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Canada •

PLAGIORCHIS ELEGANS FROM CERCARIAE TO INFECTIVE

METACERCARIAE: FACTORS AFFECTING TRANSMISSION,

REQUIREMENTS FOR DEVELOPMENT, AND BEHAVIOURAL

RESPONSES OF INTERMEDIATE HOSTS TO INFECTION.

by

CARL A. LOWENBERGER

• Institute of Parasitology l\1:cGill University, Montreal, Québec

August 1993

A Thesis submitted to the Faculty of Graduate Studies and Research in partial fulfilment of the requirements for the degree of Doctor of Philosophy

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ISBN 0-315-91709-1

Canada Plagiorchis elegans: transmission, development, and host behaviour. Il • AB8TBACT

Plagiorchis elegans is a typical digenean parasite that cycles through aquatic molluscs and insects as intermediate hosts. During emergence of P. elegans cercariae, infected snails moved to the top of the water column where they remained immobile for 2-3h. Consequently, the cercariae fOlmed a dense cloud which disp'rsed slowly. The infectivity of cercariae was <20% upon emergence and peaked at 76% 4-6h later. This delay in reaching maximum infectivity may be an adaptation to prevent superinfection and the associated mortality of insect hosts. Cercariae transformed into metacercari.ae aftE'r penetrating Aedes aegypti larvae, the experimental insect hosto Overall development oîmetacercariae, and excystment of • infective metacercariae in vitro, was temperature dependent. However, there was an initial 8-hour period of obligatory host-parasite ~ontact that was temperature independent. This may represent a period of major nutrient acquisition since young metacercariae were more active metabolically than older metacercariae, as measured by the in vitro uptake of 3H-glucosamine and 3H-Ieucine. Mosquitoes may have mechanisms to .r<::duce lasses of larvae to parasites. Oviposition by adult A. aegypti wab reduced in waters that had previously contained P. elegans-infected larvae. We propose that this selective oviposition was due to the production of an oviposition deterrent compound produced by parasitized larvae that serves

to reduce oviposition in sites detrimental to larval development. • III • ABRéGé

Le digénien Plagiorchis elegans est un parasite type qui se développe dans les gastéropodes et les insectes, ses hôtes intermédiaires. Lorsque les cercaireR de Plagiorchis elegans émergent des escargots infectés, ces derniers se déplacent vers la surface de l'eau, où ils restent immobiles de deux à trois heu.res. Ceci permet aux cercaires fonr.ant un nuage dense de se disperser lentement. Le pouvoir infectieux des cercaires à l'émergence est de moins d~ 20% et atteindra, quatre à six heures plus tard, un maximum de 76%. Cette capacité infectieuse maximale tardive est probablement une adaptation prévenant une surinfection associée à la mortalité des insectes hôtes. Par la suite, les cercaires pénètrent les larves d'Aedes aegypti, l'insecte expérimental utilisé, et se transforment en métacercaires. In vitro, le développement des métacercaires et leur capacité • de se libérer de leur kyste est relié à la température. Par contre, la période initiale de huit heures où le parasite et l'hôte sont en contact ne l'est pas. Cette période semble correspondre à une activité d'absorption en éléments nutritifs. Les jeunes métacercaires ont une activité métabolique plus élevée que les métacercaires plus vieux. Cette activité a été mesurée par l'absorption de 3H-glucosamine et de 3H-Ieucine in vitro. Les moustiques ont des mécanismes pour réduire la mortalité chez leurs larves causée par le parasite. L'oviposition des adultes Aedes aegypti est moindre dans les eaux ayant contenues préalablement des larves infectées avec Plagiorchis elegans. Cette oviposition sélective semble être le résultat de l'émission d'une substance par les larves parasitées empêchant celle-ci. De cette façon, l'oviposition est réduite aux endroits défavorables au développement des • larves. IV • SUGGESTED SHORT TITLE

Plagiorchis elegans: transmission, development, and host behaviour.

•

• " ACKNOWLEDGEMENTS l have been helpecl, prodded, pushed and restrained (ûut never bribed) by many people over the course of this study. l thank my supervisory commlttee of Drs. Manfred Rau, Marilyn Scott, and Gary Dunphy for their il'lt'lrest, suggestions, guidance, and help dù"Ïng my tenure at the Institute of Pnrasitology. l thank Dr. Kris Chadee for assistance in the design and implementation of the study using radiolabelled materials. l also thank my former supervisor Dr. John Webster for his support and comments on several aspects of the studies presented here.

My laboratory co:npanions have provided intense debate and discussion over several aspects of the rese?rch reported here. These include Charles Kimoro, Philippe Jacobs, Manon Bombardi<'r, SylvainPoirier, and Julie • Riddock. Others from the Institute who have helped me through their friendship and help over the last 4 years include (in alphabetical order) Jenny Anderson, Gord Bingham, Pierre Bourassa, Darren Campbell, Mark Fielding, Janet Forrester, Peter Gatongi, Kathy Keller, Marc LaBerge,

Silvie Labrecque, Mary LaDuke, Carlos Lanusse,. Sharon McGee, Shirley. Mongeau, Roy Nare, Christine Noronha, Siva Ranjan, Sharon Rutherford, Rosanne Seguin, Stephanie Tremblay, Christiane Trudeau, Sil-King Tse, Bernie Wright, to name but a few. In particular l am indebted to Kathy Keller and Sylvain Poirier for close friendship, inspiration, and help whenever l needed it; and sometimes when l didn't. l thank Dr. Jim Smith for help with the computers. l thank the students of the Illstitute for • allowing me the invaluable experience of representing them during staff VI • meetings and as their represent:J.tive on the Curricuhm and Computer c.omnrittees.

My parents have a.lW'iYs supported Ilie in my entieavours and 1 am indebted to them for constant enc1JUragement and love. Megan has been my mainstay during our sojourn in Montreal. ~hroughout the stormy periods she provided the voice of reason and stability. Without her support, encouragement, and recriminations, f :s thesis would never have been completed. Dr. 'Tristan ha3 become the light of our lives. His arrivaI and subsequem inclusion into my personal supervisory committee, and his constant commentary on my research has allowed me to put aspects of this reseal"ch, and life in general, in its proper perspective.

• Major funding for this research was obtained through grants from the Natural Sciences and Engineering Research Council (NSERC) and the Fonds pour 18. formation de chercheurs et l'aide à la recherche (FCAR) of Québec to M.E.R. Personal support was provided by the Walter M. Stewart Foundation, the Lynden Laird Lyster award, and the M. Gowans support fund.

. .. • VII • TABLE OF CONTENTS TITLE PAGE . ABSTRACT ...... ii ABRéGé...... iii SUGGESTED SHORT TITLE iv ACKNOWLEDGEMENTS ...... v TABLE OF CONTENTS vii LIST OF FIGURES ...... xi LIST OF TABLES ...... xv LIST OF ABBREVIATIONS xvi

CHAPI'ER 1 INTRODUCTION AND LITERATURE REVIEW ...... 1 INTRODUC'rION ...... 1 STATEMENT OF AUTHORSHIP ...... 7 LITERATURE REVIEW ...... 8 Plagiorchis elegans ...... 8 • Aedes aegypti 14 Stagnicola elodes 19 LITERATURE CITED 22

CHAPI'ER 2 PLAGIORCHIS ELEGANS: EMERGENCE, LONGEVITY, AND INFECTIVITY OF CERCARIAE, AND THE EFFECTS OF CERCARIAL EMERGENCE ON THE BEHAVIOUR OF THE MOLLUSCAN FIRST INTERMEDIATE HOST...... 36

ABSTRACT 37 INTRODUCTION ...... 38 Plagiorchis elegans ...... 41 MATERIALS AND METHODS ...... 42 BEHAVIOURAL STUDY 42 CERCARIAL EMERGENCE ...... 43 VERTICAL DISTRIBUTION OF CERCARIAE ...... 43 • CERCARIAL LONGEVITY ...... 44 viii • CERCARIAL INFECTIVITY " 44 ANALYSIS " 45 RESULTS 46 BEHAVIOUR: ACTIVITY '" ...... 46 BEHAVIOUR: VERTICAL DISTRIBUTION...... 47 CERCARIAL EMERGENCE ...... 48 VERTICAL DISTRIBUTION OF CERCARIAE ...... 49 CERCARIAL LONGEVITY ...... 49 CERCARIAL INFECTIVITY ...... 49 DISCUSSION 50 SUMMARY 56 ACKNOWLEDGEMENTS 57 FIGURES...... 58 TABLES...... 68 LITERATURE CITED 70

CONNECTING STATEMENT 1 76

CHAPTER3 PLAGIORCHIS ELEGANS: REQUmEMENTS FOR METACERCARIAL DEVELOPMENT TO INFECTIVITY, AND CONDITIONS REQUmED FOR EXCYSTMENT. 77

ABSTRACT ...... 78 INTRODUCTION ...... 79 Plagiorchis elegans ...... 79 MATERIALS AND METHODS ...... 80 RESULTS 83 DISCUSSION 84 SUMMARY 89 ACKNOWLEDGEMENTS 89 FIGURES...... 90 TABLES 96 • LITERATURE CITED 97 • ix CONNECTING STATEMENT 2 100

