 7.00 6.96 10.36 10.00 6.00 7.48 5.11 10710 7.59 7.57 6.87 6.87 10.18 9.75 5.89 7.42 4.80 10711 7.18 7.18 6.49 6.47 10.40 9.35 9.27 2.30 4.80 10713 7.14 7.18 6.30 6.40 10.53 9.60 9.33 2.20 4.86 10716 7.64 7.63 6.69 6.68 11.02 10.39 9.62 2.00 5.03 10717 7.83 7.80 6.89 7.00 10.58 10.19 9.20 1.80 5.08 10718 7.50 7.51 6.82 6.77 10.71 9.71 9.54 2.40 5.00 10721 7.50 7.39 6.66 6.60 10.47 9.89 9.11 2.07 4.71 10724 7.28 7.27 6.51 6.55 10.44 9.48 9.38 2.11 4.90 10725 7.65 7.76 6.78 6.80 10.58 9.72 9.24 2.19 5.06 10727 7.57 7.55 6.67 6.71 10.75 10.13 9.43 2.01 5.12 10728 7.76 7.74 6.86 6.84 10.75 10.04 9.28 2.31 4.96
203 10729 7.51 7.58 6.64 6.65 10.75 10.20 9.30 2.03 5.01 10730 7.53 7.59 6.65 6.67 10.77 9.97 9.69 1.80 4.86 10733 7.67 7.72 6.68 6.70 10.58 10.15 9.21 1.95 5.01 10734 7.47 7.58 6.65 6.68 10.90 10.00 9.59 1.98 5.00 10735 7.37 7.38 6.59 6.65 10.59 9.50 9.37 2.60 4.86 10736 7.43 7.48 6.58 6.60 10.80 9.70 9.65 2.70 5.00 10737 7.46 7.51 6.58 6.60 10.66 10.03 9.23 2.10 4.94 10741 5.29 5.30 4.74 4.80 7.07 7.38 5.10 1.52 3.77 10744 6.10 6.10 n/a n/a 7.70 7.70 4.75 3.93 4.00 10745 5.89 5.89 5.20 5.20 7.04 7.71 4.62 1.40 4.05 10746 5.76 5.79 5.18 5.30 6.97 7.48 3.70 3.75 3.83 10747 5.67 5.70 4.92 5.00 6.91 7.47 4.85 1.32 3.80 10749 6.07 6.00 n/a 5.51 8.37 8.25 5.50 4.04 4.30 10750 5.59 5.51 4.97 5.00 7.28 7.46 5.37 1.71 3.68 10752 5.89 5.88 5.09 5.10 7.28 7.71 5.21 1.40 3.87 10754 5.38 5.40 4.90 4.89 7.31 7.56 5.44 1.53 3.72 10755 5.69 5.70 5.00 5.10 7.42 7.46 4.41 4.30 3.79 10756 5.79 5.76 5.28 5.20 7.52 7.60 4.50 4.40 3.71 10757 5.65 5.67 4.97 4.95 7.29 7.77 5.09 1.61 3.90 10758 5.59 5.60 5.00 5.00 7.00 7.45 4.79 1.61 3.77 10760 5.82 5.88 5.20 5.21 6.01 6.97 1.94 4.33 3.71 10762 5.60 5.61 4.89 5.00 5.57 6.54 1.48 4.10 3.72 10763 5.23 5.00 4.56 4.60 5.79 6.50 3.24 1.80 3.51 10765 5.67 5.62 5.08 5.10 5.29 6.70 1.82 2.00 3.80 10766 5.79 5.72 5.19 5.20 6.19 7.33 3.04 1.99 3.80 10769 5.57 5.53 5.00 5.00 6.10 7.15 2.91 2.10 3.82 10770 5.38 5.39 4.70 4.70 5.90 7.05 2.60 2.31 3.66 10772 5.46 5.40 4.78 4.80 6.42 6.87 2.89 4.25 3.71 10773 5.78 5.70 5.09 5.02 7.08 7.32 4.70 2.42 3.95 10776 5.74 5.87 5.16 5.20 6.51 7.28 3.62 2.41 3.88 10777 5.40 5.39 4.62 4.70 6.52 6.95 4.43 1.44 3.60 10780 5.45 5.47 4.82 4.79 6.11 7.00 2.91 2.50 3.70 10781 5.70 5.69 n/a 5.01 6.97 7.49 3.35 4.06 3.60 10783 5.48 5.48 4.74 4.80 6.10 7.09 3.12 1.91 3.77 10784 5.60 5.66 5.00 5.00 6.07 7.11 2.96 2.00 3.70 10785 5.77 5.78 5.10 5.10 6.68 7.36 4.20 1.62 3.94 10786 5.59 5.60 5.10 5.09 6.24 7.11 3.02 2.61 3.89 10787 5.66 5.70 4.97 4.95 5.82 6.73 1.81 4.20 3.71 10788 5.69 5.70 5.10 5.10 6.20 7.10 2.36 3.88 3.63 10791 7.69 7.68 6.83 6.82 10.24 9.59 6.62 7.56 4.82
204 10792 8.08 8.15 7.17 7.19 10.20 9.66 6.10 7.38 4.98 10794 7.62 7.45 6.69 6.68 10.07 9.65 6.06 7.14 4.70 10795 7.38 7.40 6.60 6.64 9.93 9.38 5.77 7.23 4.71 10796 7.60 7.58 6.84 6.88 9.98 9.44 5.99 7.01 4.89 10797 7.89 7.91 7.13 7.10 10.50 9.81 6.30 7.51 5.03 10798 7.56 7.57 6.69 6.69 10.75 10.00 9.62 2.43 4.99 10799 7.96 7.95 6.80 6.90 10.09 9.66 5.86 7.33 4.93 10800 7.59 7.60 6.70 6.70 10.01 9.40 5.86 7.57 4.86 10802 7.43 7.50 6.88 6.89 10.18 9.59 6.08 7.50 4.85 10803 7.59 7.59 6.79 6.79 10.15 9.70 5.85 7.60 4.97 10804 8.15 8.15 7.24 7.21 10.46 9.76 5.94 7.23 4.75 10805 7.51 7.51 6.67 6.71 9.79 9.20 5.75 7.30 4.81 10806 7.65 7.65 6.80 6.70 10.09 9.50 5.95 7.38 5.00 10807 7.40 7.41 6.69 6.67 9.88 9.35 5.87 7.20 4.73 10808 7.57 7.56 6.66 6.67 10.10 9.77 6.02 7.22 4.80 10809 7.70 7.80 6.86 6.86 10.09 9.55 5.88 7.43 4.88 10810 7.67 7.70 6.93 6.90 10.20 9.72 5.90 7.58 4.90 10811 7.80 7.78 6.89 6.90 9.99 9.54 5.88 7.30 4.82 10812 7.50 7.54 6.76 6.78 10.02 9.50 5.79 7.40 4.98 10813 7.73 7.76 6.80 6.75 9.87 9.49 5.59 7.55 4.90 10814 7.56 7.56 6.58 6.60 10.81 9.93 9.70 2.50 4.99 10815 7.50 7.46 6.48 6.48 10.90 10.21 9.60 2.08 4.84 10816 7.60 7.62 6.77 6.78 10.83 10.00 9.40 2.21 5.10 10818 7.82 7.80 6.98 6.98 11.22 10.21 9.71 2.50 5.07 10819 7.40 7.42 6.58 6.50 10.70 10.00 9.41 2.40 5.00 10820 7.55 7.64 6.67 6.70 10.52 9.61 9.44 2.14 4.97 10821 7.45 7.50 6.56 6.55 10.82 10.35 9.43 2.20 4.97 10822 7.40 7.42 6.55 6.56 10.69 10.08 9.41 1.64 5.00 10823 7.50 7.51 6.77 6.72 10.98 10.11 9.83 2.40 4.89 10824 7.78 7.60 7.04 6.78 11.00 10.14 9.65 2.33 5.10 10825 7.26 7.27 6.50 6.48 11.21 10.22 9.70 2.30 5.23 10826 7.37 7.40 6.58 6.50 10.60 9.89 9.38 2.28 5.00 10827 7.62 7.61 6.90 6.86 10.66 10.20 9.40 1.72 4.88 10828 7.30 7.24 6.41 6.37 10.54 9.62 9.43 2.17 4.86 10829 7.55 7.57 6.63 6.61 10.91 9.94 9.80 2.40 4.97 10830 7.48 7.45 6.70 6.71 11.11 10.35 9.83 2.40 5.00 10831 7.71 7.70 6.78 6.80 10.77 9.70 9.80 2.28 4.95 10832 7.22 7.21 6.47 6.35 10.55 10.09 9.31 2.10 4.82 10833 7.59 7.70 6.70 6.66 10.73 9.96 9.63 2.16 5.10 10834 7.55 7.61 6.91 6.85 10.94 9.99 9.80 2.51 5.00
205 10835 7.55 7.57 6.68 6.75 10.72 10.02 9.39 2.22 5.09 10836 7.64 7.70 6.88 6.80 10.88 10.13 9.68 2.10 4.91 10837 7.80 7.74 6.71 6.80 10.77 10.06 9.38 2.30 5.00 10839 7.70 7.71 6.81 6.70 10.91 10.14 9.79 2.25 5.02 10840 7.87 7.80 6.99 6.82 10.87 9.95 9.80 2.23 5.00 10842 5.88 5.90 5.19 5.20 7.71 7.96 4.73 4.00 3.67 10843 5.81 5.88 5.36 5.39 7.80 8.07 5.72 1.90 4.00 10844 5.87 5.90 5.39 5.46 7.79 7.90 5.81 2.12 3.97 10845 6.07 6.10 5.51 5.50 8.02 8.01 5.10 4.22 4.00 10846 5.64 5.65 5.20 5.20 7.54 7.55 4.71 3.97 3.86 10847 5.87 5.90 5.21 5.20 7.20 7.79 4.58 2.20 3.71 10849 5.78 5.72 5.03 5.04 7.62 7.80 4.67 3.91 3.79 10850 5.67 5.66 5.10 5.10 7.24 7.68 4.05 3.73 3.57 10851 5.54 5.58 4.98 4.99 7.55 8.00 5.11 2.10 3.98 10852 5.83 5.78 5.30 n/a 7.91 8.17 5.72 2.22 4.00 10853 5.90 5.89 5.29 5.38 7.65 7.73 4.70 4.04 3.86 10854 6.22 6.20 5.55 5.64 7.98 8.06 4.97 4.10 3.91 10855 5.76 5.79 5.10 5.10 7.47 7.52 4.39 4.39 