Environmental Factors Affecting the Prevalence and

Distribution of Myxozoan Parasites and Their Aosts in

Three Lakes in Aigonquin Park, Ontario

Janet Koprivnikar

A thesis submittd in conformity with the requirernents

For the degree of Master of Science

Graduate Department of Zoology

University of Toronto

0 Copyright by Janet Koprivnikar 2001

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Environmental Factors Mecting the Prevalence and Distribution of Myxozoan

Parasites and Their Hosts in Three Lakes in Algonquin Park, Ontario

An abstract of a thesis submitted in conformity with the requirements

for the degree of Master of Science, 100 1

Janet Koprivnikar, Department of Zoology, University of Toronto

Tle oligochaete fauna and several environmental parameters of Lake Sasajewun.

Broadwing Lake and Kathlyn Lake in Algonquin Park were surveyed. The distributional

patterns of the oligochaetes, with respect to the environmental variables, were analyzed and it

was found that substrate type and the presence of certain aquatic plants were related to the

oligochaete composition. Tubificids were associated with detritus and mud, whereas sandy and

pebbly areas were dominaied by naidids. Oligochaete-plant associations rnay account for

differences in the distribution of oligochaete species among the three lakes. The findings also

indicate that the prevalence of certain of oligochaetes is congnient with the absence or presence

of particular mmyxozoan species.

A novel fom of raabeia spore was observed fiom a single specirnen of II. uncinata. This

form differs fiom those previously described by having caudal processes that gradually widen

and terminate with a single prominent branch.

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FactorieUes de l'Environnement Mectant la Fréquence et Distribution de

Parasites Myxozoa et Leurs Hôtes dans Trois Lacs du Parc Algonquin, Ontario

f ar

Janet Koprivnikar

Département de Zoologie, Université de Toronto

Résumé

Les oligochètes et plusiers factorielles de l'environnement du Lac Sasajewun, Lac

Broadwing et Lac Kathlyn dans le Parc Algonquin étaient examiné. Les patterns de distribution

des oligochètes étaient analysé et l'espèce de sédiment et de plant aquatique étaient associes avec

le distribution des vers. Les tubificids étaient associés avec la boue et la matière organique, mais

Ies naidids dominèrent les zones avec Ie sable et la pierre. Les associations entre les oligochètes

et les plants aquatiques peut-être expliquassiez les differences de la distibution des espèces des

oligochètes parmi les trois lacs. La Eréquence des oligochètes specifiques étaient associés avec

l'absence ou la presence d'espèces Myxozoa particulières.

Un nouveau formuIaire du spore raabeia était observé d'un seul spécimen d' Uncinais

rincinata. Ce formulaire est différent des autres décrits parce que les procédés élargissent

graduallement et finissent avec une seule succursale proéminent.

Acknowledgemenb

There are xnany people that 1 wish to thank for their assistance during the course of my

graduate studies. 1 th& Professor Helen Rodd for her advice, cheerfulness, and refùsal to nin

the other way when 1 o h came by crying, "Help!" 1 thank Henry Hong for somehow aiways

knowing the answer, and for makllig tirne for me when 1 really needed i t 1 also owe thanks to

Amanda Martyn for paddling diligently and keeping me amused and sane during the Great Spore

Hunt, and to Anne Koehler for her cheerfui nature in the lab, her fourth year project that was

wonderfûl to build upon, and her role as music maestro, of CO-.

1 would also Iike to thank Professor Don Jackson for bis advice and procurement of my

statistics program, Don Stacey for his advice on the identification and presewation of the

oligochaetes, and Dr. Chongxie Xiao for bis advice on detecting actinosporeans and on field

methods.

1 thank the Ontario Ministry of Natural Resources for the use of the Wildlife Resource

Station in Algonquin Park, and also the staff at the WRS.

1 owe heartfelt thanks to my supervisor, Dr. Sherwin Desser, for taking on a student who

appeared at the last minute wishing to pursue graduate studies, and having faith that 1 would do

the best work possible. 1 also thank Dr. Desser for his continued encouragement and advice.

1 must thank Eric Payne for his encouragement in my academic endeavors, and support in

those times during which 1 mut not have been pleasant to live with! 1 dso thank my parents,

who have always encourageci me to pursue my goals, and have always done what they could to

nurture my love of the naiurai sciences.

Table of Contents

. . ....................................................................................... Abstract 11 ... ...................................................................................... Resume -111

.......................................................................... Acknowledgements iv ............................................................................. Table of Contents v

................................................................................. List of Tables vi . . ................................................................................ List of Figures VII

Chapter 1 . Histoncal Review and General Introduction ................................. 1

Chapter 2 . Environmental Factors Affecting the Prevalence and Distribution of Myxozoan Parasites and Their Hosts in Three Lakes in Algonquin Park.

................................................................................. Ontario 6 ...................................................... Materials and Methods 8

........................................................................ Results 11 .................................................................... Discussion 13

......................................................................... Tables 19 ....................................................................... Figures -23

Chapter 3 . A new fom of Raabeia (Myxozoa: Actinosporea) frorn the oligochaete Uncinuis ttncinatu ................................................................... 29

..................................................... Materials and Methods 3 1 ........................................................................ Results 31

.................................................................... Discussion 32 ....................................................................... Figures -34

............................................................. Chapter 4 . General Discussion 38

................................................................................ Literature Cited 41

List of Tables

Chapter 2 Table 1. Prevalence of myxosporean infection for each species of cyprinid found in

Kathlyn Lake, Broadwing Lake and Lake Sasajewun.. ............................ 19 Table 2. Plant families identified h m Kathlyn Lake, Broadwing Lake and

..................................................................... Lake Sasajewun.. - 2 0 Table 3. Prevalences of oligochaetes identified to the fmily and species levers eom

Kathlyn Lake, Broadukg Lake and Lake Sasajewun.. .......................... - 2 1 Table 4. Pe~centages of 5 substrate types (mud, detritus, rock, pebbles and sand),

comprising the habitats in Kathiyn Lake, Broadwing Lake and Lake .......................................................................................................... Sasajewun ..23

List of Figures

Chapter 2 .......................................................... Fi0w e 1 . Aerial view of the three lakes 23

Figure 2 . CCA ordination biplot of the oligochaete farnilydata ............................. 2 5 ............................. Figure 3 . CCA ordination biplot of the oligochaete species data 27

Chapter 3 Figure 1 . Line drawing of the raabeia-type spore .............................................. 34

........................................................... Figure 2- 5 . Photographs of the spore 36

CHAPTER 1

Historical Review and General Introduction

HISTORICAL REVtEW AND GENERAL INTRODUCTION

Myxospareans were discovered in 1838 by Müller. who called them "psorosperms".