CHAPI'ER4 IN VITRO UPTAKE AND INCORPORATION OF sH· GLUCOSAMINE AND sH·LEUCINE INTO METACERCARIAL PROTEINS BY PLAGIORCHIS ELEGANS . 101

ABSTRACT . 102 INTRODUCTION . 103 MATERIALS AND METHODS . 105 sH·GLUCOSAMINE AND sH·LEUCINE UPTAKE BY METACERCARlAE . 105 sH· DISTRIBUTION IN METACERCARlAE . 106 SDS·PAGE AND FLUOROGRAPHY . 107 COLUMN CHROMATOGRAPHY . 108 RESULTS . 109 • UPTAKE OF sH·GLUCOSAMINE AND sH·LEUCINE BY P. ELEGANS METACERCARIAE . 109 PARTITIONING OF THE sH·LABELS BY METACERCARIAE . 110 SUBCELLULAR DISTRIBUTION OF sH·GLUCOSAMINE INTO GLYCOPROTEINS . 110 DISCUSSION . III SUMMARY . 116 ACKNOWLEDGEMENTS . 117 FIGURES . 118 LITERATURE CITED . 132

CONNECTING STATEMENT 3 135 x CHAPI'ER5 EVIDENCE FOR THE PRODUCTION OF AN OVIPOSITION • DETERRENT OR REPELLENT COMPOUND BY AEDES AEGYPTI LARVAE PARASITIZED BY THE DIGENEAN PLAGIORCHIS ELEGANS 136

ABSTRACT 137 INTRODUCTION...... 139 Plagiorchis elegans ...... 142 MATERIALS AND METHODS ...... 143 EXPERIMENT 1 144 EXPERIMENT 2 144 EXPERIMENT 3 145 EXPERIMENT 4: LARVAL HOLDING WATER EXPERIMENTS ...... 145 EXPERIMENT 5: MOSQUITO BEHAVIOUR ...... 148 ANALYSIS ...... 148 RESULTS 149 • EXPERIMENTS 1-3 ...... 149 EXPERIMENT 4: LARVAL HOLDING WATER EXPERIMENTS ...... 150 EXPERIMENT 5: MOSQUITO BEHAVIOUR ...... 152 DISCUSSION 153 SUMMARY 162 ACKNOWLEDGEMENTS 162 FIGURES...... 164 TABLES...... 180 LITERATURE CITED 183

CHAPI'ER 6 GENERAL DISCUSSION AND CONTRffiUTIONS TO ORIGINAL KNOWLEDGE 193 GENERAL DISCUSSION ...... 193 CONTRIBUTIONS TO ORIGINAL KNOWLEDGE ...... 200 LITERATURE CITED 203 Xl • LIST OF FIGURES CHAPTER2

Figure 2.1. Comparison of activity patterns of uninfected and Plagiorchis elegans-infected snails...... 58

Figure 2.2. Comparison of time spent at the surface of the water by uninfected and Plagiorchis elegans-infected snails prior to (pre) and following (post) the reduction in light intensity. 60

Figure 2.3. Emergence of Plagiorchis elegans cercariae in the first three hours following the reduction in light intensity. 62

Figure 2.4. Location ofPlagiorchis elegans cercariae in a 20cm water • column at various times following cercarial emergence. 64

Figure 2.5. Mean infectivity of Plagiorchis elegans cercariae of different ages post emergence. 66

CHAPTER3

Figure 3.1. In vitro excystment of Plagiorchis elegans metacercariae removed from the insect host at various times post infection. . .. 90

Figure 3.2. Excystment of Plagiorchis elegans metacercariae removed from the mosquito host at various times post infection and • maintained in Phosphate-buffered saline (PBS) until they • = reached the age required for 80% excystment as detennined from Fig 1...... 92

Figure 3.3. Excystment of infective Plagiorchis elegans metacercariae following 2h incubation in excystment medium at various temperatures. 94

CHAPTER4

Figure 4.1. 24 hOUT in vitro uptake of 3H-glucosamine and :lH-leucine (+ 1 standard error) by Plagiorchis elegans metacercariae of different ages post infection. 118

Figure 4.2. Proportional uptake of 3H-glucosamine and :lH-leucine by young (::;;8h) and old (>8h) Plagiorchis elegans metacercariae • following 24h incubation. . . 120

Figure 4.3. Cumulative uptake of 3H-glucosamine by Plagiorchis elegans metacercariae of different ages post infection incubated for different periods. 122

Figure 4.4. Cumulative uptake of 3H-leucine by Plagiorchis elegans metacercariae of different ages post infection incubated for different periods. 124

Figure 4.5. DifferentiaI incorporation of3H-glucosamine and "H­ leucine into Plagiorchis elegans juvenile worms and cyst walls. 126 XIll • Figure 4.6. Fluorograph ofSnS-PAGE (4% stacking gel; 12% running gel) of 2h- and 5d-old Plagiorchis elegans metacercarial glycoproteins labelled with 3H-glucosamine for 24h in vitro. 128

Figure 4.7. Fractionation of glycoproteins from 2h- and 5d old Plagiorchis elegans metacercariae incubated with 3H_ glucosamine for 24h in vitro using Sephacryl S-200 column chromatography. 130

CHAPTER5

Figure 5.1. Comparison of oviposition by Aedes aegypti in dishes containing 400 ml of aerated tap water to which snails, • cercariae, or larvae were introduced. 164 Figure 5.2. Comparison of oviposition by Aedes aegypti on larval holding waters (LHW) prepared with 0, 100, 300, or 600 unparasitized A. aegypti larvae/l...... 166

Figure 5.3. Oviposition by Aedes aegypti on larval holding waters (LHW) prepared with 0, 100, 300, or 600 unparasitized Aedes aegypti larvae/l. 168

Figure 5.4. Comparison of oviposition by Aedes aegypti on larval holding waters (LHW) prepared with 100 (100U) or 600 (600U) unparasitized larvae/l and 100 lightly (100L) or 100 heavily (lOOH) Plagiorchis elegans parasitized larvae. 170 • ~v Figure 5.5. Comparison of oviposition by Aedes aegypti on larval holding waters (LHW) prepared with 100 unparasitized larvaell (lOOU) and waters prepared by either concurrent (100M) or sequential (lOOSL and lOOHL) incubations of parasitized and unparasitized larvae...... 172

Figure 5.6. Comparison of oviposition by Aedes aegypti on larval holding waters (LHW) prepared with 100 unparasitized (100U), 100 lightly parasitized (100L) or 100 heavily parasitized (l00H) A. aegypti larvae, or without larvae (LHW 0). 174

Figure 5.7. Comparison of oviposition by Aedes aegypti on larval holding waters (LHW) prepared with 100 unparasitized (lOOU) or 100 heavily (lOOH) parasitized Aedes aegypti iarvae. 176

• Figure 5.8. Comparison of oviposition by Aedes aegypti on larval holding waters (LHW) prepared without larvae (LHW 0), with 100 heavily parasitized A. aegypti larvae (l00H), LHW 0 + brain heart infusion broth (BHIE), 100H + BHIE, suspensions of Flavobacterium sp...... 178 xv • LIST OF TABLES Tahle 2.1. Longevity of Plagiorchis elegans cercariae. 68

Table 2.2: Prevalence, Mean Abundance, and Location of Metacercariae in fourth instar Aedes aegypti larvae exposed for 15 minutes to Plagiorchis elegans cercariae of different ages. . .. 69

Table 3.1. Excystment of infective metacercariae of Plagiorchis elegans in various media and temperatures...... 96

Table 5.1. Summary of pupal and adult production, prevalence and intensity of infection, and estimated larval densities from oviposition sites containing Aedes aegypti larvae in the presence and absence of • Plagiorchis elegans cercariae...... 180 Table 5.2. Microbial analysis of Larval Holding Waters (LHW) prepared with 100 unparasitized (100U), 100 heavily parasitized (IOOH), or no (LHW 0) fourth instar Aedes aegypti larvae...... 181

Table 5.3. Activities of adult Aedes aegypti mosquitoes on oviposition sites containing larval holding waters (LHW) prepared with 100 unparasitized larvae (100U) or 100 larvae heavily parasitized by Plagiorchis elegans (IOOH). 182 • XVI • LIST OF ABBREVIATIONS

MBEM Modified Bock excystment medium - used for in vitro excystment. of Plagiorchis elegans metacercariae.

PBS Phosphate buffered saline

SnS-PAGE sodium dodecyl sulphate poly-acrylamide gel electrophoresis

RLI Reduction in Light Intensity; reduced light induces emergen;:e of cercariae from the snail hosto

LHW Larval holding waters

.LHWPP Larval holding waters prepared with parasitized larvae

LHWUP Larval holding waters prepared with unparasitized larvae

• LHWO Larval holding waters prepared with zere larvae

lOOU LHW prepared with 100 unparasitized larvae

GO OU LHW prepared with GaO unparasitized larvae

100L LHW prepared with 100 lightly parasitized larvae

100R LHW prepared with 100 heavily parasitized larvae

100M LHW prepared by incubating 100 unparasitized and 100 heavily parasitized larvae concurrently for 72 hours.