3.71 10856 6.19 6.20 5.49 5.59 7.88 7.95 4.91 4.11 3.86 10858 5.90 5.94 5.37 5.47 8.43 8.20 5.56 4.34 4.23 10860 5.63 5.65 5.05 5.07 7.91 7.86 5.02 4.28 3.92 10862 5.77 5.76 5.10 5.05 7.47 7.59 4.45 4.20 3.73 10863 5.59 5.60 5.01 5.01 7.64 7.70 4.63 4.13 3.80 10864 5.67 5.63 5.09 5.12 7.78 8.05 5.55 2.10 3.87 10865 5.85 5.90 5.19 5.20 7.02 7.90 4.10 2.23 3.98 10867 5.89 5.90 5.19 5.20 7.70 8.01 5.67 1.90 3.81 10868 5.89 5.86 5.09 5.10 7.98 8.02 5.04 4.30 3.88 10869 5.92 5.91 5.19 5.30 7.90 8.05 5.80 2.08 3.91 10873 5.80 5.80 5.25 5.37 8.17 8.00 5.41 4.22 4.00 10874 5.72 5.72 5.10 5.20 8.30 8.10 5.63 4.10 4.09 10875 6.05 6.07 5.46 5.49 7.55 7.91 5.17 2.10 3.91 10876 5.77 5.76 5.28 5.18 7.19 7.46 3.90 4.27 3.71 10877 5.80 5.81 5.20 5.23 6.73 7.15 3.22 4.40 3.80 10878 5.65 5.70 5.12 5.11 5.74 7.05 2.20 2.48 3.67 10880 5.64 5.70 5.00 5.10 6.78 7.32 3.25 4.01 3.69 10883 5.75 5.71 5.10 5.10 6.70 7.47 3.70 2.47 3.85 10884 5.82 5.87 5.10 5.17 6.91 7.50 4.41 1.74 3.85 10885 5.97 5.90 5.31 5.30 6.89 6.90 n/a 4.29 3.81 10886 5.76 5.79 5.11 5.13 7.07 7.44 3.65 4.19 3.80 10888 5.78 5.73 5.09 5.09 6.06 7.14 2.63 2.51 3.81
206 10891 3.73 3.75 3.28 3.30 4.62 5.55 2.24 1.50 2.78 10892 7.49 7.43 6.62 6.67 9.51 9.95 5.68 7.20 4.80 10893 7.40 7.48 6.64 6.60 9.91 9.30 5.88 7.43 4.92 10894 8.00 8.01 7.17 7.20 10.14 9.59 5.95 7.80 5.02 10895 7.50 7.52 6.69 6.70 9.89 9.13 5.93 7.51 4.91 10896 7.57 7.53 6.77 6.80 9.81 9.43 5.75 7.02 4.67 10897 7.90 7.90 7.10 7.10 10.10 9.57 5.89 7.37 4.89 10898 7.70 7.71 6.87 6.90 10.00 9.50 5.92 7.16 4.87 10899 7.81 7.85 7.10 7.15 10.13 9.39 6.00 7.70 5.00 10901 7.64 7.66 6.81 6.87 10.06 9.70 5.88 7.17 5.02 10902 7.80 7.84 6.90 6.87 9.95 9.39 5.70 7.51 5.05 10903 7.81 7.80 6.99 7.04 9.89 9.40 5.60 7.26 4.91 10904 7.85 7.87 7.05 7.05 10.41 10.09 6.03 7.60 5.01 10905 7.75 7.78 7.00 7.00 10.23 9.80 5.91 7.47 4.98 10906 7.90 7.86 7.00 7.01 10.18 9.90 5.88 7.35 4.92 10907 n/a 7.74 6.94 6.95 10.13 9.83 5.88 7.31 4.93 10908 7.80 7.81 7.03 7.07 9.90 9.74 5.61 7.00 5.00 10909 7.80 7.76 6.94 6.98 10.25 9.74 5.83 7.81 5.14 10911 7.60 7.61 6.82 6.90 10.20 9.64 5.81 7.63 5.14 10912 7.86 7.84 6.89 6.86 10.11 9.47 5.76 7.90 5.10 10913 7.60 7.64 6.90 6.90 10.12 9.40 6.05 7.60 4.78 10915 7.18 7.20 6.40 6.42 10.50 9.85 9.39 2.00 4.98 10916 7.71 7.65 6.87 6.80 10.94 10.14 9.80 2.30 5.01 10917 7.83 7.76 7.00 7.00 11.05 10.12 9.80 2.32 5.01 10920 7.68 7.68 6.89 6.90 10.61 9.80 9.22 2.42 4.98 10921 7.25 7.40 6.60 6.55 10.00 9.20 5.98 7.40 5.21 10924 7.57 7.55 6.59 6.56 9.98 9.41 5.89 7.30 4.85 10926 7.79 7.78 6.62 6.68 10.67 9.93 9.41 2.23 4.91 10927 7.52 7.50 6.66 6.67 10.47 9.80 9.12 2.11 4.81 10928 7.34 7.41 6.59 6.60 10.65 9.66 9.32 2.31 5.19 10929 7.32 7.37 6.56 6.54 10.70 9.96 9.50 2.28 4.76 10930 7.74 7.70 6.89 6.87 10.97 10.13 9.69 2.40 5.07 10932 7.41 7.40 6.57 n/a 10.38 9.92 9.10 2.00 4.82 10934 6.97 6.97 6.20 6.10 10.48 9.90 9.00 2.40 4.90 10935 7.29 7.30 6.57 6.57 10.47 9.60 9.23 2.12 5.12 10936 7.50 7.51 6.55 6.52 11.07 10.38 9.70 2.40 5.31 10937 7.40 7.35 6.46 6.45 10.70 10.10 9.43 2.20 5.00 10938 7.80 7.80 7.02 6.98 10.97 10.20 9.64 2.21 5.10 10940 7.29 7.20 6.54 6.53 10.44 9.91 9.08 2.00 5.12 10941 7.44 7.50 6.66 6.70 10.35 9.45 9.18 2.30 4.91
207 10942 7.54 7.54 6.56 6.54 10.59 9.82 9.33 2.22 5.01 10943 5.84 5.80 5.29 5.29 7.65 7.95 5.43 1.89 3.98 10947 5.80 5.80 5.10 5.10 6.89 7.44 3.19 4.38 3.81 10949 5.60 5.61 5.00 5.03 6.74 7.38 4.10 2.20 3.78 10950 5.59 5.60 5.00 5.09 7.20 7.67 3.76 4.38 3.60 10951 5.97 5.95 5.18 5.20 7.39 7.78 5.26 1.88 3.80 10952 6.00 6.01 5.26 5.26 7.08 7.71 3.47 4.20 3.79 10954 5.63 5.70 4.90 4.98 7.24 7.81 4.80 1.84 3.97 10955 5.69 5.76 5.22 5.20 7.27 7.56 3.89 4.40 3.75 10956 5.93 5.90 5.40 5.36 7.48 7.80 3.94 3.98 3.98 10957 5.68 5.67 5.13 5.05 6.97 7.60 4.14 2.38 3.95 10958 5.87 5.69 5.27 5.28 7.09 7.65 3.48 4.20 3.96 10961 5.80 5.86 5.23 5.30 5.59 6.44 1.47 4.22 3.88 10963 5.68 5.68 4.99 5.00 6.70 7.31 3.04 4.18 3.70 10964 5.68 5.70 n/a 4.95 6.81 7.34 3.14 4.50 3.78 10969 5.74 5.77 5.20 5.18 6.11 7.05 2.10 4.11 3.71 10970 5.35 5.32 4.81 4.85 5.18 6.30 1.80 2.42 3.60 10971 5.79 5.80 5.24 5.25 5.73 7.05 2.09 2.51 3.30 10972 5.55 5.50 4.89 4.92 6.42 7.29 3.60 2.14 3.40 10974 5.68 5.62 5.00 5.03 6.21 6.96 2.25 3.71 3.57 10976 5.38 5.40 4.75 4.85 5.60 6.25 1.77 3.80 3.62 10978 5.76 5.76 5.10 5.10 6.38 7.12 2.57 3.99 3.76 10979 5.56 5.50 5.04 4.95 5.72 6.56 1.72 4.27 3.75 10981 5.75 5.75 5.11 5.10 6.41 7.20 2.39 4.24 3.79 10985 5.58 5.58 4.99 4.98 5.93 6.64 1.80 4.11 3.68 10986 5.70 5.80 5.18 5.18 6.39 7.32 3.18 2.50 3.70 10988 7.46 7.49 6.57 6.57 10.08 9.54 6.00 7.48 4.78 10989 7.77 7.80 7.00 7.00 9.96 9.48 5.89 7.40 4.90 10990 7.70 7.70 6.99 6.89 9.77 9.34 5.90 6.90 4.71 10991 7.41 7.38 6.77 6.74 9.79 9.07 5.90 7.13 4.87 10993 7.51 7.50 6.80 6.82 9.71 9.19 5.70 7.30 4.67 10994 7.42 7.40 6.70 6.70 9.80 9.13 5.79 7.21 4.97 10995 7.63 7.58 6.75 6.72 10.08 9.39 5.88 7.41 5.00 10996 7.62 7.70 6.94 6.94 10.33 9.54 6.02 7.70 5.15 10997 7.17 7.14 6.47 6.42 9.83 9.30 5.83 7.17 4.64 10998 7.68 7.61 6.79 6.78 9.92 9.33 5.81 7.21 4.82 10999 7.69 7.70 6.99 6.98 9.94 9.26 5.85 7.48 4.97 11000 7.20 7.20 6.50 6.49 9.95 9.44 5.87 7.01 4.88 11001 7.39 7.40 6.69 6.57 9.97 9.30 5.85 7.40 5.10 11002 7.40 7.43 6.72 6.68 9.83 9.10 6.01 7.13 4.71