Over 1 100 rnyxosporean species have since been described, most inrecting fish but also

elasmobranchs, bryozoans, Agnatha, Holostei, Dipnoi and Chondrosrei (Lom, 1990).

Actinosporeans were first descnbed by Stolc in 1899, and were considered to be reIated to

myxosporeans. Spores of more than 50 adnosporeans, released by various species of

oligochaetes and polychaetes, have been descrîbed (see review by Marques, 1984; Bartholomew

et al., 1997; Xiao and Desser 1998% b).

Until the mid-1 Bo 's , it was generaliy accepted that the classes Myxosporea and

Actinosporea constituted the phylum Myxozoa Grassé 1960. In 1984, WoIf and Markiw

demonstrated that a myxosporean species, It&.robolirs cerebralis, underwent part of its

development in an oligochaete, Tr1bife.r trtb@~. Numerous studies have confirmed a hvo-host

life cycle for other species of myxosporeans (Ruidisch et aI., 199 1; El-Matbouli et al., 1992;

Bartholomew et al., 1997). As a result ofthese findings, Kent et al. (1994) proposed that the

class Actinosporea be merged into the class Myxosporea. Siddall et al. (1995) proposed that the

phylum Myxozoa be eliminated and that myxozoans be transferred to the phylum Cnidaria.

EIucidation ofthe two-host life cycle led &O an increased interest in myxozoans and many

studies have described the life cycle and uitrastnicture of various species (Desser and Paterson,

1978; Desser et al., 1983a, b; see review by Kent et al., 2001).

Aquatic oligochaetes were recognized and described by the ancient Greeks (Brinkhurst

and Gelder, 1991). The ability of tubificids to develop dense colonies in organically polluted

waters led to these worms being used as indicators of pollution and, subsequently, to many

studies involving the role of ~Iigochaetes in pollution ecology. Certain oligochaete species have

3

been used to assess organic pollution (Milbrink, 1980; Piygiel et al., 2000) and heavy metal

contamination (Chapman et al., 1980; Rosso et al., 1994).

While numerous studies have examined the effects of pollutants on oligochaetes, the

microhabitats and densities of these worms in natural conditions are not weli understood.

Brinkhurst (1999) highlighted the need to study factors that are directly related to worm biology,

such as food, rather than physical and chernical factors related to the water column, which

exphin littIe of the variance in oligochaete distribution. Physicochemical parameters, such as

oxygen content, pH. and the concentration of different ions, often cannot sufficiently explain the

distribution of oligochaetes in natural settings (Smith, 1985; Martinez-Ansemil and Collado,

1996). Oligochaetes graze on microorganisms associated with plants and also on organic matter

in sediment (Brinkhurst and Gelder, 1991). It has been suggested that the precise nature of

organic matter in sedirnents, and the microflora that it supports. determine the outcome of

interspecific cornpetition among oligochaete species (Brinkhurst and Gelder, 199 1). A

correlation between oligochaete abundance and Escherichia coli bacteria counts has been

documented (Brinkhurst and Jamieson, 1971). The succession of various naidid species has been

observed to be consistent with seasonal changes in periphytic algal conununities on reed stems,

indicating that the succession of population peaks of dominant naidids rnay be the result of

changes in the composition of available food (Lohlein, 1996). The availability and nature of the

food supply may also cause considerable local variation in the life cycles of various origochaete

species (Brùikhurst and Gelder, 199 1).

The discovery of the role of oligochaetes in the myxozoan life cycle has stimulated

interest in the study of these worms. Field studies on the dynamics of infection in oligocnaetes

have been targeted as areas of hi& prionty in the research of diseases caused by myxozoan

irifection (Nickum, 1996; Kent et ai., 2001). The impact of myxozoan infections in fish will

continue to drive investigations into the biology of these parasites and their hosts, particdarly in

4

natural settings. To date, with the exception of some field observations (Burtie et al., 1991; Styer

et al., 1991; Yokoyama et al., 1991,1993; Rogdie and Knapp, 1998; El-Mansy et ai., 1998c),

there have been few studies focused on the ecological associations of myxozoan parasites and

their hosts in natural conditions.

The myxosporean parasites of fish in Lake Sasajewun, Broadwing Lake and Kathlyn

Lake in Algonquin Park, Ontario, Canada, have been studied extensively and over 50 species

have been descnbed (Gowen, 1983; Li and Desser, 1985; Xiao and Desser, 1997; Salim and

Desser, 2000). A comprehensive study, one of the first of its kind, was conducted in Lake

Sasajewun in order to examine the oligochaete fauna for corresponding actinosporean stages and

also to examine the habitat preferences of the woms (Xiao and Desser, 1998~). Twenty-two

new forms of actinosporean spores were descnbed (Xiao and Desser, 1998a, b), and the

associations of certain species of oligochaetes with depth and sediment type were noted. Despite

this extensive survey, there remains a large discrepancy between the nurnber of myxosporeans

and actinosporeans described frorn this lake (Xiao and Desser, 1998c), and the biology of

oligochaetes in their natural environment remains largeiy unknown (Brinkhurst, 1999).