100SL LHW prepared by incubating 100 light~y parasitized larvae for 72 hours in lOOU.

100HL LH"# prepared by incubating 100 heavily parasitized larvae for 72 hours in lOOU. • 1

CHAPTER1

INTRODUCTION AND LITERATURE REVIEW

INTRODUCTION

Parasitism may be defined as a relationship between two organisms in

which one species, the parasite, gains its livelihooù from, and has a • physiologicai dependency on the host for the essentials oflife (Œsen 1974, Marquardt and Demaree 1985). A major problem facing parasites with

different hosts at different stages of their lives is the dispersal of propagules

to new hosts (Smith Trail 1980). Adults of free living parasite groups such

as parasitoids have the ability to actively seek'out and discriminate between

potential hosts (McBrien and Mackauer 1991) whereas parasites with multi­

host life cycles but no free living stages have adopted other means to ensure

transmission. Sorne of these parasites act through their intimate

physiological contact with their intermediate hosts, eliciting host

behavioural changes which serve to increase the rate of parasite 2 • transmission (Dobson 1988). [For a review of such interactions see Rolmes and Bethel (1972) and Rurd (1990)].

The Digenea, a member of which is the focus of this thesis, have indirect

life cycles characterized by the inclusion of molluscs as first intermediate

hosts (Olsen 1974, Shoop 1988). Life cycles with increasing numbers of

intermediate hosts are generally considered to be evolutionary

advancements of the species and serve to facilitate transmission (Shoop

1988).

In digenean transmission timing is of the essence; infective stages and • potential hests must overlap both temporally and spatially. From the parasite's perspective this is provided by the sustained dissemination of

eggs and cercariae through space and time by the definitive and molluscan

intermediate hosts respectively (Shoop 1988). A comprehensive knowledge of

the ecology of both host and parasite is required to understand transmission

dynamics. Digenean transmission occurs through ingestion of, or

penetration by, infective stages. Thus in essence, the transmission ecology of

the Digenea is the study of the conditions under which the two organisms,

parasite and host, are found tLgether temporally :md spatially, and the • factors that contribute to successful transmission. 3 • Anautogenous female mosquitoes interrnittently parasitize their hosts during the process of blood feeding. They are the most important, and the

most prominent, of the blood sucking arthropods both as a nuisance and as

vectors of disease (Harwood and James 1979). Mosquitoes can disrupt

successful nesting of birds, influence the migratory activity of reindeer

(Terent'ev 1972), and can reduce livestock production (Steelman 1976).

These losses must be added to the widespread suffering and death due LO

the severity of the diseases spread by mosquitces (Harwood and James

1979). For a summary of the effects of mosquito-borne diseases on the

affairs of humans see Gillett (1972) and Mattingly (1969).

• Mosquitoes and man have had a long and antagonistic relationship. Eradication programmes have attempted to kill adults and larvae with

chemicals, by destroying larval habitat, or by employing naturally existing

predators, pathogens, and parasites. The gains made in mosquito control

through the use of insecticides have been reversed by the development of

insecticide resistance by the mosquitoes (McEwan and Stephensen 1979).

New strategies based on an understanding of the population dynamies of

mosquitoes have shifted the emphasis from eradication to population

management and have integrated conventional insecticides into programmes

using biologieal insecticides such as Bacillus thuringiensis israeliensis (BtÏ), • insect growth regulators, and biological control agents. 4 • Parasites, predators and pathogens already kill 90-95% of the pre-ims.go stages of mosquitoes (Hagstrumm 1971, Service 1983). M

progra=es must, therefore, l,e oriented to the surviving 5-10%. Classical

biological conkol relies on agents which establish, cycle through, and

maintain the target species pt acceptable levels. Fish (Wickramasinghe and

Costa 1986), nematodes (Petersen 1973, 1984), and fungi (Federici et al.

1985) have all been used successfully under very ~pecific conditions

(Chapman et al. 1972). However, a central problem in conventional

biological control is one of recycling: having enough of the controlling agent

present at all times. Since control agents which cycle through the target

population are often unable to react quickly to outbreaks, obligate parasites • may produce occasional epizootics but generally are unable to control mosquito populations over time (Hominick and Tingley 1984).

Entomophilic digeneans represent a somewhat different class of potential

biological control agents. While they have complex life cycles and long

generation times compared with mosquitoes, the completion of their life

cycle is not dependent on mosquitoes. Due to the sustained asexual

production of cercariae in the molluscan intermediate host, large numbers of

cercariae are produced daily, whether or not the target species is present.

Laboratory studies have shown that cercariae from the genus Plagiorchis

kill mosquito larvae (Dempster and Rau 1991), inhibit pupation (Dempster 5 • et al. 1986), and reduce the fecundity and longevity of surviving adults (Kimoro 1990).

Although these studies have shown that cercariae can kill mosquito larvae,

we know little of the interactions between the parasite and its intermediate

hosts and the developmental requirements of the parasite within the hosts.

This thesis examines several aspects of these interactions between the

mollusc Stagnicola elodes, the mosquito Aedes aegypti, and the cercariae

and metacercariae of the digenean Plagiorchis elegans, to better understand

the ecology of their interactions.

• Chapter 1, therefore introduces the rationale for the studies carried out, as weIl as the participants: the parasite, the snail, and the mosquito.

Chapter 2 examines the behaviour of snails during the period of cercarial

emergence, the pattern of cercarial emergence, the distribution of cercariae

in the water column, their longevity and changes in infectivity over time.

Chapter 3 evaluates the influence of environmental temperature on the

development of the metacercariae to infectivity within the insect host. The

excystment ofinfective metacercariae is examined in relation to different

thermal regimes and chemical stimuli. 6 • Chapter 4 assesses the metabolic activities of metacercariae as measured by the in vitro uptake of radio-Iabelled nutrients to qualify the observations

from chapter 3. Metacercariae are assessed in terms of total nutrient uptake

and subsequent distribution of the label in the juvenile worm or the cyst

wall. Column chromatography and gel electrophoresis are used to compare

and contrast the incorporation of the radiolabelled nutrients into proteins by

metacercariae ofdifferent ages.

Chapter 5 evaluates the oviposition response of gravid adult mosquitoes to

determine ifoviposition site selection is affected 1) by the presence of the

snai! intermediate hosts, either shedding cercariae or uninfected controls, 2) • by the presence of cercariae in the absence of snails, or 3) by the presence of lightly infected or moribund larvae. Oviposition site selection is also

evaluated in waters which had previously held parasitized or unparasitized

larvae to determine whether there is a mechanism by which adults can

distinguish between suitable or unsuitable habitats for larvaJ growth and

development in the absence of larvae.

Chapter 6 is a general discussion and summary of the results and

conclusions of the earlier chapters. The elements in the thesis that are

considered to be "...contributions to original knowledge..." are indicated in

this chapter. • 7

STATEMENT OF AUfHORSHIP

1 have elicited comments and have held discussions on the experimental

design with a wide array of people. However, the overall experimental

design, data collection and statistical analyses as described in this thesis

were carried out by the author. Ms. Kathy Keller of Dr. Chadee's laboratory

assisted with the experiments using gel electrophoresis and column

chromatography. Ms.•Joan Kearvell cultivated, identified, and enumerated

the bacteria from LHW and prepared the suspensions of Flavobacterium sp.

in Chapter 5. These details on authorship are presented to conform with the

regulations of the Faculty of Graduate Studies and Research, McGill

University. McGill university also states that "In the case of manuscripts

co-authored by the candidate and others, the candidate is required to make

an explicit statement in the thesis of who contributed to such work, imd to

what extent; supervisors must attest to the accuracy of such claims at the

PhD oral defence. Since the task of the examiners is made more difficult in

these cases, it is in the candidates best interests to make perfectly clear the

responsibilities of the different authors of co-authored papers." • 8 • LITERATURE REVIEW

Plagiorchis elegans (Rudolphi)

Plagiorchis elegans is a cosmopolitan and ubiquitous intestinal parasite of

birds and mammals. Monoecious adults are found in the upper regions of

the small intestine where they adhere by means of the ventral sucker and

browse on the mucosal tissue and food material undigested by the hosto The

size of adults (2.2-3.7 mm)( Styczynska-Jurewicz 1962, Genov and

8amnaliev 1984) is dependent on their age (Styczynska-Jurewicz 1962) and

on the definitive host from which they are taken (Blankespoor 1974,

MacKenzie and MacKenzie 1980). Similarly, longevity of adult worms varies

with the definitive host, ranging from 10-60 days in birds to about a month

in man (McMullen 1937). For the morphological descriptions of various

species of the genus Plagiorchis see Genov and Samnaliev (1984).