208 11003 7.84 7.80 6.90 6.90 9.81 9.40 5.69 7.20 4.62 11004 7.31 7.39 6.65 6.68 9.73 9.30 5.59 7.20 4.82 11005 7.66 7.63 6.90 6.91 9.89 9.50 5.54 7.41 4.75 11006 7.25 7.37 6.67 6.60 9.70 9.08 5.79 7.22 4.75 11007 7.66 7.65 6.99 7.00 9.92 9.40 5.69 7.37 4.90 11009 7.50 7.54 6.84 6.94 9.91 9.50 5.71 7.42 4.90 11012 7.48 7.51 6.67 6.61 10.74 10.01 9.45 2.18 4.97 11013 7.36 7.40 6.42 6.48 10.53 10.04 9.18 2.02 5.00 11014 7.07 7.05 6.20 6.19 10.32 9.19 8.91 2.13 4.91 11015 7.88 7.80 7.21 7.17 11.00 10.11 6.90 7.80 5.42 11016 7.30 7.35 6.43 6.48 10.53 9.94 9.15 2.26 4.79 11017 7.48 7.51 6.59 6.68 11.02 9.96 9.90 2.50 5.02 11018 7.48 7.50 6.59 6.70 10.78 9.84 9.49 2.31 5.01 11019 7.51 7.59 6.68 6.70 10.88 10.42 9.45 2.11 4.91 11020 7.52 7.48 6.42 6.40 10.67 10.17 9.56 1.89 5.01 11021 7.15 7.22 6.48 6.57 10.32 9.68 8.78 2.10 4.77 11022 7.70 7.70 6.75 6.80 10.44 9.84 9.16 1.90 5.00 11023 7.59 7.59 6.71 6.79 10.53 9.85 9.25 2.30 4.90 11024 7.73 7.77 6.70 6.88 10.75 10.21 9.11 2.10 5.09 11025 7.47 7.44 6.58 6.60 10.32 9.70 9.12 2.10 4.83 11026 7.41 7.46 6.46 6.51 10.27 9.51 9.20 2.21 4.84 11027 7.47 7.50 6.70 6.70 10.46 9.91 9.24 1.92 4.98 11028 7.32 7.40 6.50 6.50 10.33 9.34 9.14 2.58 4.86 11030 7.35 7.37 6.40 6.47 10.50 9.80 9.52 1.98 4.89 11031 7.43 7.47 6.50 6.49 10.59 9.93 9.16 2.30 5.01 11033 7.18 7.17 6.21 6.40 10.52 9.81 9.27 2.31 4.91 11034 7.70 7.74 6.66 6.67 10.79 9.96 9.50 2.23 4.91 11035 7.57 7.58 6.65 6.67 10.90 9.95 9.51 2.62 5.07 11036 7.25 7.26 6.36 6.38 10.30 9.66 8.95 2.25 4.96 11037 7.33 7.30 6.60 6.50 10.53 9.85 9.20 2.33 4.96 11039 5.90 5.90 5.41 5.41 7.47 7.73 4.39 4.00 3.81 11040 5.76 5.75 5.12 5.13 7.45 7.65 5.60 1.70 3.78 11042 5.70 5.70 5.00 5.00 7.61 7.55 4.80 4.05 3.88 11043 5.62 5.63 5.10 5.10 7.67 7.62 4.88 3.91 3.80 11044 5.91 5.90 5.20 5.20 7.75 7.90 4.65 4.28 4.00 11046 5.80 5.80 5.22 5.20 6.99 7.52 4.42 2.20 3.70 11048 5.75 5.79 5.10 5.20 7.67 7.72 4.81 4.02 3.90 11049 5.40 5.41 4.90 4.98 7.11 7.40 4.30 3.85 3.57 11052 5.79 5.80 5.30 5.30 7.55 7.59 4.76 4.10 3.95 11053 5.73 5.78 5.10 5.18 7.58 7.60 4.33 3.67 3.82
209 11055 5.70 5.70 5.00 5.00 7.23 7.64 4.75 2.39 3.98 11057 5.73 5.70 5.18 5.17 7.68 7.82 5.62 2.15 3.84 11058 5.58 5.57 4.99 4.97 6.98 7.70 4.36 2.93 3.76 11060 5.87 5.90 5.10 5.20 7.84 8.10 5.77 1.90 3.90 11061 5.91 5.98 5.20 5.34 8.46 8.52 6.18 2.57 4.24 11063 5.74 5.70 5.10 5.12 7.70 7.81 4.90 3.97 3.78 11064 5.79 5.80 5.11 5.10 7.38 7.38 4.55 4.00 3.70 11066 5.67 5.68 5.17 5.11 7.58 7.70 5.67 1.90 3.79 11068 5.74 5.60 5.10 5.10 7.50 7.77 5.50 1.90 3.70 11071 5.71 5.71 5.24 5.30 8.18 7.84 5.20 4.60 4.09 11073 5.48 5.55 4.96 4.90 6.11 6.91 3.46 1.50 3.86 11075 5.66 5.80 5.00 5.00 5.20 6.50 1.12 2.23 3.80 11076 5.42 5.48 4.80 4.80 6.81 7.24 3.31 4.21 3.80 11077 5.63 5.60 5.11 5.10 7.42 7.60 4.48 4.00 3.22 11079 5.70 5.77 5.08 5.12 6.43 7.32 2.58 4.21 3.66 11083 3.68 3.68 3.11 3.10 3.99 5.35 1.59 1.51 2.70 11084 3.80 3.80 3.32 3.32 3.87 n/a 1.22 1.49 2.77 11086 6.96 6.99 6.19 6.20 9.88 8.91 5.10 6.78 4.83 11087 7.04 7.03 6.10 6.14 9.19 8.74 5.39 6.50 4.71 11088 6.62 6.60 5.99 5.92 9.16 8.50 5.48 6.90 4.64 11089 6.72 6.76 6.14 6.23 9.30 8.74 5.76 6.71 4.50 11090 6.95 6.97 6.20 6.20 8.98 8.60 5.41 6.04 4.69 11091 6.88 7.00 6.20 6.22 9.00 8.53 5.20 6.53 4.58 11093 6.51 6.50 5.83 5.83 8.80 8.31 5.05 6.67 4.56 11094 7.13 7.21 6.24 6.24 9.30 8.98 5.36 6.80 4.70 11095 7.00 7.10 6.37 6.40 9.41 8.81 5.69 6.67 4.55 11096 6.80 6.87 6.14 6.09 9.18 8.69 5.60 6.62 4.58 11097 7.30 7.30 6.55 6.60 9.77 9.79 5.74 6.75 4.86 11098 6.90 6.91 6.29 6.30 9.58 9.05 5.76 7.95 4.75 11099 7.05 7.10 6.41 6.45 9.53 9.01 5.03 7.00 4.93 11100 7.38 7.40 6.59 6.60 9.70 9.23 5.82 7.03 4.84 11101 7.09 7.08 6.44 6.40 9.52 8.95 5.79 6.71 4.70 11102 6.59 6.59 5.79 5.80 9.77 9.04 8.54 2.18 4.75 11103 7.10 7.36 6.38 6.46 9.59 9.33 5.55 6.89 4.80 11104 7.06 7.01 6.30 6.33 9.48 8.85 5.66 7.12 4.92 11105 6.78 6.75 6.17 6.18 9.34 8.85 5.58 6.60 4.62 11106 6.80 7.00 6.20 6.20 9.05 8.81 5.23 6.52 4.70 11107 6.67 6.70 5.90 6.01 9.02 8.49 5.44 6.51 4.55 11108 7.06 7.07 6.25 6.28 9.44 8.80 5.60 7.11 4.61 11109 6.56 6.63 5.89 5.90 9.36 8.83 5.63 6.71 4.60
210 11110 6.77 6.71 6.19 6.16 9.24 8.84 5.42 6.80 4.47 11111 7.00 7.07 6.29 6.28 9.30 8.60 5.56 7.10 4.72 11112 7.19 7.19 6.39 6.40 10.48 9.79 9.00 2.18 5.42 11113 7.18 7.20 6.20 6.20 10.37 9.71 9.35 1.90 4.80 11114 6.80 6.88 5.98 6.00 9.74 8.91 8.60 1.80 4.79 11115 7.29 7.28 6.41 6.50 10.53 9.59 9.55 2.22 5.12 11116 7.00 7.02 6.30 6.33 10.03 9.35 8.80 2.21 4.91 11117 6.87 7.00 6.10 6.10 10.12 9.49 9.13 1.82 4.70 11118 6.87 6.88 6.10 6.10 9.89 9.14 8.85 1.99 4.71 11119 6.87 6.83 6.13 6.12 10.00 9.47 8.83 1.80 4.75 11120 7.25 7.23 6.30 6.30 10.00 9.40 9.11 1.52 4.85 11121 7.21 7.20 6.45 6.40 10.08 9.40 9.01 1.78 4.81 11122 6.64 6.60 5.84 5.80 9.56 9.06 8.59 1.30 4.71 11123 6.68 6.67 5.90 5.90 10.02 9.11 9.05 2.11 4.79 11124 7.74 7.76 6.91 6.90 10.70 10.04 9.32 2.19 5.00 11125 7.15 7.16 6.45 6.43 10.20 9.50 9.11 1.73 4.76 11126 6.84 6.92 6.10 6.13 10.12 9.34 8.92 2.00 4.95 11127 7.97 7.95 7.00 6.95 10.73 10.30 9.39 2.18 5.09 11128 7.38 7.38 6.51 6.50 10.47 9.66 9.28 2.00 4.97 11129 6.53 6.54 5.80 5.84 9.88 9.23 8.17 1.91 4.72 11130 6.80 6.82 6.09 6.09 10.07 9.49 8.91 2.00 4.72 11131 7.48 7.55 6.60 6.59 10.68 9.86 9.54 2.10 5.18 11132 6.79 6.80 6.03 6.00 9.96 9.33 8.70 2.00 4.80 11133 6.90 6.90 6.09 6.10 10.12 9.36 8.83 2.15 4.91 11135 6.39 6.41 5.65 5.60 9.39 9.81 8.41 2.18 4.53 11136 7.00 7.05 6.19 6.17 10.18 9.48 9.18 1.90 4.76 11142 7.11 7.22 6.49 6.47 9.47 9.14 5.59 6.68 4.88 11143 7.59 7.70 6.83 6.85 9.68 9.29 5.70 7.19 4.80 11144 8.13 8.11 7.39 7.40 10.18 9.88 5.73 7.63 5.18 11145 7.65 7.60 6.77 6.81 10.10 9.65 5.92 7.33 5.09 11146 6.92 6.90 6.14 6.14 9.36 8.89 5.42 6.81 4.73 11147 7.60 7.60 6.77 6.76 10.08 9.49 5.91 7.20 5.20 11148 7.48 7.50 6.60 6.69 10.31 9.50 9.02 1.99 4.98 11149 7.42 7.40 6.72 6.77 9.80 9.38 5.78 7.00 4.91 11150 7.60 7.61 6.85 6.80 9.70 9.38 5.59 6.45 5.45 11151 7.70 7.75 6.90 6.96 10.02 9.53 5.89 7.50 4.83 11152 7.25 7.25 6.46 6.50 9.76 9.10 5.96 7.10 4.91 11153 7.45 7.50 6.70 6.70 10.00 9.60 5.71 7.32 5.00 11154 7.72 7.78 6.88 6.88 9.63 9.25 5.69 6.88 4.96 11155 6.94 6.96 6.20 6.20 9.66 9.15 5.88 6.90 4.64