The purpose of this research was to conduct further investigations into environmental

factors affecting the prevalence and distribution of rnyxozoan parasites and their oligochaete

alternate hosts by re-exarnining and expanding the study site used by Xiao and Desser (1998~) in

Algonquin Park to include Broadwing Lake and Kathlyn Lake. Oligochaetes were also to be

monitored for the release of actinosporean spores. This thesis was designed to test the

hypothesis that since the three Iakes present very different environments, their oligochaete fauna

would Iikely Vary in composition and density among the lakes, as wouId the rnyxozoan parasites

observed.

The oligochaetes and environmental parameters, including sediment type and vegetation,

were surveyed in the three lakes and the distribution pattern of the oligochaete fauna, with

5

respect to the environmental variables, were analyzed (Chapter 2). The prevalences of certain

myxosporean species infécting four species of cyprinids in the three lakes were also related to

the prevalences of particular oligochaete species.

Examination of oligochaetes for the release of actinosporean spores resulted in the

description of a new form (Chapter 3).

This thesis is organized into self-contained chapters, each with an abstract of its contents,

introduction, materials and methods, results, and discussion.

Environmental Factors Affecting the Prevalence and

Distribution of Myxozoan Parasites and Their Hosts in

Three Lakes in Algonquin Park, Ontario

(Adapted from Koprivnikar, J., Koehler, A., Rodd, F. H., and S. S. Desser. 2001.

Environmental Factors Affecting the Prevalence and Distribution of Myxozoan Parasites

and Their Hosts in Three Lakes in Aigonquin Park, Ontario. J. Parasitol. 87: In Press)

ABS'TRACT:

In 1999,4 species of cyprinids were surveyed for myxozoan parasites in a watershed in

Algonquin Park, Canada, comprised of Kathlyn Lake, Broadwing Lake and Lake Sasajewun.

Eight species of myxozoans were found which diMered in their prevalence and distribution

arnong the 3 Iakes. The oligochaetes and environmental parameters, including sediment types

and aquatic plants, ofthese 3 lakes were surveyed the fotlowing year. Oligochaetes belonging to

17 species were cokcted hom the 3 lakes. The distribution patterns of the oiigochaete fauna,

with respect to the environrnenral variables, were analyzed using canonical correspondence

analysis. Tubificids were predominant in the detritus and muddy substrate of Broadwing Lake,

whereas naidids were predominant in the sandy and pebbly sediment of Lake Sasajewun. Our

findings indicate that the prevalence of certain oligochaetes is congruent with the absence or

presence of particular myxozoün species, and that environmental factors influence the

distribution of certain oligochaete species.

8

INTRODUCTION

Since the discovery of the 2-host myxozoan life cycIe by WoIf and Markiw (1984), there

has been a renewed interest in the Myxozoa. Nurnerous studies describing new species, their

development and transmission have been conducted (Etuidisch et al., 199 1 ; El-Matbouli and

Hoffman, 1998; Xiao and Desser, 1998a, 1998b; Salim and Desser, 2000), however, there has

been little focus on the ecological associations of these parasites and their hosts. One of the first

comprehensive studies was conducted in Lake Sasajewun, Algonquin Park, (Xiao and Desser

1998a,b, c), where over 14 000 oligochaetes were exarnined between 1995 and 1997, and several

new foms of actinosporeans, as well as the habitat preferences of their oligochaete hosts, were

described.

In this study, the original site in Algonquin Park was re-examined and expanded to

include Broadwing Lake and Kathlyn Lake, which are parts of the same watershed. These 3

lakes present very different environments with respect to sediment and aquatic vegetation, both

of which infiuence the presence and distribution of oligochaetes (Barnes, 1968; Brinkhurst and

Jarnieson, 1971). The environmental factors associated with oligochaete abundance and

distribution, as well as associations arnong oligochaete species and myxosporean prevalence in

cyprinids in these lakes, are presented.

MATERIALS AND h.iETHODS

Study Sites

The watershed in Algonquin Park consisteci of the following 3 lakes: Kathlyn Lake (45'

36' 00" N, 78" 32' 20" W) with a surface area of 0.26 km' and a maximum depth of 18 m;

Broadwing Lake (45' 35' 50" N, 78" 32' 00" W) with a surface area of 0.074 km2 and a

maximum depth of 3 m; and Lake Sasajewun (45' 35' 30" N, 7g0 3 1' 30" W), an artificid Iake

created by the damming of the Madawaska River, with a surface area of 0.44 km' and a

maximum depth of 9 m. Shailow streams link the 3 Iakes, with Kathlyn Lake draining into

9

Broadwing Lake, and Lake Sasajewun receiving inflow fiom the other two lakes as well as fiom

the Madawaska River (Fig. 1).

Sampüng

Cyprinids were captured fiom the 3 lakes during May to August 1999, using Ge&

rninnow traps baited with Purina@ Puppy Chow. Cornmon Shiner (Lr~rilris cornutru), Golden

Shiner (Notemigonus crysolezicas), Northern Redbelly Dace (Pho-rinus eus), and Creek Chub

(Semotillis atromacrilatiis) were examined for cyst-forming species of Mvxoboitis. Cysts were

isolated and ruptured on slides, air-dried, stained with Diff-QuikQ, and coverslipped with

Diatex@ mounting media, The spores and polar capsules of 20 specimens of each myosporean

species were measured and taxonomic determinations were made with reference to previous

studies done in the 3 lakes (Gowen, 1983; Li and Desser, 1985; Lom et al., 1989; Salim and

Desser, 2000).