Eggs (0.034-0.40 X 0.017-0.024 mm) are produced as early as 6 days post

infection (Genov and Samnaliev 1984) and are released into the external

environment with the faeces of the hosto Contrary to adult features, egg size

is relatively constant; Blankespoor (1974) found that egg size in Plagiorchis

noblei was one feature that varied little between different definitive hosts • and age of adults. Following ingestion by a lymD'l.eid snail the miracidium 9 • hatches from the egg, penetrates the gut liIÙng, and forms an elongated mother sporocyst on the outer surface of the intestine. Asexually produced

daughter sporocysts (1.07-2.14 X 0.28-0.41 mm)(Styczynska-Jurewicz 1962)

migrate and establish in large numbers in the hepatopancreas.

Polyembryony gives rise to xiphidiocercariae which first emerge from the

snail approximately 40 days following egg ingestion (Blankespoor 1974,

Œsen 1974). The cercariae (0.214 X 0.28 mm) (Styczynska-Jurewicz 1962,

Genov and Samnaliev 1984) have a strong stylet (0.028-0.03 X 0.004-0.006

mm), well developed suckers, a large pharynx, intestinal caeca which reach

the end of the body, and a Y shaped excretory system

the attention of many researchers, yet our understanding of the

phenomenon is severely limited (Erasmus 1972). Cercarial emergence by P.

elegans is largely nocturnal (Styczynska-Jurewicz 1962). Similarly

Blankespoor (1977) showed a marked nocturnal and seasonal periodicity of

emergence in P. noblei, and Webber et al. (1986) found that >91% ofP.

noblei cercariae emerged from Stagnicola elodes within the first 5 hours

following a reduction in light intensity. 10 • Many cercariae show photocyc1e-dependent emergence patterns CRees 1947, Macy 1960, Asch 1972, Cable 1972, Wagenbach and Alldridge 1974).

Different strains of the same species can vary in their time of peak

emergence (Théron 1984) and hybrids of two species can show intermediate

peaks of emergence (Théron 1989). Other factors such as temperature, host­

parasite interactions and the time course of the infection can a11 affect

cercarial emergence patterns (Pfluger 1980, Rojo-V:izquez and Simon-Martin

1985).

The question which arises is not only how the photocyc1e affects cercarial

emergence but must also address the adaptive significance of emergence at • a specific time. Shostak and Esch (1990) proposed 3 theories to expiain the periodicity of cercarial emergence: i) periodic emergence may increase

transmission efficiency if cercariae exit the snail to coincide with the

presence of the next hosto This concept is adaptive when the next host has a

daily activity cycle and the cercariae are active but short lived (Cable 1972,

Lewis et al. 1989); ii) periodic emergence a110ws cercariae to emerge at a

particu1ar location. This concept is adaptive in species that activeiy alter the

behaviour of their mo11uscan hosts such as Gynaecotyla adunca (Curtis

1987), species that depend exc1usively on the mo11usc for dispersal of

cercariae, or species with weak or sessile cercariae; iii) periodic emergence

can reduce cercarial mortality by predation or unfavourable conditions. 11 • Species that have frequent contact with or are unable ta escape from predatars may use this strategy ta avoid the peak feeding time of predatars

or by overwhelming the predators' ability to handle prey items.

In general cercariae serve to disperse the parasite over time and space

(Erasmus 1972, Shoop 1988) but Plagiorchis cercariae are ineffectual

swimmers (Kavelaars and Boums 1968, personal observation). The cercariae

maintain their position in the water column by rapid tail movements but

show little directed lateral movement and settle in the water column. The

longevity of cercariae is inversely related to temperature (Blankespoor 1977,

Styczynska-Jurewicz 1962). However, cercariallongevity exceeds the period • of iniectivity (Blankespoor 1977).

Plagiorchis elegans penetrates a number of aquatic insect orders as weIl as

other aquatic invertebrates (Williams 1963, Daniel and Ulmer 1964,

Blankespoor 1974). Once a suitable host is found the cercariae attach by

means of suckers and actively penetrate the host using the stylet and

histolytic enzymes (Bock 1986). Taft (1990) followed the penetration

sequence ofPlagiorchis sp. cercariae using cinephotomicrography and

reported leech like crawling over the host's cuticle, random probing with the

stylet, loss of the tail, penetration, and subsequent encystment. Encystment • begins with the cercliria rotating around itselfand the secretion of an 12 • elastic cyst wall (Taft 1990, personal observation). The rate of rotation decreases over the first 10 minutes during which time two cyst walls of

parasite origin are deposited; the first cyst wall comprises mucosubstances

and a low proportion of proteins, whereas the inner cyst wall comprises a

layer of carbohydrates and proteins (Bock 1988). A third layer of host origin

consisting of disintegrating haemocytes and non·cellular haemolymph

components is laid down later (Taft 1990).

Metacercariae (0.09-0.13 X 0.08-0.11 mm)(Genov and Samnaliev 1984,

Styczynska-Jurewicz 1962) are immobile within the insect and show little

growth over time. The Y shaped excretory vesicle progressively fills with • dark globular excretory bodies, increasing in both size and conspicuousness (8tyczynska-Jurewicz 1962, Williams 1963, personal observation).

Metacercariae require 2-7 days to become infective to the definitive host

(Williams 1963, Blankespoor 1974, Genov and Samnaliev 1984) and the life

cycle is completed when the infected intermediate host is ingested by a

suitable definitive hosto The metacercariae of Plagiorchis excyst intrinsically

on receiving the appropriate environmental stimuli. Bock (1986, 1989) found

that under appropriate environmental conditions metacercariae of

Plagiorchis spec. 1 became very active; the juveniles egested stored caecal

fluid against the inner walls of the cyst and actively emerged through this

area. This "explosive expulsion" ofjuveniles has been reported by other 13 • authors (Howell 1970, Bass and LeFlore 1984) and allows rapid excystment and early attachment by the parasite to the intestine (Bock 1989). 14 • Aedes aegypti (L)

Aedes aegypti is a typical container breeding mosquito known best as a

vector ofyellow fever and dengue fever. It is widely distributed between

40 0 N and 40°8 but may be excluded in regions of great temperature

fluctuations or extreme drought. Aedes aegypti is a holometabolouf insect;

the life cycle consists of an egg, 4 larval stages, a pupa, and the adult stage

(Christophers 1960). Eggs are laid in and around water and hatch when

submerged in waters with reduced levels of dissolved oxygen (Judson 1960,

Christophers 1960).

• The growth rate oflarvae, as weIl as the subsequent size of adults, is a function of nutrition and temperature. The duration of larval instars !Tom

eclosion to pupation ranges !Tom 7-14 days. During the moults the heavily

sclerotized regions of the integument increase in size immediately following

ecdysis, but not between ecdyses. The abdomen and thorax which are

covered with a thin, extensible cuticle grow continuously throughout larval

life (Clements 1963). The larvae are phytophagous, using bacteria and

microorganisms as their main food source. However, waters overly

contaminated with bacteria can kill the larvae (Lewis 1933) and pupation

ceases in waters with inadequate food. Excessive larval competition for food,

or overcrowding in the presence of an adequate food supply (>1000 15 • larvaellitre) can prevent moulting or produces smaller larvae, pupae, and adults (Christophers 1960, Clements 1963, Wada 1965).

While larval growth rates are directly temperature dependent, temperatures

>39C for even short periods are lethal (Headlee 1942). Larvae can

recuperate from low temperatures but will not mature under continuous

temperatures <12C or >39C. The optimum temperature range for growth

and development ofA. aegypti is 27-30C, and several models have been used

to construct a developmentJtemperature curve (Christophers 1960).

Pupation follows ecdysis of the fourth instar larva. The pupa is an active, • non-feeding stage in which larval tissues are replaced by adult structures. As the pupa matures a layer of air appears below the cutic1e, separating the

adult from the pupal skin. The top of the cephalothorax breaks through the

surface film and the adult emerges. For a detailed description of these

processes see Christophers (1960).

Following emergence the activities offeeding, mating, host selection, and

oviposition ..ite selection follow temporal and hormonally-regulated

behavioural patterns. Many of these stereotyped behaviours are genetically

controlled and are released by various endogenous or exogenous factors • (Klowden 1990). Nectar provides nutrition; a blood meal is required by Hi • females to obtain the protein required for oogenesis (Harwood and James 1979, Briegel 1985, Handel and Lea 1984). Males are sexually mature and

begin mate searching within 15-24h following emergence while females have

a post-emergent period in which they are refractory to insemination

(Hausermann and Nijhout 1975). While copulation is independent of parous

status and continues throughout their lives, A. aegypti females are

monogamous; all ofi'spring result from the first copulation (Craig 1967,

Williams and Hagen 1977). Repeated copulation provides the female with

compounds from the male accessory reproductive glands. These compounds

can alter the behaviour of the female, inducing monogamy (Craig 1967),

autogeny (Q'Meara and Evans 1976), and altered circadian flight activity. • Gravid virgin females do not show characteristic pre-oviposition behaviour and do not oviposit due to an endogenous oviposition inhibitor (Fuchs and

Kang 1978). This inhibiti<,ll may be inactivated by mating or by

transplantation of the male substance. Interspecific transplants of male

accessory glands have demonstrated a non-specificity between several

mosquito species for stimulating oviposition and mating inhibition

(Ramalingham and Craig 1976).