211 11156 7.70 7.65 6.83 6.90 9.71 9.20 5.71 7.02 4.98 11157 7.73 7.70 6.89 6.97 10.25 9.86 6.01 7.48 5.00 11158 7.84 7.80 7.00 6.96 9.94 9.60 5.59 6.90 5.14 11160 7.19 7.20 6.48 6.47 9.36 8.99 5.40 6.99 4.80 11161 7.64 7.62 6.80 6.80 10.02 9.69 5.89 7.20 4.92 11162 7.73 7.80 6.90 6.93 9.79 9.40 5.58 n/a n/a 11163 7.00 7.10 6.32 6.42 9.28 8.19 5.44 6.81 4.82 11164 7.52 7.34 6.59 6.59 9.71 9.29 5.70 7.15 4.73 11165 7.60 7.62 6.70 6.80 9.80 9.24 5.71 7.20 5.10 11166 7.27 7.29 6.49 6.48 9.70 9.40 5.58 7.28 4.79 11167 6.78 6.79 6.20 6.10 9.41 9.00 5.51 6.80 4.74 11168 7.57 7.60 6.76 6.77 10.00 9.40 5.96 7.30 4.98 11169 7.89 7.86 6.84 6.90 10.23 9.60 6.16 7.60 4.91 11170 7.86 7.89 6.96 6.95 10.14 9.78 5.85 7.22 5.11 11171 7.59 7.58 6.80 6.78 10.00 9.63 5.93 7.10 4.91 11172 7.20 7.20 6.38 6.35 9.41 9.04 5.50 6.41 4.71 11173 7.89 7.92 7.14 7.10 10.34 10.09 5.98 7.23 5.02 11174 7.71 7.73 7.04 7.06 9.90 9.61 5.62 7.01 5.09 11175 7.70 7.79 6.99 6.95 9.91 9.50 5.74 7.28 4.90 11176 7.69 7.76 6.90 6.97 10.36 10.11 5.90 7.43 4.90 11177 6.97 7.09 6.39 6.43 9.70 9.58 5.48 7.08 4.81 11178 7.41 7.47 6.69 6.70 10.33 9.94 8.91 2.12 4.18 11179 7.50 7.50 6.69 6.67 10.62 9.90 9.31 2.02 5.19 11180 7.08 7.10 6.23 6.19 9.78 9.19 8.65 1.80 4.62 11181 7.20 7.25 6.40 6.40 10.35 9.60 9.22 1.80 4.81 11182 7.59 7.59 6.73 6.77 10.24 9.90 8.91 2.19 5.00 11183 6.90 7.00 6.08 6.10 9.96 9.08 8.67 2.01 5.25 11184 7.37 7.40 6.50 6.56 10.25 9.84 9.05 1.99 4.16 11185 7.46 7.46 6.49 6.48 10.51 10.10 9.04 2.20 5.12 11186 7.60 7.55 6.57 6.55 10.43 9.76 9.06 2.30 4.90 11187 7.36 7.34 6.49 6.46 10.51 9.95 9.35 2.18 4.91 11188 7.67 7.70 6.88 6.90 10.88 10.39 9.37 2.18 5.19 11189 7.46 7.44 6.60 6.68 10.50 9.78 9.30 2.00 4.92 11190 6.90 6.91 6.18 6.20 10.13 9.43 9.01 1.94 4.68 11191 7.53 7.57 6.60 6.65 10.27 9.68 8.93 2.00 5.10 11192 7.48 7.47 6.54 6.55 10.62 10.00 9.30 2.04 5.01 11193 6.20 6.10 5.59 5.55 9.18 8.31 8.40 1.70 4.51 11194 7.42 7.50 6.58 6.51 10.65 10.00 9.35 2.23 5.20 11195 7.80 7.80 6.46 6.46 10.24 9.50 9.00 n/a n/a 11196 7.59 7.57 6.59 6.56 10.42 9.85 9.00 2.22 5.00
212 11197 7.34 7.45 6.60 6.65 10.38 9.77 9.20 2.77 4.95 11198 6.98 7.05 6.26 6.30 9.97 9.38 8.68 2.08 4.80 11199 7.36 7.39 6.72 6.71 10.57 9.82 9.23 2.22 5.01 11200 7.54 7.54 6.70 6.77 10.56 10.00 9.18 2.10 4.99 11201 7.44 7.40 6.55 6.58 10.02 9.28 8.80 2.18 4.91 11202 7.50 7.53 6.69 6.70 10.57 10.00 9.19 2.19 5.00 11203 7.70 7.71 6.70 6.76 10.36 9.84 8.94 2.12 5.10 11207 7.68 7.73 7.00 6.95 10.01 9.97 5.65 7.61 4.97 11208 7.43 7.44 6.70 6.65 10.84 10.11 9.57 2.40 4.85 11209 7.60 7.60 6.74 6.70 10.78 10.00 9.13 2.74 5.45 11328 7.31 7.48 6.60 6.64 9.73 9.39 5.79 7.13 4.90 11329 7.10 7.04 6.25 6.19 9.36 9.03 5.39 6.80 4.68 11330 6.49 6.50 5.74 5.70 8.66 8.19 4.97 6.61 4.51 11331 6.79 6.85 6.07 6.08 9.07 8.29 5.35 7.01 4.80 11332 7.69 7.75 6.94 6.90 9.77 9.62 5.57 7.10 4.93 11333 7.57 7.52 6.74 6.81 9.82 9.52 5.55 7.20 4.89 11334 7.44 7.47 6.60 6.60 9.77 9.36 5.67 7.18 4.91 11335 7.14 7.12 6.41 6.40 9.37 8.83 5.34 6.80 5.27 11336 7.11 7.13 6.30 6.31 9.04 8.36 5.43 6.30 5.21 11337 7.41 7.47 6.73 6.70 9.59 9.35 5.57 6.76 4.76 11338 7.59 7.53 6.82 6.80 9.88 9.31 5.87 7.25 4.93 11339 7.87 7.87 6.99 7.00 10.17 9.78 5.83 7.70 5.00 11340 6.73 6.72 5.86 6.05 9.30 8.98 5.44 6.90 4.65 11341 7.37 7.41 6.60 6.56 9.58 9.21 5.62 6.89 4.80 11342 7.61 7.60 7.00 6.90 9.71 9.39 5.89 6.76 4.78 11343 7.31 7.37 6.58 6.60 9.71 9.19 5.77 7.30 5.30 11344 7.29 7.23 6.55 6.60 9.86 9.40 5.83 7.28 4.88 11345 7.08 7.08 6.30 6.30 9.37 9.06 5.40 6.73 4.78 11346 7.74 7.70 7.02 7.01 9.86 9.56 5.74 6.98 5.01 11347 7.07 7.08 6.25 6.24 9.15 8.80 5.25 6.71 4.56 11348 6.70 6.69 5.95 5.95 8.89 9.45 5.19 6.50 4.51 11349 7.60 7.56 6.84 6.71 10.04 9.65 5.90 7.20 4.88 11350 7.45 7.45 6.69 6.70 9.56 9.20 5.56 6.91 4.97 11351 7.27 7.21 6.48 6.45 9.47 9.08 5.60 6.72 4.70 11352 7.45 7.44 6.80 6.78 9.80 9.51 5.69 6.85 4.73 11353 6.84 6.90 6.20 6.20 9.10 8.69 5.39 6.63 4.53 11354 6.90 6.90 6.20 6.20 9.29 8.87 5.44 6.84 4.70 11355 7.50 7.50 6.68 6.70 9.95 9.65 5.78 7.22 4.80 11356 6.95 7.00 6.28 6.28 9.07 8.60 5.37 6.61 4.52 11357 7.53 7.56 6.80 6.87 9.62 9.12 5.79 6.81 4.80
213 11358 6.95 6.98 6.20 6.20 9.13 8.74 5.24 6.61 4.70 11359 7.55 7.53 6.73 6.68 10.13 9.70 5.90 7.49 5.03 11360 7.47 7.48 6.75 6.78 9.57 9.15 5.68 6.78 4.93 11361 7.05 7.09 6.34 6.30 9.34 9.00 5.48 6.70 4.65 11362 7.20 7.19 6.36 6.40 9.38 8.60 5.67 6.76 5.13 11363 6.52 6.50 5.90 5.89 8.81 8.31 5.19 6.68 4.34 11364 7.11 7.11 6.30 6.32 9.78 9.30 5.82 6.94 4.98 11365 7.82 7.81 6.90 6.90 9.88 9.50 5.78 7.14 4.90 11366 7.30 7.30 6.58 6.53 9.76 9.40 5.52 7.23 4.89 11367 7.30 7.35 6.73 6.66 9.67 9.24 5.67 7.00 4.81 11368 6.92 7.03 6.07 6.09 10.28 9.60 9.20 1.78 5.10 11369 6.30 6.26 5.50 5.55 9.51 8.80 8.40 1.91 4.59 11370 6.59 6.63 5.73 5.74 9.20 8.58 8.03 1.77 4.51 11371 7.81 7.77 6.80 6.80 10.95 10.28 9.70 2.00 5.13 11372 7.40 7.45 6.60 6.59 10.65 10.31 9.39 1.90 5.00 11373 7.20 7.22 6.20 6.21 10.15 9.50 8.92 1.90 4.89 11374 7.49 7.54 6.60 6.60 10.94 10.43 9.61 2.00 4.98 11375 7.49 7.47 6.70 6.74 10.46 9.78 9.28 1.81 5.20 11376 7.10 7.30 6.37 6.40 10.17 9.55 9.12 1.62 4.80 11377 7.36 7.31 6.36 6.40 10.06 9.50 9.00 1.69 4.70 11378 6.86 6.82 5.84 5.80 9.89 9.44 8.81 1.50 4.74 11379 7.15 7.15 6.33 6.37 10.00 9.57 8.90 1.70 4.78 11380 7.26 7.30 6.45 6.43 10.27 9.57 9.04 1.97 4.88 11381 7.53 7.46 6.51 6.57 10.50 10.06 9.23 2.09 5.02