In order to survey the lakes for oligochaetes, sediment and plants, transect lines

separated by approximately 25, 10 m, and 15 m were marked along the shorelines of Lake

Sasajewun, Broadwing Lake, and Kathlyn Lake respectively (Fig. 1). Sediment samples fiom

each of the 3 Iakes were collected fiom June to August 2000 using a device consisting of a 120-

mL plastic container attached to a garden hoe. At each sampling site, sediment samples were

coIlected at water depths of0.5 m and 1 m. Four samples were taken and combined for each

water depth. Sediment samples were not taken at water depths greater than 1 m as oligochaete

abundance decreases sharply beyond this point (Xiao and Desser, 1998~). ln total, 108 sites in

Lake Sasajewun, 52 in Broadwing Lake and 52 in Kathlyn Lake were sampled, with the order of

sampling being random. Sediment samples were sieved through a 0.3 mm rnesh and classified

accordmg to their substrate type. Oligochaetes isolated îÎom the sediment sarnples were fixed in

Kahle's solution, dehydrated through an alcohol series, cleared with xylene, and mounted in

Canada Balsam. Otigochaetes were identified to the family level, and to the species levei when

1 O

possible, following the guidelines of Brinkhurst (1986). Aquatic plant species within a 2 m

radius of each sampling site were identified following the guidelines of Crins et ai. (1998) and

Newmaster et al. (1997).

Data Analysis

The cyprinid data set was examined for differences among the 3 lakes in the overd1

prevalence of infection using linear logistic regression models (PROC LOGISTIC, SAS institute

Inc., 1989), and also for differences in the prevalences of the mwosporean species using

nominal logistic regression (MI?, 1995).

The faunistic and environmental data were analyzed to detemine the distribution patterns

of the oligochaete fauna and their relation to the environmental variables using canonicaf

correspondence analysis (CCA) in the program CANOCO V. 4.0 (Ter B m k , 1987; Ter Braak

and Verdonschot, 1995). Twenty-three environmental factors were included in the analyses,

among them the 3 lakes, 5 sediment types, water depth, shore distance, and 13 aquatic plant

fmilies (see Tables 2 and 4).

Analyses were performed with data combined from al1 3 lakes, and separately with data

fiom each lake, using the oligochaete fauna classified at both the family and species level.

Oligochaete abundances were log-transformed afler adding a vatue of O. 1 to each value in the

data sets. Rare species were downweighted and the Enchytraeidae, represented by a single

specimen of Cognerria glanddosa, was given a weight of O.

RESULTS

Myxosporean data

Eight species of Myxosporea were identified fiom 1271 cypnnids colIected h m the 3

!&es (Table 1). in the 4 cyprinid species, the prevalence of infection was 15% in Lake

Sasajewun, 1 1% in Broadwing Lake, and 7% in Kathlyn Lake. Significant differences were

found among the 3 lakes in the overall prevalence of infection (chi-square statistic= 15.790,

I I

p< 0.0001), but there was no evidence that infection rate varied across time. The prevalence of

infection also differed significantly among the lakes for 3 of the cyprinid species: C o m o n

Shiner (chi square= 6.361, p < 0.0416). Golden Shiner (chi-square= 14.418, p < 0.0007), and

Creek Chub (chi-square= 15.333, p <0.0005). Three of the myxosporean species, h[v.roboitls

algonquinensis, 12.L xiaoi, and M. barrai, were not found in cyprinids fiom Kathlyn Lake.

General Patterns of Oligochaete Distribution

A total of 991 oligochaetes belonging to 17 species were collected fiom the 3 lakes

(Table 2). Lake Sasajewun had the highest percentage of sites with oligochaetes (8 1?6),

consisting mainly of Ripisres parasita and Iiyodriius rempleroni. The oligochaete fauna in

Broadwing Lake was less abundant (worms present at 58% of sites) than in the other 2 lakes, and

was dominated by Ttrbife.~ trrbif~r, Lhnodriius hofmeisreri, and Dero digitara. Kathlyn Lake

had worms at 68% of the sarnpling sites. Vejdovs~ella contara, Uncinais uncinara. and

Arcteonais lomondi were abundant in Kathlyn Lake but were rare in the other 2 Iakes. The

majority of worms were found within 6 metres of shore, and the distribution was fairly even

between the 2 sarnpling depths.

Twenty-three aquatic plant species beIonging to 13 families were identified from the

sampling sites (Table 3). Five substrate types were observed, tvith the highest percentage of sites

having sandylsilty sediment in Lake Sasajewun, and mudidetritus in Broadwing Lake (Table 4).

A CCA conducted with 01igochaete and environmental data fiom al1 3 lakes (number of

sites-212) indicated a broad distribution of oligochaetes over the study area, with onIy 3.5% of

the variance in species composition expIained by the £ k t a i s , and 3.8% by the second.

Major Factors Innuencing Species Distribution

A CCA conducted with oligochaetes classified to the family level eqlained 58.4% of the

species-environment relation with the first axis, and 40% with the second. Both axes appeared to

12

be prirnady related to the nature of the substrates (Fig. 2). Axis 1 was related to sofi substrates,

particularly rnud, and the aquatic plants associated with those substrates. Hard substrates, such

as rock and pebbles, and their associated flora were related to Axis 2. Mud, shore distance,

pondweed, water shield, heath and Broadwing Lake were strongly correlated factors and showed

a positive correlation with tubificids. ui contrat, naidids were positively conelated with

pebbles, water milfoil, burreed and Lake Sasajewun, which were factors strongly correlated with

one another.

A second overall CCA, conducted with oligochaetes classified at the species level,

resulted in a first avis (23.3% of the species-environment relation) that appeared to be related to

shore distance and the aquatic plant families associated with this factor (Fig.3). The second avis

(19.2% of the species-environment relation) separated Lake Sasajewun and its major substrate

cornponent, sand, from the factor detritus, whicb was the dominant substrate type in Broadwing

Lake and Kathlyn Lake.

The oligochaete species associated with Lake Sasajewun and sand were Ripistes parasita,

I[vodriltcs templetoni, N. commlrrris and Nais variabilis. Shore distance, plantain, grass,

pondweed, lobelia and Broadwing Lake were highIy correlated with each other, and showed a

positive correlation with the oligochaete species Slavina apperrdiciitata, Trrbife.1: rubife.1: and

Rhyacodnltcs coccineus. A cornparison of the CCA results for each lake revealed that some

environmental factors explained negligible variance in 2 of the 3 lakes. This occurred when a

factor was rare in a lake or was not present at dl. Although the grass family was a variable in the

CCA performed for Broadwing Lake data, the variance was negligible in the other 2 lakes,

therefore this factor was not included in their analyses. Pipewort and lobelia were included as

factors in only the Kathlyn Lake analysis, whereas sand and milfoil were included in only the

Lake Sasajewun analysis.