Host seeking behaviour is dependent on parous status (Klowden et al. 1987);

females which have fed, are developing eggs, or are in oviposition mode are • not receptive to blood-feeding cues. ThiR inhibition of host seeking is lost 17 • following oviposition (Davis 1984). A single blood meal is usually sufficient for a complete gonotrophic cycle but multiple host feeding ruay be affected

by the nutritional status of the larvae (MacDonald 1956), water supply

(Khan and Maibach 1970), or by the interruption ofblood feeding by the

host (Edman et al. 1975). Gonotrophic cycles are initiated by the blood meal

and the number of eggs produced per cycle depends largely on the volume of

the blood meal, initial body weight of mosquitoes (Colless and Chellapah

1960), and genetics (Briegel 1985).

Aedes aegypti shows an endogenously regulated diel pattern in oviposition

(Haddow and Gillett 1957, KJowden 1990) that may be influenced by the • length of the photophase (Chadee and Corbet 1987) and by a light activated hormone (Haddow and Gillett 1957). Oviposition site selection is governed

by visual cues, substrate texture, reflectivity ofwater, and odours (Wood

1961, Russo 1978, Adham 1979, Mclver 1982) and by aquatic microbes and

their metabolites (Gjullin and Johnsen 1965, Ikeshoji et al. 1979, Benzon

and Apperson 1988). While gravid females are attracted to waters

containing conspecific larvae (Soman and Reuben 1970), they oviposit

preferentially in sites containing eggs of other conspecifics over sites

containing their own eggs from previous gonotrophic cycles (Chadee et al.

1990). Since the flight range of females may be as short as 25-30m from

their site of emergence (Soper 1935), the probability ofgenetically related 18 • females using the same oviposition sites is high. Following oviposition pre- oviposition behaviours are ended and females resume host seeking for more

blood with which to develop a subsequent batch of eggs.

•

• 19 • Stagnicola elodes (Say)

Sto.gnicola elodes is a typical herbivorous, freshwater, pulmonate snail

ubiquitously distributed in Canada, especially in habitats with thick aquatic

vegetation on muddy substrates (Clarke 1981). The shell is a dextral conical

spire composed of tubular whorls containing the visceral mass of the animal

(Malek and Cheng 1974). The apex contains the oldest and the smallest

whorls, which coi! around a central axis. The last and biggest whorl (the

body whorl) terminates at the aperture from which the head and foot of the

animal protrude. The shell itself consists of 4 layers; the outer

periostracum-quinone-tanned homy protein called conchin and the inner • shelllayers consisting of calcium carbonate. The shell colour results from pigments in the periostracum or in the calcareous layers (Malek and Cheng

1974).

Development of eggs and growth of adult snails is temperature dependent.

The growth rate oflymnaeid snails increases with temperatures from 6-24C.

Temperatures of 18-22C resu1t in maximum longevity and fecundity but

temperatures >30C may be lethal (Schelie and Berry 1973). Lymnaeid

snails come to the surface of the water to breathe; respiration is

accompiished by arching and flattening the floor of the mantle cavity and • opening and closing the pneumostome which is usually closed while the 20 • animal is submerged. Stagnicola elodes is monoecious, the gonad being an ovotestis (Malek and Cheng 1974) and individuals are capable of self or

cross fertilization (Clarke 1981).

The life cycles and trl .amission dynamics of digenean parasites depend

upon molluscs. However, the hosts are also directly affected by the

parasites. Digenean infections can produce reduced thermal tolerances

(Etges and Grasso 1965, Lee and Cheng 1971), abnormal or enhanced

growth ofboth shell and soft tissues (Wesenberg-Lund 1934, Wilson and

Denison 1980, Cheng et al. 1983), sex reversaI, reduced growth (Zischke and

Zischke 1965) reduced longevity (Pan 1965), reduced fecundity (Wilson and • Denison 1980), and increased oxygen uptake and heart rate (Lee and Cheng 1971).

Digenean infections also cause parasitic castration in which the parasite

inhibits either partially or completely the formation of gametes by the host

(Malek and Cheng 1974). Castration may vary from a direct ingestion of the

gonads and parasite-induced atrophy of gonadal tissue (Wright 1966; 1971,

Noble and Noble 1976, Cheng 1986) to partial cessation of egg production

and eventual recovery (Hurd 1990). Castration may be caused by

competition between host and parasite for nutrients (physiological

starvation) and similarities have been shown between the effects of 21 • starvation and infections by digeneans {Becker 1980). Alternatively the parasites can interrupt host reproduction by interfering with the production

of reproductive hormones (Malek and Cheng 1974, Sluiters 1981, de Jong­ .{ Brink et al. 1988).

Parasitic castration is not limited to trematode-mollusc relationships. Kuris

(1974) compares digeneans with parasitoids and argues that parasitic

castration is a specialized mode of predation which results in the

reproductive death ofhosts. Beaudoin (1974) considers parasitic castration

as an evolutionary strategy ofparasite-induced manipulation ofhost

reserves from host reproduction to increased host longevity, which leads to • increased parasite fitness as a result of an improved environment.

In the case ofS. elodes, parasitic castration by P. elegans is probably the

result of a physiological interruption of the host's reproductive potential.

The result of parasitic castration of hosts infected with P. elegans is the

replacement of egg production by the daily production of thousands of

cercariac; the cercariae and subsequent metacercariae which form the focus

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Shostak, A.W. and G.W. Esch. 1990. Photocycle-dependent emergence by cercariae ofHalipegus occidualis from Helisoma anceps, with special reference to cercarial emergence patterns as adaptations for transmission. J. ParasitoI. 76: 790-795.

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•

,".'

• • 36 CHAPTER2

PLAGIORCHIS ELEGANS: EMERGENCE, LONGEVITY, AND

INFECTIVITY OF CERCARIAE, AND THE EFFECTS OF

CERCARIAL EMERGENCE ON THE BEHAVIOUR OF THE

MOLLUSCAN FIRST INTERMEDIATE HOST.

• Carl A. Lowenberger! and Manfred E. Rau2

!Institute of Parasitology and 2Department ofEntomology

Macdonald Campus of McGill University,

21,111 Lakeshore Rd, Ste-Anne-de-Bellevue

Quebec, Canada, H9X 3V9

A revision of this chapter will be submitted to

Parasitology 37 • AB8TRACT: We investigated changes in the behaviour ofStagnicola elodes associated with the emergence of Plagiorchis elegans cercariae. Within 15

min of the reduction in light intensity, which triggered the onset of cercarial

emergence, infected snails moved to the top of the water column and

remained there for 2-3h. 79% of aH cercariae that emerged from the snail

did so during this period. Uninfected snails showed no such behavioural

changes foHowing the change in light intensity. Cercariae were released in a

dense cloud around the snail at the water surface and dispersed passively.

Within 3-4h >80% of aH cercariae had settled to the bottom 5cm of the

water column. The infectivity of cercariae increased from <20% upon

emergence from the snail to >75% 4-6h post emergence, and then declined

steadily to <5% by 24h post emergence. Cercariallongevity was >30h and

exceeded the period of infectivity. This delay in reaching maxÏ'J"t'.ID

infectivity may represent an adaptive mechanism to disseminate the

cercariae, thus reducing superinfection of intermediate hosts and

subsequent parasite-associated mortality of second intermediate hosts. • 38 INTRODUCTION

A major problem facing parasites is the dispersal of propagules to new hosts

(Smith Trail 1980). Adults of free living groups such as the parasitoids

actively seek out and discriminate between potential hosts (McBrien and

Mackauer 1991), whereas many parasite species are transmitted passively

through ingestion of one host by another. Parasites w,th multiple host life

cycles but without free living stages may interact physiologically with their

hosts to modify host behaviour in ways to increase the rate of parasite • transmission (Dobson 1988). The role of such behavioural changes in parasite transmission has received

considerable attention. Holmes and Bethel (1972) distinguished between

host behavioural changes that resulted in accidentaI ingestion, and those

resulting in increased predation of parasitized intermediate hosts by

definitive hosts. They al!'\o recognized different parasite strategies used to

increase the likelihood of predation of infected intermediate hosts, including

reduced stamina and locomotion, host disorientation, increased

conspicuousness, and an altered response to external stimuli. 39 • The complexities of digenean life cycles are weIl documented, and are characterized by the inclusion of several taxa as hosts. A major challenge to

digeneans is the localization and infection of the proper host at the

appropriate time. The effects ofDicrocoelium dendriticum on the behaviour

of its insect intermediate host that increase the probability of transmission

to the definitive host are weIl documented (Carney 1969). Swennen (1969)

reported that Macoma balthica infected with gymnophallid trematodes left

tracks in the sand and were more conspicuous to predators. Pearre (1979)

reported that chaetognaths infected with digeneans were more conspicuous

and more susceptible to predation than non-infected individuals, and

Carney (1969) reported that carpenter ants infected with the digenean

Brachylecithum lost their photophobie behaviour and became more

vulnerable to the avian definitive hosts of the parasite. Webber et al. (1986)

showed that Aedes aegypti larvae infected with Plagiorchis elegans

(identified as Plagiorchis noblei) metacercariae spent more time at the

surface and were more vulnerable to predation by mammali'ill definitive

hosts.