214 Appendix B2: Collection data to accompany morphological measures. Number Sex Stage Year Month Site Stream Mating Location Infection 10392 M A 2002 may 8 Harker Y U N 10393 F A 2002 may 8 Harker Y U N 10394 M A 2002 may 8 Harker Y U N 10395 F A 2002 may 8 Harker Y U N 10396 M A 2002 may 8 Harker Y U N 10397 F A 2002 may 8 Harker Y U N 10398 M A 2002 may 8 Harker Y U N 10399 F A 2002 may 8 Harker Y U N 10400 M A 2002 may 8 Harker Y U N 10401 F A 2002 may 8 Harker Y U N 10402 M A 2002 may 8 Harker Y U N 10403 F A 2002 may 8 Harker Y U N 10404 M A 2002 may 8 Harker Y U N 10405 F A 2002 may 8 Harker Y U N 10406 M A 2002 may 8 Harker Y U N 10407 F A 2002 may 8 Harker Y U N 10409 F A 2002 may 8 Harker Y U N 10413 F A 2002 may 8 Harker Y U N 10415 F A 2002 may 8 Harker Y U Y 10416 M A 2002 may 8 Harker N U N 10417 M A 2002 may 8 Harker N U N 10421 M A 2002 may 8 Harker N U N 10422 M A 2002 may 8 Harker N U Y 10424 M A 2002 may 8 Harker N U Y 10425 M A 2002 may 8 Harker N U Y 10426 M A 2002 may 8 Harker N U N 10429 M A 2002 may 8 Harker N U N 10430 M A 2002 may 8 Harker N U N 10431 M A 2002 may 8 Harker N U N 10433 F A 2002 may 8 Harker N U N 10434 F A 2002 may 8 Harker N U N 10436 F A 2002 may 8 Harker N U N 10440 F A 2002 may 8 Harker N U N 10441 F A 2002 may 8 Harker N U Y 10447 M A 2002 may 1 Darr Y D N 10448 F A 2002 may 1 Darr Y D N 215 10449 M A 2002 may 1 Darr Y D N 10450 F A 2002 may 1 Darr Y D N 10452 F A 2002 may 1 Darr Y D N 10453 M A 2002 may 1 Darr Y D N 10454 F A 2002 may 1 Darr Y D N 10455 M A 2002 may 1 Darr Y D N 10456 F A 2002 may 1 Darr Y D N 10458 F A 2002 may 1 Darr Y D N 10459 M A 2002 may 1 Darr N D N 10464 M A 2002 may 1 Darr N D N 10465 M A 2002 may 1 Darr N D Y 10466 M A 2002 may 1 Darr N D N 10468 M A 2002 may 1 Darr N D N 10469 M A 2002 may 1 Darr N D N 10470 M A 2002 may 1 Darr N D Y 10472 M A 2002 may 1 Darr N D Y 10473 M A 2002 may 1 Darr N D N 10474 M A 2002 may 1 Darr N D N 10483 M A 2002 may 1 Darr N D N 10484 M A 2002 may 1 Darr N D N 10485 M A 2002 may 1 Darr N D N 10486 F A 2002 may 1 Darr N D N 10487 F A 2002 may 1 Darr N D N 10488 F A 2002 may 1 Darr N D N 10489 F A 2002 may 1 Darr N D N 10491 F A 2002 may 1 Darr N D Y 10492 F A 2002 may 1 Darr N D N 10493 M A 2002 may 5 Collin Y D N 10494 F A 2002 may 5 Collin Y D N 10495 M A 2002 may 5 Collin Y D N 10496 F A 2002 may 5 Collin Y D N 10497 M A 2002 may 5 Collin Y D N 10498 F A 2002 may 5 Collin Y D N 10499 M A 2002 may 5 Collin Y D N 10500 F A 2002 may 5 Collin Y D N 10502 F A 2002 may 5 Collin Y D N 10503 M A 2002 may 5 Collin Y D Y 10504 F A 2002 may 5 Collin Y D N 10505 M A 2002 may 5 Collin N D N 10506 M A 2002 may 5 Collin N D N
216 10507 M A 2002 may 5 Collin N D Y 10508 M A 2002 may 5 Collin N D N 10509 M A 2002 may 5 Collin N D Y 10510 M A 2002 may 5 Collin N D Y 10511 M A 2002 may 5 Collin N D N 10512 M A 2002 may 5 Collin N D N 10513 M A 2002 may 5 Collin N D N 10514 M A 2002 may 5 Collin N D N 10515 M A 2002 may 5 Collin N D N 10516 M A 2002 may 5 Collin N D Y 10517 F A 2002 may 5 Collin N D N 10518 F A 2002 may 5 Collin N D N 10519 F A 2002 may 5 Collin N D N 10520 F A 2002 may 5 Collin N D N 10521 F A 2002 may 5 Collin N D Y 10522 F A 2002 may 5 Collin N D Y 10523 F A 2002 may 5 Collin N D N 10524 F A 2002 may 5 Collin N D N 10525 M A 2002 may 7 Harker Y D N 10526 F A 2002 may 7 Harker Y D N 10527 M A 2002 may 7 Harker N D Y 10528 M A 2002 may 7 Harker N D N 10529 M A 2002 may 7 Harker N D N 10530 M A 2002 may 7 Harker N D N 10531 M A 2002 may 7 Harker N D Y 10532 M A 2002 may 7 Harker N D N 10533 M A 2002 may 7 Harker N D N 10534 M A 2002 may 7 Harker N D N 10535 M A 2002 may 7 Harker N D N 10536 M A 2002 may 7 Harker N D N 10537 M A 2002 may 7 Harker N D N 10539 M A 2002 June 1 Darr Y D Y 10540 F A 2002 June 1 Darr Y D N 10541 M A 2002 June 1 Darr N D N 10543 M A 2002 June 1 Darr N D Y 10544 M A 2002 June 1 Darr N D N 10545 M A 2002 June 1 Darr N D N 10546 M A 2002 June 1 Darr N D Y 10547 M A 2002 June 1 Darr N D Y 10548 M A 2002 June 1 Darr N D Y
217 10549 M A 2002 June 1 Darr N D Y 10550 M A 2002 June 1 Darr N D Y 10551 M A 2002 June 1 Darr N D Y 10552 M A 2002 June 1 Darr N D N 10553 F A 2002 June 1 Darr N D N 10554 M N5 2002 June 1 Darr N D N 10559 F N5 2002 June 1 Darr N D N 10560 F N5 2002 June 1 Darr N D N 10561 M N5 2002 June 1 Darr N D N 10563 F N5 2002 June 1 Darr N D N 10565 F N4 2002 June 1 Darr N D N 10566 M N3 2002 June 1 Darr N D N 10567 M N4 2002 June 1 Darr N D N 10568 F N4 2002 June 1 Darr N D N 10570 F N4 2002 June 1 Darr N D N 10574 F N4 2002 June 1 Darr N D N 10577 F N4 2002 June 1 Darr N D N 10588 M N3 2002 June 1 Darr N D N 10589 F N3 2002 June 1 Darr N D N 10590 M N3 2002 June 1 Darr N D N 10591 M N3 2002 June 1 Darr N D N 10592 F N3 2002 June 1 Darr N D N 10593 M N3 2002 June 1 Darr N D N 10596 M N3 2002 June 1 Darr N D N 10598 M N3 2002 June 1 Darr N D N 10599 M N3 2002 June 1 Darr N D N 10600 n/a N3 2002 June 1 Darr N D N 10601 n/a N3 2002 June 1 Darr N D N 10603 M N3 2002 June 1 Darr N D N 10604 F N3 2002 June 1 Darr N D N 10609 M A 2002 June 8 Harker Y U N 10610 F A 2002 June 8 Harker Y U Y 10611 M A 2002 June 8 Harker Y U Y 10612 F A 2002 June 8 Harker Y U Y 10613 M A 2002 June 8 Harker Y U N 10614 F A 2002 June 8 Harker Y U N 10615 M A 2002 June 8 Harker Y U N 10616 F A 2002 June 8 Harker Y U Y 10617 M A 2002 June 8 Harker N U Y 10618 M A 2002 June 8 Harker N U N