DISCUSSION

Oiigochaete Habitat Associations

Most of the oligochaete species found in the 3 lakes occuned in a wide range of habitats;

however, general trends were observed for farnilies and for those species especially abundant in

only 1 lake. Lake Sasajewun had the most oligochaete species, probably as a result of the geat

diversity in habitats, particularly with respect to sediment composition. Al15 substrate types

were present, including a large number of areas with stony and pebbly sediments that are well

suited for naidids (Brinkhurst and Jarnieson, 1971 ). The latter authors showed that larger lakes

have a greater number of oligochaetes, probably a result of the greater diversity of microhabitats

present. While Kathlyn Lake shares a number of characteristics with Lake Sasajewun that make

it suitable for naidids, Lake Sasajewun may have a greater abundance of oligochaetes due to the

extensive gently sloping shoreIine and the factors reIated to this variable, such as the aquatic

flora. Xiao and Desser (1998~) found that the number of species and overall prevalence of

oligochaetes declined with increased water depth.

There was a high prevalence of tubificids in Broadwing Lake, likely due to the 0.25- 1 m

thick layer of sofl sediment consisting of mud and decomposing organic matter. Not only do

tubificids prefer to live in these types of sol? sediments (Brinkhurst and Jarnieson, 1971), but che

thick layer of mud and decomposing matter mates an anaerobic environment which can be

tolented by only certain species of tubificids (Bames, 1968).

Oligochaete species rankings for each environmental factor were calculated h m the

combined data ordination diagrams by projecting the species points ont0 the environmental

factor arrows. Upon examination of the environmental factors that had significant variance in

only 1 of the lakes, relationships emerged that may explain differences among the lakes in

oligochaete species prevalences. Tub& mbifer and L. hofieisteri were most abundant in

Broadwing Lake and ranked k t and fourth, respectively, for the p s variable, which was

14

included as a factor in only the Broadwing Lake and overall CCAs, due to its negligable

explanatory power in the analyses for Lake Sasajewun and KathIyn Lake. The oligochaete

species V. comata and W. uncinara were most prevalent in Kathlyn Lake and were each ranked

first for the 2 respective factors ofpipewort and lobelia. These 2 plant famiIies were

environmenta1 factors exctusive to the Kathlyn Lake and overail analyses, and only had

negiigible variance in the other 2 lakes. Ripisres parasita was most abundant in Lake Sasajewun

and ranked second for the environmental factor sand, which was included Ui only the Lake

Sasajewun and overall CCAs. Ilyodriliu rempietorii was aiso most prevalent in Lake Sasajewun,

and ranked third for the rnilfoil variable, which was only a factor in the Lake Sasajewun and

overai1 analyses.

The associations among the plant and oIigochaete communities are important, as the

majority of oligochaete species feed on dead organic matter, especially vegetation (Barnes,

1968). It has been demonstrated that oligochaetes wilI move into an area of sediment with the

highest nutritional potential in terms of organic carbon and nitmgen, regardiess of the texture of

the sediment (Bnnkhmt and lamieson, 1971). It is likely that the precise nature of organic

rnatter in sediment and the microflora it supports determines the outcorne of interspecific

cornpetition, and thus niche discrimination, among oligochaetes (Brinkhurst and Gelder, 1991).

Certain species of oligochaetes may have become specialized feeders of specific microflora, the

presence of which is heavily influenced by the species of aquatic plants present. Such

associations between certain oligochaete and plant species may account for the differences in the

distribution of oligochaete species among the 3 Iakes in this study.

The information gathered in this study confimis the oiigochaete-habitat associations in

Lake Sasajewun found by Xiao and Desser (I998c), who observed that the shoregrass, Linorella

americana, served as a reIiable indicator of fine sik and c1ay sediments in the in-shore areas,

15

which had a hi& prevalence of burrowing tubificid oligochaetes. They also found that shallow

areas with sandy and stony sedirnents were dominated by naidids.

Aost, parasite, and habitat associations

Although the life cycles of many myxozoan species remain unknown, certain species

involved in the myxosporean-oligochaete associations found in this study would be excellent

candidates for experirnental transmission studies.

hQxobolus pseudokoi and the oligochaete i. renlpletoni displayed sirnilar patterns of

prevalence in the 3 lakes. Both species were most prevaient in Lake Sasajewun, followed by

Kathlyn Lake and then Broadwing Lake, suggesting that I. rempleroni rnay be the oligochaete

host for M. psetldokoi. Other patterns of oligochaete and myxosporean prevalence aIso suggest

possible host-parasite relationships. Myxobolus smithi and the oligochaete N. cornmunis both

had the lowest prevalence in Broadwing Lake. Myxobolrrs sp. was present only in Kathlyn Lake,

which had by far the highest prevalence of the worm Il. uncinata, in contrast to the prevalences

in Lake Sasajewun (1%) and Broadwing Lake (0%). It is also interesting to note that 3

myxosporeans, M. barrai, M. algonqtrinensis and M. xiaoi, and the oligochaete L. hoflmeisteri

were not present in Kathlyn Lake but were found in the other 2 Iakes, suggesting that this worm

may be the alternate host for at least 1 of these myxosporeans.

It is possible that the relatively high prevaience of infection observed in the cypnnids

fiom Lake Sasajewun may be partially influenced by the habitat preferences of the fish, in

addition to the higher abundance of oligochaetes as compared to the other 2 lakes. The gently

sloping shailow areas charactenstic of Lake Sasajewun are prime feeding sites for fish that

becorne infected by ingesting infected woms and by coming into contact with waterborne

actinosporeans (Xiao and Desser, 1998~). OIigochaetes are most abundant in such shallow areas

and overlap in these habitats increases the exposure of fish to actinosporeans.