Parasite-induced behavioural changes in invertebrate intermediate hosts are

not restricted ta infect;ons with digeneans. Lester (1971) showed a reduction

in stamina and locomotion in sticklebacks infected with Schistocephalus

solidus. Bethel and Holmes (1974) showed that amphipods infected with 40 • Polymorphus paradoxus reduced their evasive tactics and rose to the surface where they were more visible to predators. Moore (1983) found that

cockroaches infected with Moniliformis became more active during the

foraging period of the Norway rat, the definitive host of this parasite. For a

review of interactions between invertebrate hosts and parasites see Hurd

(1990).

AlI of the above studies have dealt with the last juvenile stage of the

parasite and the behavioural changes elicited in the last intermediate hosto

Less weIl documented are the effects of these parasites on earlier

intermediate hosts. Although digeneans produce cercariae that are released • from a molluscan first intermediate host, there are few reports of parasite­ induced behavioural changes in these hosts (Curtis 1987, de Jong-Brink

1990). Conceivably, a single digenean parasite may employ more than one

strategy and modify the physiology, energy allocation, and behavioural

patterns of each ofits intermediate hosts that increase the probability of its

transmission (Smith Trail1980).

The activity patterns ofinfected snails during cercarial emergence may

influence the distribution of cercariae in the water column, and

consequently may affect parasite transmission. The present study assessed • the impact of emerging Plagiorchis elegans cercariae on the behaviour of the 41 • molluscan first intermediate host, Stagnicola elodes. Furthermore, the study examined changes in the vertical distribution of cercariae within the water

column and changes in the infectivity of cercariae over time.

Plagiorchis elegans

The biology of the various stages ofP. elegans has been described (Macy

1960, Styczynska-Jurewicz 1962, Genov and Samnaliev 1984, Lowenberger

and Rau 1993). Briefly, the monoecious adults of P. elegans are intestinal

parasites of a variety of mammals and birds. Eggs are released into the

external environment with the faeces of the definitive host and are ingested

by lymnaeid snails (Genov and Samnaliev 1984). The miracidium hatches • from the egg, penetrates the gut lining, and forms a mother sporocyst (Blankespoor 1977). Asexually produced daughter sporocysts establish in

the hepatopancreas and give rise to xiphidiocercariae which first emerge

from the snail approximately 40 days following egg ingestion (Daniell and

ffimer 1964, Blankespoor 1974). Cercarial emergence is elicited by the

change from light to dark (Genov and Samnaliev 1984, Webber et al. 1986).

The cercariae penetrate a wide range of aquatic insects (Williams 1963,

Blankespoor 1974) and encyst in the haemocoel !lS metacercariae. Overall

development of metacercariae to infectivity is temperature dependent

following a obligatory temperature independent period of parasite-host 42 • contact (Lowenberger and Rau 1995). Metacercariae excyst and transforrn into adults in the intestine of the definitive hosto

MATERIALS AND METHOnS

BEHAVIOURAL STUDY

A cone shaped observation chamber (S5crn diameter, depth 10cm at centre,

volume SOOO ±25rnl) was used to study snail behaviour. The cone was filled

with aerated tap water containing O.Sg finsly gl-ound Tetramin® fish food. A

single snail was introduced into the cone at 18:00 and allowed 2h to

acclirnatize. The scotophase, in which light was provided by a 40W red light, • began at 22:00 and ended at 06:00. A Panasonic AG 6010 video record.er in conjunction with a Hitachi CVCC camera with an automatic, light sensitive

diaphragm was used to film snail activities from 20:00-02:00 (2h light:4h

dark). The sarne 10 infected and 6 uninfected snails were videotaped at

three temperatûres (15, 20, anà 25C) in a temperature controlled

environrnent. AlI snails were acclimated to each experimental ternperature

for 8d prior to each trial. Additionally, snails were paired in the cone:

uninfected with uninfected, uninfected with infected, and infected with

infected to determine ifinteractions between snails affected their behaviour

during cercarial emergence. We measured the distance travelled by snails

during each 15 min period and determined the vertical position of snails in 43 • the water column at 2 min intervals using a Panasonic WV5470 monitor to which were fixed acetate overlay templates that corrected for camera

distortion. The experiments were carried out from 15-25C, as this is the

range over which mosquito larvae and cercariae are most likely to come into

contact under normal conditions.

CERCARIAL EMERGENCE

The emergence of cercariae from each of the 10 infected snails was assessed

over 5 successive nights at each of 15, 20, and 25C. The snails were placed

individually into plastic cups containing 50 ± 3.0ml of aemted tap water 30

min before the end of the 16h photophase. At 30 or 60 min intervals • following the reduction in light intensity, snails were removed from the cups, rinsed with 5.0 ± 2ml distilled water to remove lightly adhering

cercariae, and transferred to new containers. After the third transfer the

snails remained in the saIne cups for the duration of the scotophase. The

number of cercariae emerging from each snail during each period was

assessed by volumetrie subsample estimation.

VERTICAL DISTRmUTION OF CERCARIAE

In order to assess the vertical distribution of freshly emerged cercariae over

time, 10 lots of 100 cercariae were placed in vertical columns 20cm high and • 5.0cm diameter that had been siliconized

at 30 min intervals. This was repeated at temperatures.of 15, 20, and 25C

(10 replicates at each temperature).

CERCARIAL LONGEVITY

Cercariallongevity was determined using cercariae released during the first

30 minutes following the reduction in light intensity. Groups of 100

cercariae (5 groups/day replicated over 5 consecutive days) were placed in

glass dishes (60 x 15mm) containing 20ml water from aquaria used to house

the snails. At 2h intervals up to 12h post emergence, and subsequently at • 4h intervals, the numbers of cercariae that were active were cOW1ted. An active cercaria was one either swimming or crawling by itself. Cercariae

which moved only after being touched with a needle were considered

inactive.

CERCARIAL INFECTIVITY

Cercarial infectivity was assessed using cercariae released during the first

30 min following the reduction in light intensity. At 30 min intèrvals from

0.5-12h post emergence, and at hourly intervals from 13-36h post

emergence, 5 randomly selected active cercariae were placed in each of 10

wells (Falcon 24 weil, Becton Dickson,New Jersey, USA) containing 2.0 ± 45 • 0.5ml aerated tap water. One fourth instar Aedes aegypti larva was introduced to each weIl. The small volume of water ensured contact between

the cercariae and a potential hosto Following 15 min exposure to the

cercariae, the larvae were removed from the wells, washed to remove lightly

adhering cercariae, and transferred to plastic cups containing 50ml of

aerated tap water. The prevalence, abundance, and location ofinfection

were determined 24h post exposure (ô replicates/cercarial age).

ANALYSIS

Statistical analyses were performed using SYSTAT 5.0 software, using

Student's t-test to compare distances travelled by infected and uninfected • snails at the same temperature. Analysis of variance on repeated measures (Anova RPT) was used to compare distances travelled by infected and

uninfected snails prior to and following the change in light intensity and the

time spent at the surface by infected and uninfected snails prior to and

following the reduction in light intensity. The Mann-Whitney U-test (MWU)

was used to compare the time spent at the surface by infected and

uninfected snails. The Kruskal Wallis test for multiple comparisons was

used to compare the number of cercariae that emerged from the snails at

different temperatures. The level of significance (a) was set at P=0.05 in all

tests. 46 • RE8ULT8

BEHAVlOUR: ACTIVITY

Prior to the reduction in light intensity (RLI) there was no significant

difference in the distances travelled by infected and uninfected snails at

each of the experimental temperatures (Student's t-test; 15C, P=0.291; 20C,

P=O.071; 25C, P=0.512). However, following RLI there was a significant

reduction in the activity of infected snails. Within 15 min of RLI, infected

snails significant1y reduced the distance travelled (Fig 2.1). These distances

were significant1y lower than those of uninfected snails over the same

periods at 15C and 20C, but not at 25C (Student's t-test; 15C, P=0.035; 20C, • P=0.006; 25C, P=0.345). When the post RLI period was divided into 90 min segments and activities in these periods were compared with similar

segments for UIÙnfected snails we found that, regardless of temperature,

infected snails showed significant1y reduced activity during the first 90 min

following RLI (Student's t-test; 15C, P=0.017; 20C, P=0.021; 25C, P=0.019).

In the second 90 min period the activity patterns of the snails at 15C and

25C were not significantly different from the uninfected snails (Student's t­

test; 15C, P=0.055; 20C, P=0.020; 25C, P=0.841). Infected snails returned to

pre-RLI levels ofactivity faster at 15 and 25C than at 20C. 47 • Analysis of repeated measures of the distances travelled by uninfected snails did not reveal signHicant differences over time regardless of light

intensity (Anova RPT; 15C, P=0.274; 20C, P=0.894; 25C, P=0.997) whereas

infecteù :mails showed a significant reduction in distance travelled following

the RLI (Anova RPT; 15C, P=0.04,; 20C, .0;0.026; 25C, P=0.037) (Fig 2.1).