218 10619 M A 2002 June 8 Harker N U N 10620 M A 2002 June 8 Harker N U N 10621 M A 2002 June 8 Harker N U N 10622 M A 2002 June 8 Harker N U Y 10623 M A 2002 June 8 Harker N U Y 10624 M A 2002 June 8 Harker N U Y 10625 M A 2002 June 8 Harker N U N 10626 M A 2002 June 8 Harker N U Y 10627 M A 2002 June 8 Harker N U N 10628 M A 2002 June 8 Harker N U Y 10629 M A 2002 June 8 Harker N U N 10630 M A 2002 June 8 Harker N U N 10631 M A 2002 June 8 Harker N U N 10632 M A 2002 June 8 Harker N U N 10633 M A 2002 June 8 Harker N U N 10634 M A 2002 June 8 Harker N U Y 10635 M A 2002 June 8 Harker N U Y 10636 M A 2002 June 8 Harker N U N 10637 M A 2002 June 8 Harker N U N 10638 M A 2002 June 8 Harker N U Y 10639 M A 2002 June 8 Harker N U Y 10640 M A 2002 June 8 Harker N U Y 10641 M A 2002 June 8 Harker N U Y 10642 F A 2002 June 8 Harker N U Y 10643 M N5 2002 June 8 Harker N U N 10644 M N5 2002 June 8 Harker N U N 10645 M N5 2002 June 8 Harker N U N 10646 F N4 2002 June 8 Harker N U N 10650 M N4 2002 June 8 Harker N U N 10651 F N4 2002 June 8 Harker N U N 10652 M N4 2002 June 8 Harker N U N 10653 M N4 2002 June 8 Harker N U N 10654 F N4 2002 June 8 Harker N U N 10655 F N4 2002 June 8 Harker N U N 10658 M N3 2002 June 8 Harker N U N 10659 F N3 2002 June 8 Harker N U N 10660 F N3 2002 June 8 Harker N U N 10661 F N3 2002 June 8 Harker N U N 10662 F N3 2002 June 8 Harker N U N 10663 M N3 2002 June 8 Harker N U N
219 10664 M N3 2002 June 8 Harker N U N 10665 M N3 2002 June 8 Harker N U N 10666 F N3 2002 June 8 Harker N U N 10667 M N3 2002 June 8 Harker N U N 10668 n/a N3 2002 June 8 Harker N U N 10670 F A 2002 June 7 Harker Y D N 10671 M A 2002 June 7 Harker N D Y 10672 M A 2002 June 7 Harker N D Y 10673 M A 2002 June 7 Harker N D N 10674 M N4 2002 June 7 Harker N D N 10675 F N4 2002 June 7 Harker N D N 10677 F N4 2002 June 7 Harker N D N 10679 M N3 2002 June 7 Harker N D N 10681 M N3 2002 June 7 Harker N D N 10682 F N3 2002 June 7 Harker N D N 10684 F N3 2002 June 7 Harker N D N 10685 M N3 2002 June 7 Harker N D N 10690 M A 2002 July 8 Harker N D N 10691 M A 2002 July 8 Harker N D N 10692 M A 2002 July 8 Harker N D N 10693 M A 2002 July 8 Harker N D N 10694 M A 2002 July 8 Harker N D N 10695 M A 2002 July 8 Harker N D N 10696 M A 2002 July 8 Harker N D N 10698 M A 2002 July 8 Harker N D N 10699 M A 2002 July 8 Harker N D N 10705 M A 2002 July 8 Harker N D N 10707 M A 2002 July 8 Harker N D N 10710 M A 2002 July 8 Harker N D N 10711 F A 2002 July 8 Harker N D N 10713 F A 2002 July 8 Harker N D N 10716 F A 2002 July 8 Harker N D N 10717 F A 2002 July 8 Harker N D N 10718 F A 2002 July 8 Harker N D N 10721 F A 2002 July 8 Harker N D N 10724 F A 2002 July 8 Harker N D N 10725 F A 2002 July 8 Harker N D N 10727 F A 2002 July 8 Harker N D N 10728 F A 2002 July 8 Harker N D N 10729 F A 2002 July 8 Harker N D N
220 10730 F A 2002 July 8 Harker N D N 10733 F A 2002 July 8 Harker N D N 10734 F A 2002 July 8 Harker N D N 10735 F A 2002 July 8 Harker N D N 10736 F A 2002 July 8 Harker N D N 10737 F A 2002 July 8 Harker N D N 10741 F N5 2002 July 8 Harker N D N 10744 M N5 2002 July 8 Harker N D N 10745 F N5 2002 July 8 Harker N D N 10746 M N5 2002 July 8 Harker N D N 10747 F N5 2002 July 8 Harker N D N 10749 M N5 2002 July 8 Harker N D N 10750 F N5 2002 July 8 Harker N D N 10752 F N5 2002 July 8 Harker N D N 10754 F N5 2002 July 8 Harker N D N 10755 M N5 2002 July 8 Harker N D N 10756 M N5 2002 July 8 Harker N D N 10757 F N5 2002 July 8 Harker N D N 10758 F N5 2002 July 8 Harker N D N 10760 M N4 2002 July 8 Harker N D N 10762 M N4 2002 July 8 Harker N D N 10763 F N4 2002 July 8 Harker N D N 10765 F N4 2002 July 8 Harker N D N 10766 F N4 2002 July 8 Harker N D N 10769 F N4 2002 July 8 Harker N D N 10770 F N4 2002 July 8 Harker N D N 10772 M N4 2002 July 8 Harker N D N 10773 F N4 2002 July 8 Harker N D N 10776 F N4 2002 July 8 Harker N D N 10777 F N4 2002 July 8 Harker N D N 10780 F N4 2002 July 8 Harker N D N 10781 M N4 2002 July 8 Harker N D N 10783 F N4 2002 July 8 Harker N D N 10784 F N4 2002 July 8 Harker N D N 10785 F N4 2002 July 8 Harker N D N 10786 F N4 2002 July 8 Harker N D N 10787 M N4 2002 July 8 Harker N D N 10788 M N4 2002 July 8 Harker N D N 10791 M A 2002 July 7 Harker N U N 10792 M A 2002 July 7 Harker N U N
221 10794 M A 2002 July 7 Harker N U N 10795 M A 2002 July 7 Harker N U N 10796 M A 2002 July 7 Harker N U N 10797 M A 2002 July 7 Harker N U N 10798 F A 2002 July 7 Harker N U N 10799 M A 2002 July 7 Harker N U N 10800 M A 2002 July 7 Harker N U N 10802 M A 2002 July 7 Harker N U N 10803 M A 2002 July 7 Harker N U N 10804 M A 2002 July 7 Harker N U N 10805 M A 2002 July 7 Harker N U N 10806 M A 2002 July 7 Harker N U N 10807 M A 2002 July 7 Harker N U N 10808 M A 2002 July 7 Harker N U N 10809 M A 2002 July 7 Harker N U N 10810 M A 2002 July 7 Harker N U N 10811 M A 2002 July 7 Harker N U N 10812 M A 2002 July 7 Harker N U N 10813 M A 2002 July 7 Harker N U N 10814 F A 2002 July 7 Harker N U N 10815 F A 2002 July 7 Harker N U N 10816 F A 2002 July 7 Harker N U N 10818 F A 2002 July 7 Harker N U N 10819 F A 2002 July 7 Harker N U N 10820 F A 2002 July 7 Harker N U N 10821 F A 2002 July 7 Harker N U N 10822 F A 2002 July 7 Harker N U N 10823 F A 2002 July 7 Harker N U N 10824 F A 2002 July 7 Harker N U N 10825 F A 2002 July 7 Harker N U N 10826 F A 2002 July 7 Harker N U N 10827 F A 2002 July 7 Harker N U N 10828 F A 2002 July 7 Harker N U N 10829 F A 2002 July 7 Harker N U N 10830 F A 2002 July 7 Harker N U N 10831 F A 2002 July 7 Harker N U N 10832 F A 2002 July 7 Harker N U N 10833 F A 2002 July 7 Harker N U N 10834 F A 2002 July 7 Harker N U N 10835 F A 2002 July 7 Harker N U N