The habitat preferences and spawning behaviours of the 4 cyprinid species rnay

contribute to the arnong-iake differences in the prevaience of myxosporean infection for each

species of fish (Table 1). Common Shiner spawn on gravelIy shoaIs (Scott and Crossman, 1973),

which are charactenstic of Lake Sasajewun. This behaviour probably increases contact with

infected oligochaetes in shallow areas, especially naidids that prefer pebbly sediments, and may

account for the differing prevaiences of infection in Common Shiner in Lake Sasajewun (43%),

Kathlyn Lake (3 1%), and Broadwing Lake (25%). The diet of Creek Chub consists largely of

plant matter (Scott and Crossman, 1973) that is most abundant in shailow areas in which

oligochaetes are nurnerous. Time spent feeding in these areas allows for a greater chance of

contacting actinosporean spores. This could account for the infection prevalence of 10% in

Creek Chub in Lake Sasajewun and Broadwing Lake, compared to the 1% prevalence of

infection in Kathlyn Lake, as the former 2 lakes have substantiaIly more aquatic vegetation in the

shallow areas. Golden Shiners prefer clear, weedy, still shallow areas (Scott and Crossman,

1973). This ideal oligochaete habitat, which is characteristic of Lake Sasrijewun, may account

for the relatively hi& prevalence of infection in Golden Shiners (IO%), as opposed to

prevalences of 1 and 3% in Broadwing Lake and Kathlyn Lake, respectively.

The host-parasite associations described here are also of interest as there is a large

discrepancy between the numbers of myxosporeans and actinosporeans described in these 3 lakes

in Algonquin Park. The number of actinosporean species in Lake Sasajewun is less than half of

the number of myxosporeans recorded in the same lake (Gowen, 1983; Li and Desser, 1985;

Lom et al., 1989; Xiao and Desser, 1997,1998q 1998b, 1998~; SaIim and Desser, 2000). This

discrepancy may be due to several factors, including a direct Iife cycle for some myxosporeans

(Diamant, 1997), polymorphism of myxosporeans in different fish hosts, and the enormous

sarnple size that would be required for a thorough survey of the actinosporeans, considering the

low prevalence of infkction in oligochaetes (Xiao and Desser, 1998~).

17

An understanding of the complete iife cycles of myxozoan species and habitat

preferences of particular oligochaete species would enhance efforts to conmi the spread of

myxozoan infections. This information is particularly important considering many oligochaete

species have lifespans of 2 to 4 years (Poddubnaya, 1980), and a recent siudy has demonstrated

that T. iitbifeir is capable of releasing spores throughout its entire lifespan (Gilbert and Granath,

2001). Myxozoan infections can easily be spread fiom 1 body of water to another through the

transport of myxosporean cysts by piscivorous birds, by Iive m i ~ o w s used as fishing bait, and

aIso by mud on fishing gear which may also contain oligochaete cocoons, thus understanding

which bodies of water may be at nsk due to ideal oligochaete habitats is beneficial to ongoing

efforts to rninimize the spread of myxozoans. The determination of environmental factors that

may discourage the abundance of oligochaetes that are potential hosts rnay also facilitate the

control of infections caused by myxozoans.

Table 1 : Prevalence of myxosporean infection for each species of cyprinid found in Kathlyn Lake, Broadwing Lake and Lake Sasajewun

Kathiyn Lake Common Shiner

Creek Chub Golden Shiner Redbelly Dace

Bmrdwing Lake Common Shiner

Creek Chub Golden Shiner

Redbelly Dace

Lake Saiajewun Common Shiner

Creek Chub Golden Shiner

Redbelly Dace

Prevalence of infection

Myxobolw pseudnkoi M. siddalli M. pendufa

- M. smithi M. sp.

M. bartai M. pseudokoi M. siddalli M. pendula M. algonquinensis M. xiaoi M. smithi

M. bartai M. pseudokoi M. siddulli M. pendulu M. afgonqitinensis M. xiaoi M. srnithi

Previlence oibfection witb eacb myxosporean species

Table 2: Plant families identified fiom Kathlyn Lake, Broadwing Lake and Lake Sasajewun, + indicates presence, - indicates absence.

Aster (Asteraceae) Bladderworî (Lentibuiuriaceae) Burreed (Sprganiaceae) Grass (Poaceae) Heath (Ericaceae) Lobelia (hbeliuceae) Pipewort (Eriocaulaceae) Plantain (Plm~aginaceae) Pondweed (Potamogetonaceae) Sedge (Slperaceae) Water lily (Nymphaeaceae) Water milfoil (Haloragaceae) Water plantain (Alismaiaceae) Water-shield (Cabombaceae)

Kathlyn b k e

Broadw ing Lake

Lake Sasajewun

Table 3: Prevalences of oligochaetes identified to the farnily and species levels fiom Kathlyn Lake, Broadwing Lake and Lake Sasajewun

Naididae Arcteonais lomondi Der0 digitata h r o nivea Nais cornmunis Nais variabilis Ripisres parasita SIavim appendicuiata SNaria lacustris Uncinais unctnata Yejdovs~ella comata

Tubiflcidre IIyodrifus tempiefoni Limnodrilus huflmeisteri Rhyacodrilw coccineus Tubijèx tubvex

Lumbriculidae Lumbriculus vuriegatus

Encbyt raeidae Cognetria glanulosa

Katblyn Lake

68 7 13 7 3 9 2 O O 15 1 O

30 9 O 6 13

3 3

O O

Broadwiog L i ke

58 2 22 11 O 14 O 3 O O 3

4 1 6 8 6 19

2 2

O O

Lake Sasajewun

76 2 16 8 2 8 35 O 1 1 2

22 13 2 1 6

2 2

0.2 0.2

Table 4: Percentages of 5 substrate types (mud, detriius, rock, pebbles and sand), comprising the habitats in Kalhlyn Lake, Broadwing Lake and Lake Sasajewun. (multiple types of sediment possible at the sites, thus sums are greater than O )

Mud Detritus Rock Pebbles Sand

Katbiyn Lake 8 73 15 O 42

Broidwing Lake 3 7 8 1 O 2 42

Lake Sisrijewun 28 74 1 4 47

22

Figure 1: Aerial view of the three lakes, Lake Sasajewun (LS), Broadwing Lake (BL), and Kathlyn Lake (KL). The Madawaska River (MR) and Lake Sasajewun dam (D) are also indicated. The sampling transect lines are shown for each Me. The shaded areas drawn for each lake were unsuitable for samphg.