When pairs of infected and uninfected snails were introduced to th,:>

observation chamber the activity patterns ofinfected snails showed a rapid

and significant reduction in activity following RLI (P<0.05) whereas

uninfected snails continued ta travel throughout the observation arena

(P>0.05) (data not shown).

BEHAVIOUR: VERTICAL DISTRmUTION

Regardless of experimental temperature there was no significant difference

(MWU; 15C, P=0.097; 20C, P=0.230; 25C, P=0.252) in the time spent at the

surface by infected or uninfected snails during the photophase (Fig 2.2), and

previous studies (data not shown) showed that snail activities in the last 4h

of the photophase did not differ significantly from earlier periods of the

photophase. However, following the RLI, infected sHails spent significantly

more time at the surface than uninfected snails at the same temperature

(MWU; 15C, P=0.026; 2!lC, P=0.007; 25C, P<û.OOl) (Fig 2.2). Analysis of

repeated measures showed no significant difference in time spent at the

surface of t.he water before and folIowing the RLI by uninfected snails

"- -" ,- 48 • (Anova RPT; 15C, P=0.75; 20C, P=0.61; 25C, 'P=0.899). However infected snails spent significantly more time at the surface following the RLI (Anova

RPT; 15C, P=0.012; 20C, P=0.007; 25C, P=0.004) (Fig 2.2). Both activity

patterns and the time spent at the surface by infected snails became

indistinguishable from their pre-RLI levels or those of uninfected snails 4­

5h following RLI.

CERCARIAL EMERGENCE

T:':1e emerge.lce of cercariae in terms of numbers and pattern was

temperature dependent (Fig 2.3). Significantly more cercariae emerged at

20C than at 15 or 25C (MWU; P<0.05) whether expressed as total emerged

cercariae or cercariae emerged perhour. While there was no significant

difference in wtal numbers of cercariae that emerged in the first 3h

following RU at 15 and 25C, cercr;.ial emergence at 25C was significantly

higher t1lan at 15C in hour 1 post RLI (MWU; P=0.023), and significantly

lower than 15C in hours 2 (MWU; P=0.019) and 3 (MWU; P=0.038) post

RLI. The period of peak emergence occurred earlier as the temperature was

increased from 15 ta 20C, while the pattern of emergence, ie. maximwn

peak in the first hour followed by a decline in cercarial emergence in

successive hours, was similar at 20 and 25C (Fig 2.3). Cercarial emergence

within the first three hours accounted for 79% of the total number of • emerged cercariae over the entire 8 hour scotophase. 49 • VERTICAL DISTRmUTION OF CERCARIAE Cercariae showed a similar vertical distribution in the water column at ail

experimental temperatures (15, 20, 25C). At each temperature, 80% of the

cercariae had settled to the middl", of the 20cm column within 1.5-2h. By

3h, 80% of cercariae were found in the lower half of the column and by 3.5­

4h >80% of ail cercariae we:e found in the lowest 5cm of the column (Fig

2.4). By 3-4h post emergence cercarial activities had changed from

predominantly swimming in the water column to crawling on the substrate.

CERCARlAL LONGEVITY

AlI cercariae were active for the first 10h following emergence. The number • of active cercariae subsequently decreased over time such that by 24h post em,~rgencc, almost 50% of cci(;Jriae had died, and only 5% of cercariae were

active 40h post emergence (Table 2.1).

CERCARIAL INFECTIVITY

Infectivity of cercariae was defined as the percentage of cercariae which

actively penetrated and successfully formed metacercariae within the

mosquito larvae. Within the first 30 min following emergence cercariae

showed a level ofinfectivity <20% (Fig 2.5). Infectivity rose to a peak of 78%

by 4-6h post emercence. This peak was followed by a graduaI decliue such

that infectivity essentially reached zero by 24h post emergence (Fig 2.5), 50 • although >50% of aU cercariae lived >24h (Table 2.1). The prevalence of infection in mosquitoes exposed to cercariae <12h post emergence was >75%

(Table 2.2). Approximately 31% of aU metacercariae were found in larvae

exposed to cercariae 4-6h post emergence, and progressively greater

proportions of metacercariae were found in the head, thorax, and abdomen

(Table 2.2).

DISCUSSION

Snails infected with P. elegans showed significant alterations in their

activity patterns foUowing the reduction in light intensity. Within 15-30 min • of RLI aU infected snails had moved to the surface of the water where they remained for the next. 2-3h. Neither the mechanism of this alteration in

snail behaviour nor the actualmr;ans of exit ofP. elegans cercariae from the

snai! host has been determined. Direct microscopical observations of snails

withinl hour ofRLJ suggest that cercariae exit the snail through the

pneumostome via the pulmonary cavity. Cercarial emergence is

accompanied by extensive mucous secretions, possibly indicating damage to

the mucosal lining of the host's lung. Changes in the activity patterns by

infected snails may be a direct result of cercarial migration and emergence

through host tissues to the external environment. As almost 80% of the

cercariae emerge in the first 3h foUowing the dark period, there may be an 51 • accumulation of fully developeà cercariae near the point of exit awaiting the stimulus(i) to emerge. The fact that cercarial emergence continues over

severaI hours, albeit at extremely reduced levels, suggest.s that some

cercariae may move considerable distances, are slowed during their

migration through host tissues, or do net receive or react equally to the

stimulus(i).

While the reduction in snail activity patte..ns is similar at al! temperatures

the rate of recovery differs. At 25C the snails retumed to pre RLI levels

much faster than at lower temperatures. The rapid onset of cercarial

emergence and the low numbers ofcercariae released at 25C compared with

20C may affect the speed and duration of the recovery phase of infected

snails. This would explain why activity patterns of infected snails at 25C

were sign::ficantly~educed from pre RLI levels during the first 90 min

following RLI, but were not significantly different from pre RLI levels in the

1.5-3h post RLI period.

The role of the molluscan host in cercarial emergence has not been clearly

defined. Sorne (Asch 1972, Anderson et al. 1976) believe that the snail plays

a direct role in the chronobiology ofcercarial emergence while others

(Théron 1984, 1985, Théron and Combes 1983) consider the influ.ence of the • snail to be very limited. The timing of cercarial emergence in relation to 52 • host ::ctivity patterns also varies. AndeTson et al. (1976) report that the peak of emergence of Trichobilharzia ocellata from .ï,ymnaea stagnalis

occurs in the 2nd-6th hours fol1owing the return to light which coïncides

with the period ofgreatest snail activity. In contrast we have shown that

the nocturnal emergen~e of P. elegans cercariae coincides with a severe

reduction in host activity. Thus, the role of the host in cercarial emergence

remains unclear.

The periodicity of cercarial release from molluscan hosts has been studied

for many digeneans. These cycles of cercarial emergence correspond to daily

changes in light intensity and. temperature regimes, and may correlate with • activitie;; of the next intermediate host (Ginetsinskaya 1968, Cable 1972, Théron 1984). An absence of periodicity has been reported for species that

do not use a second intermediate host but encyst in the environment

following emergence (Kendall and McCullough 1951, Ginetsinskaya 1968).

There is a circadian emerger.ce rhythm in schi~tosomes (Théron 1989, Pages

and Théron 1990), with peaks of emergence during the light whereas

Wagenbach and Alldredge (1974) have shown a circadian, nocturnal

emergence for Plagiorchis micracanthos. Similar nocturnal shedding

patterns have been found for P. noblei (Blankespoor 1977), Plaj;"iorchis

verspertilionis (Macy 1960) and Schistob:Jma douthitti (Olivier 1951). 53 • The 3ct.ivity patt.ems of P. elegans-infected mailG at the time of shedding influenr;e the spatial pattern of cercarial deposition. As the snails are

essentially immobile at the surface during the first 3h following the RLI,

approximately 80% of all cercariae are released in a dense cloud arour.d~he

snail hosto Conceivably, an insect entering such a. cloud would be invaded by

a large number of cercariae causing rapid death. This would he counter­

adaptive to parasite transmission.

Shostak and Esch (1990) presentrid three hypotheses for the adaptive

significance of periodic mass emergence of cercariae: 1) cercariae emerge to

coincide with the presence of the next hosto This concept is adaptive ifthe • next host has a daily activity cycle or ifthe cercariae are short lived (Cable 1972, Lewis et al. 1989). As P. elegans parasitizes a wide array of aquatic

arthropods, cercarial emergence is prohably not timed to coincide with the

presence of a particular second intermediate hosto 2) Periodicity of cercariat

emergence enhances dispersai from the host at a specifie location and is

adaptive in species that depend exclusively on the mollusc for cercarial

dispersal or species with weak or sessile cercariae. Since P. elegans itself

appears to alter host behaviour this process could occur at any time, and

cercarial emergence is probably not tied to the circadian rhythms of the

snail hosto 3) Periodic mass emergence of cercariae may impair the ability of

predators to detect the cercariae. Alternately, predators may he absent at 54 • thiptilm" or may be unable to reduce significantly the numbers of cercariae that emerge en masse. While we cannot discount this third possibility, the

first two hypotheses do not appear to be highly relevant adaptive

mechanisms for P. elegans. Plagiorc.tis elegans actively manipulates the

behaviour of the snail host which results in cercariae emerging at the

surface from where they are passively dispersed prior to reaching maximum

infectivity. This may be particularly advantageous for transmission.