222 10836 F A 2002 July 7 Harker N U N 10837 F A 2002 July 7 Harker N U N 10839 F A 2002 July 7 Harker N U N 10840 F A 2002 July 7 Harker N U N 10842 M N5 2002 July 7 Harker N U N 10843 F N5 2002 July 7 Harker N U N 10844 F N5 2002 July 7 Harker N U N 10845 M N5 2002 July 7 Harker N U N 10846 M N5 2002 July 7 Harker N U N 10847 F N5 2002 July 7 Harker N U N 10849 M N5 2002 July 7 Harker N U N 10850 M N5 2002 July 7 Harker N U N 10851 F N5 2002 July 7 Harker N U N 10852 F N5 2002 July 7 Harker N U N 10853 M N5 2002 July 7 Harker N U N 10854 M N5 2002 July 7 Harker N U N 10855 M N5 2002 July 7 Harker N U N 10856 M N5 2002 July 7 Harker N U N 10858 M N5 2002 July 7 Harker N U N 10860 M N5 2002 July 7 Harker N U N 10862 M N5 2002 July 7 Harker N U N 10863 M N5 2002 July 7 Harker N U N 10864 F N5 2002 July 7 Harker N U N 10865 F N5 2002 July 7 Harker N U N 10867 F N5 2002 July 7 Harker N U N 10868 M N5 2002 July 7 Harker N U N 10869 F N5 2002 July 7 Harker N U N 10873 M N5 2002 July 7 Harker N U N 10874 M N5 2002 July 7 Harker N U N 10875 F N4 2002 July 7 Harker N U N 10876 M N4 2002 July 7 Harker N U N 10877 M N4 2002 July 7 Harker N U N 10878 F N4 2002 July 7 Harker N U N 10880 M N4 2002 July 7 Harker N U N 10883 F N4 2002 July 7 Harker N U N 10884 F N4 2002 July 7 Harker N U N 10885 M N4 2002 July 7 Harker N U N 10886 M N4 2002 July 7 Harker N U N 10888 M N4 2002 July 7 Harker N U N 10891 n/a N3 2002 July 7 Harker N U N
223 10892 M A 2002 July 1 Darr N D N 10893 M A 2002 July 1 Darr N D N 10894 M A 2002 July 1 Darr N D N 10895 M A 2002 July 1 Darr N D N 10896 M A 2002 July 1 Darr N D N 10897 M A 2002 July 1 Darr N D N 10898 M A 2002 July 1 Darr N D N 10899 M A 2002 July 1 Darr N D N 10901 M A 2002 July 1 Darr N D N 10902 M A 2002 July 1 Darr N D N 10903 M A 2002 July 1 Darr N D N 10904 M A 2002 July 1 Darr N D N 10905 M A 2002 July 1 Darr N D N 10906 M A 2002 July 1 Darr N D N 10907 M A 2002 July 1 Darr N D N 10908 M A 2002 July 1 Darr N D N 10909 M A 2002 July 1 Darr N D N 10911 M A 2002 July 1 Darr N D N 10912 M A 2002 July 1 Darr N D N 10913 M A 2002 July 1 Darr N D N 10915 F A 2002 July 1 Darr N D N 10916 F A 2002 July 1 Darr N D N 10917 F A 2002 July 1 Darr N D N 10920 F A 2002 July 1 Darr N D N 10921 M A 2002 July 1 Darr N D N 10924 M A 2002 July 1 Darr N D N 10926 F A 2002 July 1 Darr N D N 10927 F A 2002 July 1 Darr N D N 10928 F A 2002 July 1 Darr N D N 10929 F A 2002 July 1 Darr N D N 10930 F A 2002 July 1 Darr N D N 10932 F A 2002 July 1 Darr N D N 10934 F A 2002 July 1 Darr N D N 10935 F A 2002 July 1 Darr N D N 10936 F A 2002 July 1 Darr N D N 10937 F A 2002 July 1 Darr N D Y 10938 F A 2002 July 1 Darr N D N 10940 F A 2002 July 1 Darr N D N 10941 F A 2002 July 1 Darr N D N 10942 F A 2002 July 1 Darr N D N
224 10943 F N5 2002 July 1 Darr N D N 10947 M N5 2002 July 1 Darr N D Y 10949 F N5 2002 July 1 Darr N D N 10950 M N5 2002 July 1 Darr N D N 10951 F N5 2002 July 1 Darr N D N 10952 F N5 2002 July 1 Darr N D N 10954 F N5 2002 July 1 Darr N D N 10955 M N5 2002 July 1 Darr N D N 10956 M N5 2002 July 1 Darr N D N 10957 F N5 2002 July 1 Darr N D N 10958 M N5 2002 July 1 Darr N D N 10961 M N4 2002 July 1 Darr N D N 10963 M N4 2002 July 1 Darr N D Y 10964 M N4 2002 July 1 Darr N D N 10969 M N4 2002 July 1 Darr N D N 10970 F N4 2002 July 1 Darr N D N 10971 F N4 2002 July 1 Darr N D N 10972 F N4 2002 July 1 Darr N D N 10974 M N4 2002 July 1 Darr N D N 10976 M N4 2002 July 1 Darr N D N 10978 M N4 2002 July 1 Darr N D N 10979 M N4 2002 July 1 Darr N D N 10981 M N4 2002 July 1 Darr N D N 10985 M N4 2002 July 1 Darr N D N 10986 F N4 2002 July 1 Darr N D N 10988 M A 2002 July 5 Collin N D N 10989 M A 2002 July 5 Collin N D N 10990 M A 2002 July 5 Collin N D N 10991 M A 2002 July 5 Collin N D N 10993 M A 2002 July 5 Collin N D N 10994 M A 2002 July 5 Collin N D N 10995 M A 2002 July 5 Collin N D N 10996 M A 2002 July 5 Collin N D N 10997 M A 2002 July 5 Collin N D N 10998 M A 2002 July 5 Collin N D N 10999 M A 2002 July 5 Collin N D N 11000 M A 2002 July 5 Collin N D N 11001 M A 2002 July 5 Collin N D N 11002 M A 2002 July 5 Collin N D N 11003 M A 2002 July 5 Collin N D N
225 11004 M A 2002 July 5 Collin N D N 11005 M A 2002 July 5 Collin N D N 11006 M A 2002 July 5 Collin N D Y 11007 M A 2002 July 5 Collin N D N 11009 M A 2002 July 5 Collin N D N 11012 F A 2002 July 5 Collin N D N 11013 F A 2002 July 5 Collin N D Y 11014 F A 2002 July 5 Collin N D N 11015 M A 2002 July 5 Collin N D N 11016 F A 2002 July 5 Collin N D N 11017 F A 2002 July 5 Collin N D N 11018 F A 2002 July 5 Collin N D N 11019 F A 2002 July 5 Collin N D N 11020 F A 2002 July 5 Collin N D N 11021 F A 2002 July 5 Collin N D N 11022 F A 2002 July 5 Collin N D N 11023 F A 2002 July 5 Collin N D N 11024 F A 2002 July 5 Collin N D Y 11025 F A 2002 July 5 Collin N D N 11026 F A 2002 July 5 Collin N D N 11027 F A 2002 July 5 Collin N D N 11028 F A 2002 July 5 Collin N D N 11030 F A 2002 July 5 Collin N D N 11031 F A 2002 July 5 Collin N D N 11033 F A 2002 July 5 Collin N D N 11034 F A 2002 July 5 Collin N D N 11035 F A 2002 July 5 Collin N D N 11036 F A 2002 July 5 Collin N D N 11037 F A 2002 July 5 Collin N D N 11039 M N5 2002 July 5 Collin N D N 11040 F N5 2002 July 5 Collin N D N 11042 M N5 2002 July 5 Collin N D N 11043 M N5 2002 July 5 Collin N D N 11044 M N5 2002 July 5 Collin N D N 11046 F N5 2002 July 5 Collin N D N 11048 M N5 2002 July 5 Collin N D N 11049 M N5 2002 July 5 Collin N D N 11052 M N5 2002 July 5 Collin N D N 11053 M N5 2002 July 5 Collin N D N 11055 F N5 2002 July 5 Collin N D N