24

Figure 2: CCA ordination biplot of the oligochaete famiIy data, displaying 98.4% of the variance with respect to the environmental variables. The eigenvalues of axis 1 (horizontaily) and axis 2 (vertically) are 0.074 and 0.05 1 respectiveIy. Physical parameters of Kathlyn Lake (KL), Broadwing Lake (BL) and Lake Sasajewun (LS) are indicatcd by arrows and are abbreviated as follows: depth= de, detritus= d, mud= m, pebbles= p, rock= r, sand= s, shore distance= sd. Plant families identified LTom the three lakes are also indicaied by anows and are abbreviated as follows: bladdenvort= bl, burreed= bu, gras= g, heath= h, lobelia= 1, f: of plant species= ps, pipewort= pi, plantain= pl, pondweed= po, sedge= se, water My= wl, water plantain= wp, water milfoil= wm, watershield= W. Oligochaete families are abbreviated as follows: Enchytraeidae= E, Lumbriculidae= L, Naididae= N, Tubificidae= T.

I I I I I I 1 1 I 1 1 I 1 I I I I 1 1 I I 1 1 I

1

Figure 3: CCA ordination biplot of the oligochaete species data, displaying 42.5% of the variance with respect to the environmeniai variables. The eigenvalues of axis 1 (horizontally) and axis 2 (vertically) are 0.093 and 0.077 respectively. Physical parameters of Kathlyn Lake (KT.,), Broadwing Lake (BL) and Lake Sasajewun (LS) are indicated by arrows and are abbreviated as folfows: 5= 0.5 m depth, l= lm depth, detritus= d, mud= m, pebbles= p, rock= r, sand= s, shore distance= sd. Plant families identified from the three iakes are also indicated by arrows and are abbreviated as follows: bladdenvort= bl, burreed= bu, p s = g, heath= h, Iobelia= I, # of plant species= ps, pipewort= pi, plantain= pl, pondweed= po, sedge= se, water My= wl, water plantain= wp, water milfoil= wm, watershield= W.

Oligochaete species are represented as follows: 1= Dero nivea, 2= Dero digitata, 3= Yejdovskyeila comata, 4= Siavina appendimlata, 5= Srylaricr lacmtris, 6= Nuis variabdis, 7= Nais comntunis, 8= Uncinais uncinata, 9= Arcteonais lumondi, 10= Ripistes parasita, 1 l= Ilyodrilus templetoni, 11= Tubfex rtrbife-r, 13= Limnodriltis hofnieisreri, 14= Rhyacodrilus coccineus, 15= Lumbricttltrs variegatus.

CHAPTER 3

A New Form of Raabeia (Myxozoa: Actinosporea)

from the Oligocbaete Uncinais uncinata

(Adapted h m Koprivnikar, J. & S. S. Desser. 2001. A New Fonn of Raabeia (Myxozoa: Actinosporea)

fiom the Oligochaete Uncinais ~rnciriofu. Folia Parasitologica 48: In Press)

ABSTUCT:

In a study of the otigochaete fauna and their actinosporean parasites in 3 lakes in

Algonquin Park, a nover form of raabiea was observed in a singte specimen of Uncinais

uncinata. This form differs tiom those previously described by having caudal processes that

gradually widen and teminate with a single prominent branch.

3 O

INTRODUCTION:

Actinosporean parasites of oligochaetes have been studied extensively since the 2-host

mylrozoan Iife cycle was elucidated by Wolf and Markiw (1984). The life cycles of

rnyxosporeans in their oligochaete and polychaete hosts as well as the ultrastructure of

actinosporeans have been described in sevenl studies (Ruidisch et al. 1991; El-Matbouli et al.

1992; El-Matbouli and Mandok 1995; Bartholomew et al. 1997; Lom et al. 1997; Xiao and

Desser 1998a, b; Molnar et al. 1999; Eszterbauer et al. 2000).

As part of an investigation of environmental factors influencing the prevalence and

distribution of myxozoans and their hosts (Koptivnikar et al. 2001), oligochaetes in 3 contiguous

lakes in Algonquin Park, Canada, were exarnined for actinosporean spores. A single specimen

of Utrcinais tlncinata released a novel form of raabeia, which is descnbed in this chapter.

TE TE RIALS AND METHODS:

A total of991 oligochaetes belonging to 17 species were collected From 228 sites in Lake

Sasajewun, Broadwing Lake and Kathlyn Lake fÏom May to August 2000. The worms were

kept individually in cell-well plates and checked periodically over 2 weeks for actinosporean

spores (Yokoyama et ai. 1991). Smears of waterborne spores were air dried, stained with Diff-

Quik@, observed by phase-contrast microscopy, and photographed using a Zeiss Universal 1

photomicroscope. Measurernents of spores were based on 5 specimens and are given as an

average followed by the range in micrometers.

RESULTS:

A single specimen of Uncinais uncinata released raabeia-type actinosporean spores.

Taxonomie summary

Spores: Composed of a styleless epispore with 3 equal-sized caudal processes, each of which

gradually widens and tenninates in one prominent branch (Fig. 1,2); polar capsules pyiform in

shape; extruded polar filaments obsemed (Fig. 2,4,5); cyhdrical spore body (length= 11.21 1 4

3 1

0.781, width= 4.923 fl.434) contains 17 e l ) g e m cells (Fig. 3,4,5); nuclei were not visible;

length of spore from anterior tip of spore body to tip of caudal process is 34.637 + 1.845.