226 11057 F N5 2002 July 5 Collin N D N 11058 F N5 2002 July 5 Collin N D N 11060 F N5 2002 July 5 Collin N D Y 11061 F N5 2002 July 5 Collin N D N 11063 M N5 2002 July 5 Collin N D N 11064 M N5 2002 July 5 Collin N D Y 11066 F N5 2002 July 5 Collin N D N 11068 F N5 2002 July 5 Collin N D N 11071 M N5 2002 July 5 Collin N D Y 11073 F N4 2002 July 5 Collin N D N 11075 F N4 2002 July 5 Collin N D N 11076 M N4 2002 July 5 Collin N D Y 11077 M N4 2002 July 5 Collin N D N 11079 M N4 2002 July 5 Collin N D N 11083 F N3 2002 July 5 Collin N D N 11084 F N3 2002 July 5 Collin N D N 11086 M A 2002 Sept 5 Collin N D N 11087 M A 2002 Sept 5 Collin N D Y 11088 M A 2002 Sept 5 Collin N D N 11089 M A 2002 Sept 5 Collin N D N 11090 M A 2002 Sept 5 Collin N D N 11091 M A 2002 Sept 5 Collin N D N 11093 M A 2002 Sept 5 Collin N D N 11094 M A 2002 Sept 5 Collin N D N 11095 M A 2002 Sept 5 Collin N D N 11096 M A 2002 Sept 5 Collin N D Y 11097 M A 2002 Sept 5 Collin N D Y 11098 M A 2002 Sept 5 Collin N D N 11099 M A 2002 Sept 5 Collin N D N 11100 M A 2002 Sept 5 Collin N D Y 11101 M A 2002 Sept 5 Collin N D N 11102 F A 2002 Sept 5 Collin N D N 11103 M A 2002 Sept 5 Collin N D N 11104 M A 2002 Sept 5 Collin N D N 11105 M A 2002 Sept 5 Collin N D N 11106 M A 2002 Sept 5 Collin N D N 11107 M A 2002 Sept 5 Collin N D N 11108 M A 2002 Sept 5 Collin N D N 11109 M A 2002 Sept 5 Collin N D N 11110 M A 2002 Sept 5 Collin N D N
227 11111 M A 2002 Sept 5 Collin N D N 11112 F A 2002 Sept 5 Collin N D N 11113 F A 2002 Sept 5 Collin N D N 11114 F A 2002 Sept 5 Collin N D N 11115 F A 2002 Sept 5 Collin N D N 11116 F A 2002 Sept 5 Collin N D N 11117 F A 2002 Sept 5 Collin N D N 11118 F A 2002 Sept 5 Collin N D N 11119 F A 2002 Sept 5 Collin N D N 11120 F A 2002 Sept 5 Collin N D N 11121 F A 2002 Sept 5 Collin N D N 11122 F A 2002 Sept 5 Collin N D Y 11123 F A 2002 Sept 5 Collin N D N 11124 F A 2002 Sept 5 Collin N D N 11125 F A 2002 Sept 5 Collin N D N 11126 F A 2002 Sept 5 Collin N D N 11127 F A 2002 Sept 5 Collin N D N 11128 F A 2002 Sept 5 Collin N D N 11129 F A 2002 Sept 5 Collin N D N 11130 F A 2002 Sept 5 Collin N D N 11131 F A 2002 Sept 5 Collin N D Y 11132 F A 2002 Sept 5 Collin N D N 11133 F A 2002 Sept 5 Collin N D N 11135 F A 2002 Sept 5 Collin N D N 11136 F A 2002 Sept 5 Collin N D N 11142 M A 2002 Sept 1 Darr N D N 11143 M A 2002 Sept 1 Darr N D N 11144 M A 2002 Sept 1 Darr N D N 11145 M A 2002 Sept 1 Darr N D N 11146 M A 2002 Sept 1 Darr N D N 11147 M A 2002 Sept 1 Darr N D Y 11148 F A 2002 Sept 1 Darr N D N 11149 M A 2002 Sept 1 Darr N D N 11150 M A 2002 Sept 1 Darr N D N 11151 M A 2002 Sept 1 Darr N D N 11152 M A 2002 Sept 1 Darr N D N 11153 M A 2002 Sept 1 Darr N D N 11154 M A 2002 Sept 1 Darr N D N 11155 M A 2002 Sept 1 Darr N D N 11156 M A 2002 Sept 1 Darr N D N
228 11157 M A 2002 Sept 1 Darr N D N 11158 M A 2002 Sept 1 Darr N D N 11160 M A 2002 Sept 1 Darr N D N 11161 M A 2002 Sept 1 Darr N D N 11162 M A 2002 Sept 1 Darr N D N 11163 M A 2002 Sept 1 Darr N D N 11164 M A 2002 Sept 1 Darr N D N 11165 M A 2002 Sept 1 Darr N D N 11166 M A 2002 Sept 1 Darr N D N 11167 M A 2002 Sept 1 Darr N D N 11168 M A 2002 Sept 1 Darr N D N 11169 M A 2002 Sept 1 Darr N D N 11170 M A 2002 Sept 1 Darr N D N 11171 M A 2002 Sept 1 Darr N D N 11172 M A 2002 Sept 1 Darr N D N 11173 M A 2002 Sept 1 Darr N D N 11174 M A 2002 Sept 1 Darr N D N 11175 M A 2002 Sept 1 Darr N D N 11176 M A 2002 Sept 1 Darr N D N 11177 M A 2002 Sept 1 Darr N D N 11178 F A 2002 Sept 1 Darr N D N 11179 F A 2002 Sept 1 Darr N D N 11180 F A 2002 Sept 1 Darr N D N 11181 F A 2002 Sept 1 Darr N D N 11182 F A 2002 Sept 1 Darr N D N 11183 F A 2002 Sept 1 Darr N D N 11184 F A 2002 Sept 1 Darr N D N 11185 F A 2002 Sept 1 Darr N D N 11186 F A 2002 Sept 1 Darr N D N 11187 F A 2002 Sept 1 Darr N D N 11188 F A 2002 Sept 1 Darr N D N 11189 F A 2002 Sept 1 Darr N D N 11190 F A 2002 Sept 1 Darr N D N 11191 F A 2002 Sept 1 Darr N D N 11192 F A 2002 Sept 1 Darr N D N 11193 F A 2002 Sept 1 Darr N D N 11194 F A 2002 Sept 1 Darr N D N 11195 F A 2002 Sept 1 Darr N D N 11196 F A 2002 Sept 1 Darr N D N 11197 F A 2002 Sept 1 Darr N D N
229 11198 F A 2002 Sept 1 Darr N D N 11199 F A 2002 Sept 1 Darr N D N 11200 F A 2002 Sept 1 Darr N D N 11201 F A 2002 Sept 1 Darr N D Y 11202 F A 2002 Sept 1 Darr N D N 11203 F A 2002 Sept 1 Darr N D N 11207 M A 2002 Sept 1 Darr N D N 11208 F A 2002 Sept 1 Darr N D N 11209 F A 2002 Sept 1 Darr N D N 11328 M A 2002 Oct 8 Harker N U N 11329 M A 2002 Oct 8 Harker N U Y 11330 M A 2002 Oct 8 Harker N U N 11331 M A 2002 Oct 8 Harker N U N 11332 M A 2002 Oct 8 Harker N U N 11333 M A 2002 Oct 8 Harker N U N 11334 M A 2002 Oct 8 Harker N U N 11335 M A 2002 Oct 8 Harker N U N 11336 M A 2002 Oct 8 Harker N U N 11337 M A 2002 Oct 8 Harker N U N 11338 M A 2002 Oct 8 Harker N U N 11339 M A 2002 Oct 8 Harker N U N 11340 M A 2002 Oct 8 Harker N U N 11341 M A 2002 Oct 8 Harker N U N 11342 M A 2002 Oct 8 Harker N U N 11343 M A 2002 Oct 8 Harker N U N 11344 M A 2002 Oct 8 Harker N U Y 11345 M A 2002 Oct 8 Harker N U Y 11346 M A 2002 Oct 8 Harker N U N 11347 M A 2002 Oct 8 Harker N U N 11348 M A 2002 Oct 8 Harker N U N 11349 M A 2002 Oct 8 Harker N U N 11350 M A 2002 Oct 8 Harker N U N 11351 M A 2002 Oct 8 Harker N U N 11352 M A 2002 Oct 8 Harker N U N 11353 M A 2002 Oct 8 Harker N U N 11354 M A 2002 Oct 8 Harker N U N 11355 M A 2002 Oct 8 Harker N U Y 11356 M A 2002 Oct 8 Harker N U N 11357 M A 2002 Oct 8 Harker N U N 11358 M A 2002 Oct 8 Harker N U Y
230 11359 M A 2002 Oct 8 Harker N U N 11360 M A 2002 Oct 8 Harker N U N 11361 M A 2002 Oct 8 Harker N U N 11362 M A 2002 Oct 8 Harker N U N 11363 M A 2002 Oct 8 Harker N U N 11364 M A 2002 Oct 8 Harker N U N 11365 M A 2002 Oct 8 Harker N U N 11366 M A 2002 Oct 8 Harker N U N 11367 M A 2002 Oct 8 Harker N U N 11368 F A 2002 Oct 8 Harker N U N 11369 F A 2002 Oct 8 Harker N U N 11370 F A 2002 Oct 8 Harker N U N 11371 F A 2002 Oct 8 Harker N U N 11372 F A 2002 Oct 8 Harker N U N 11373 F A 2002 Oct 8 Harker N U N 11374 F A 2002 Oct 8 Harker N U N 11375 F A 2002 Oct 8 Harker N U N 11376 F A 2002 Oct 8 Harker N U N 11377 F A 2002 Oct 8 Harker N U N 11378 F A 2002 Oct 8 Harker N U Y 11379 F A 2002 Oct 8 Harker N U N 11380 F A 2002 Oct 8 Harker N U N 11381 F A 2002 Oct 8 Harker N U N
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