Type host: Uncinais uncinata (Oligochaeta: Naididae)

Type locality: Lake Sasajewun, Algonquin Park, Canada (45" 35' N, 78" 30' W)

Prevalence: 1 of 991 worms (0.09%)

Remarks: Differs from previously described raabeia-type spores in that the caudal processes

gradually widen and terminate in a prominent branch.

Drscussro~:

Eleven forms of the conventional genus Rnabeia Janiszewska, 1955, have been described

(Janiszewska 1955,1957; Janiszewska and Krzton 1973; Yokoyama et al. 1995; Xiao and Desser

l998a; Molnar et al. 1 999). These are Raabeia golice~zsis Janiszewska, 1957, found in Tubfex

ritbifx; R. magna Janiszewska 1957, and R.jiirciligera Janiszewska and Krzton, 1973, both

recorded in Limnodrilus homeisteri; a raabeia- type actinospore in Brancliirira sowerbyi

(Yokoyma et al. 1995); 6 forms of raabeia found in either T. tubifer or L. Iiofmeisteri (Xiao and

Desser 1998a); and a raabeia-type spore in T. arbifer described by Molnar et al. (1999). Spores

ofnabeia with bifurcated tails have been described by Molnar et al. (1999) and by Janiszewska

and Krzton (1973), but these forms have caudal processes that branch more than once,

sometimes in an irreguiar pattern. The processes of raabeia- type spores previously described

also differ in that they gradually taper before branching, whereas the processes of the form

described here widen before branching.

The overall prevaience of infection in the oligochaetes fiom the 3 lakes was 0.09%,

corresponding with prevalences observed in previous studies in these lakes (Xiao and Desser

1998a). The naîural prevalence, however, is likely higher as not ail woms may have released

spores during the observation period and recent evidence indicates that large numbers of spores

3 2 may be contained in the fecal packets of oiigochaetes (Gilbert and Granath 2001), thereby

decreasing the chance that waterbome spores would be obsewed.

While the alternate stage for the form of raabeia in this study is u h o w n , it is rnost likely

a species of Myxobolus, as this association has been described by Lom et al. (1997), and

demonstrated by Molnar et al. (1 999). Aithough more than 40 species of My.robolus have been

described fiom cyprinids in the 3 lakes in Algonquin Park (Gowen 1983; Li and Desser 1985;

Xiao and Desser 1997; Salim and Desser 2000), only 7 forms of raabeia have been observed so

far (Xiao and Desser I998a), thus providing considerable scope for further research.

Fig. 1: Line drawing of the raabeia-type spore.

35

Fig. 2: Spore with bifurcated caudal processes and 3 discharged polar filaments (arrows). Fig.

3: Spore body with intact polar capsules and 19 germ cells in the sporoplasrnic mass. Fig. 4:

Spore body with 1 discharged polar filament (arrowhead) and 16 germ cells. Fig. 5: Spore body

with 2 discharged polar filaments (arrowheads) and 17 germ cells. Scale bars: Figs. 3-5 = IO

CHAPTER 4

General Discussion

GENERAL DISCUSSION

Prior to the study detailed in this thesis, investigations of myxozoan-oligochaete

interactions in natural settings were limited to a few field observations (Burtle et al., 1991, Styer

et al., 1991; Yokoyama et al., 1991, 1993; Rognlie and Knapp, 1998; El-Mansy et al., 1998~) and

one major comprehensive study (Xiao and Desser, 1998~). The study of oligochaete habitats

waç largely limited to the effect of various physicochcmical factors (Smith, 1985; Martinez-

Ansemil and Collado, 1996; see review by Brinkhurst, 1999).

Substantial data in this study support other observations that substrate type is associated

with oligochaete distribution. It waç observed that tubificids prefer to inhabit areas with muddy

sediment, and that naidids ofien dwell in pebbly or stony areas, corresponding with previous

findings (Brinkhurst and Jarnieson, 1971; Xiao and Desser, 1998~). The data aIso support the

idea put forth by Brinkhurst (1999) that food plays an important role in oligochaete distribution,

as associations were found among various oligochaete and aquatic plant species. The following

oligochaete-plant associations were discovered: Tubifet tub* and Limnodrilrts hofmeisreri

wih grass, Vejdovskyella comata with pipewort, Uncinais tincinara with lobelia, and I(vodri1rcs

templetorii with milfoil.

The new fom of raabeia described adds to the number of types of actinosporeans

described from Lake Sasajewun, however, there is still a large discrepancy between the number

of actinosporeans and myxosporeans described from this lake. Over 50 myxosporeans having

been described but only half as many actinosporeans (Gowen, 1983; Li and Desser, 1985; Xiao

and Desser, 1997; Xiao and Desser, 1998a, b; Salim and Desser, 2000). Raabeia foms are

associated with species of Mpobolus (Lom et al., 1997), over 40 of which have been described

in Lake Sasajewun, giving considerable material for possible life cycle research.

The oligochaete and myxosporean prevaiences detailed in this study may aiso provide a

starting point for attenpts to elucidate the life cycles of the myxozoans in Lake Sasajeunm.

3 9 Certain oligochaete and myxosporean prevalence patterns lend themselves to the suggestion that

the following oligochaete species may be the alternate hosts for certain species of My.?robolrrs: I.

templetoni and M. psardokoi, Nais commirnis and M. smithi, Li. uncinata and M. sp., 1;.

hofi~eisteri and one of M. barrai, hi. xaoi or M. algonqrrinensis.

The severe ecological and economic impacts of myxozoan infections wiU continue to

stimulate research on the interactions among myxozoans and their hosts. The determination of

factors controlling the distribution and abundances of oligochaetes is an area of focus for

researchers concerned with the effects of myxozoan infections, particularly Whirling Disease

(Kent et al., 2001), but research will no doubt further intensify as aquaculture becomes more

prevalent. in particular, there is a paucity of information about marine myxozoans and iheir

hosts that must be addressed (Kent et al., 2001). While numerous insights into the phylum

Myxozoa have been made since Wolf and Markiw's landrnark discovery in 1984, further

investigations in tropical and marine environments will surely enhance our knowIedge of this

enigmatic group of parasites.

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