While cercariae are generally considered to be dispersal stages of digenean

parasites (Erasmus 1972, Shoop 1988) the cercariae of Plagiorchis sp. are

notgood swimmers (Kavelaars and Boums 1968). Swimming maintains the • position of cercariae in the water column rather than providing directed lateral movement. Cercariae are passively dispersed throughout the water

column and quickly settle to the substrate. Dispersal prior to attaining

maximum infectivity may lead to lower densities of infective cercariae,

lower parasite burdens and reduced mortality of bottom dwelling hosts in

which metacercariae overwinter (Karpenko and Zaika (1979) cited in Genov

and Samnaliev 1984). Although mosquitoes have served well as laboratory

hosts for P. elegans, they may not play a primary role in the transmission of

P. elegans in the field. • 55 • The cercariae of P. noblei are reported to be infective for up to ] 2h (Blankespoor 1977). Dempster and Rau (1989) evaluated cercarial infectivity

of P. elegans (identified as P. noblei) by exposing mosquitoes to cercariae in

relatively deep water. While infectivity was reported to be initially high, the

subsequent decline was probably due to the spatial separation between the

parasite at the bottom of the water column and hosts active in the upper

regions of the water column. Our data suggest that the infectivity of P.

elegans cercariae, as defined by the ability of the cercariae to penetrate a

host when host-parasite contact is ensured, is Lw upon emergence, peaks 4­

6h post emergence, and is followed by a steady, graduaI decline. Not all

digenean cercariae show this pattern. Transuersotrema patialense is highly • infective upon emergence followed by an exponential decline in infectivity (Anderson et al. 1977) while the ability of8chistosoma mansoni cercariae to

penetrate hosts declines only slightly 1-5h post emergence, and they can

still penetrate murine hosts 24h later (Stirewalt and Fregeau 1968).

A delay in reaching maximum infectivity may represent an obligatory

dispersal phase which disseminates cercariae over a greater area. The

concept of an obligatory dispersal phase is not new. Evans and Gordon

(1983) have shown that Echinoparyphium recuruatum reaches a peak of

infectivity 2-3h post emergence, followed by a steady decline. A dispersal • phase would reduce the risk of superinfection of second intermediate hosts 56 • and consequently parasite-induced host mortality and reduced rates of transmission to the definitive hosto As cercariul infectivity declines with

time, the longevity of cercariae may exceed their period of jnfectivity. Evans

and Gordon (1983) reported that while E. recurvatum cercariae have an

average lifespan of 30.5h, they lose the ability to infect a second

intermediate host after 19h. Blankespoor (1977) reports that longevity and

infectivity of P. noblei cercariae are temperature dependent and that the

ability of cercariae to penetrate a second intermediate host is lost

approximately 12h post emergence, although they remain active for more

than 30h. Similarly, we have shown that P. elegans cercariae reach

maximum. infectivity 4-6h post emergence, and although they can live for • more than 30h, their ability to infect mosquito larvae is minimal 16-20h following emergence. The brevity of the infective cercarial stage may be

characteristic of a highly active, non-feeding organism which exhausts its

finite energy reserves (Anderson and Whitfield 1975) but continues to

survive beyond its ability to penetrate and encyst within a potential hosto

SUMMARY

The behaviour of Stagnicola elodes is strongly affected by the emergence of

Plagiorchis elegans cercariae. As light intensity is reduced, cercarial

emergence begins and infected snails m e to the top of the water column.

They remain there, essentially motionless, for 2-3h. During this period 57 • about 80% of cercariae emerge from the snail. Due to the immobility of the snails, the emerging cercariae form ? dense cloud around the snail. The

cercariae settle to the bottom of the water column within 1.5-2.5h.

Infectivity of cercariae increases from <20% upon emergence to >75% by 4­

6h following emergence, and then declines to <5% by 24h post emergence.

Due to cercarial settling and a delay in reaching maximum infectivity, the

cercariae may be passively dispersed prior to reaching maximum infectivity

and will, therefore, predominantly parasitize bottom dwelling arthropods.

This dispersal phase of the cercariae may reduce superinfection, and

subsequent parasite-induced mortality, of second intermediate h03ts.

• ACKNOWLEDGEMENTS We thank Sylvain Poirier for assistance in setting up and calibrating the

videorecording system and the design of acetate overlays used to correct the

effects of camera distortion. Funding was provided by the Natural Sciences

and Research Council of Canada (NSERC) (OGP 0007606) to MER a'nd the

Fonds pour la formation de Chercheurs et l'aide à la recherche (FCAR) (ER

0129) to MER. Personal funding for CAL was provided by a Walter M.

Stewart Award and a Lynden Laird Lyster Award. Research at the Institute

of Parasitology of McGill University is supported by NSERC and FCAR. • 58

Figure 2.1. Comparison of activity patterns of uninfected and Plagiorchis

elegans·infected snails. The values represent the mean distances travelled

by snails (+1 standard error) measured over 15 minute periods under

different temperature regimes. Cercarial emergence began within 15

minutes of the reduction in light intensity which is indicated by the arrow.

• • 120 15C ! '100 1 • -control ,..... :: T!~\l. 1 1:1 -infected ~ 40 ~ ,r, ~ ~ 'I-i-l'i~i-l-~~I-I-1~I + ~---ll.- ...:f:!:!:!i!:!!~~~~~~ ::: 2: ...... a 0 30 80 80 120 150 110 210 240 270 306 C) -....; • 40 ~ 200t·· _,"--1=...&--a-r;t=C;:::....;a~. __.1.'_ ....' :e...... ---a.'_...L.'_.l.'----l' o 30 80 80 120 150 110 210 240 270 300

80

40

20

Ol...-..&...-...... ------:;I::!:!:!~~~_J..__L___I .' C 30 80 80 ~20 150 110 210 240 270 300 Time (minutes) • 60

Figure 2.2. Comparison of the mean time spent at the surface of the water

(+1 standard error) by uninfected and Plagiorchis elegans-infected snails

prior to (pre) and following (post) the reduction in light intensity.

Significant differences (Mann-Whitney U-test P

uninfected snails are indicated by *.

• • • •

100 Q) _ INFECTED * ~ 80 1 m CONTROL ..... r * M ~ 60

~ «S 40 Q) E! E:: 20 ~ o -'- PRE POST PRE POST PRE POST 15C 20C 25C • 62

Figure 2.3. Emergence of Plagiorchis elegans cercariae in the first three

hours following the reduction in light intensity. Values represent mean

cercarial emergence (+1 standard error) by 10 infected snails over S

consecutive days. Witô.in the same hour, bars headed by different letters

indicate significantly different levels of cercarial emergence (Kruskal Wallis

multiple comparison P

DJI~ •

o • • 64 Figure 2.4. Location of Plagiorchis elegans cercariae in a 20cm water

column at various times foliowing cercarial emergence. The values represent

pooled samples from 15, 20, and 25C, as there were no differences in the

vertical distribution of cercariae at these temperatures.

•

• •

. {' . . , ....•...•••• . --- . ~ .. , . 1• ••••••~;,...... •••••• +.+ .. • •••••••••••••• ••••••• ••...... ••••••••••••••••••• CD ...... 8...... ••••••••••• C"4 E-li

....

i i o o o o o ....o ID ID aVl.lVO.l90• JO • 66 Figure 2.5. Mean infectivity (+1 standard error) ofPlagiorchis elegans

cercariae of different ages post emergence. Ten fourth instar Aedes aegypti

larvae were each exposed for 15 minutes to 5 cercariae in 2.0 ml aerated tap

water (6 replicates/ cercarial age). Following exposure, the larvae were

washed to remove loosely adhering cercariae and were examined for

metacercariae. Infectivity was defined as the percentage of cercariae that

successfully fonned metacercariae within the insect. • •••

100 1 t 1 l T_._ r:I 75 0 • .-1 ~ () Q) 50 t+-I r:I ...... 1 Tt l \± T ~ IJ- l '-., f 26 1 1. 1 • .I.~ T r r

o -1 1 lit- 1 1 1 liT ...... ~ o 2 4 6 8 10 12 14 16 18 20 3640 Time (Hours) post emergence • 68

Time (h) post Mean Number1 (SE) emergence of Active Cercariae

2 100 (0) 4 100 (0) 6 100 (0) 8 100 (0) 10 100 (0) 12 94.1 (1.1) 16 86.4 (1.3) 20 70.6 (1.6) 24 52.9 (l.8) 28 38.8 (2.2) 32 22.7 (l.3) 36 12.9 tl.4) 40 5.1 (1.9)

Table 2.1. Longevity ofPlagiorchis elegans cercariae. Cercariae were collected within 30 minutes ofemergenc:'! from the snail host and placed in groups of 100 in glass dishes (5 dishe~night) and examined for activity ilt various times post emergence. This was replicated over 5 consecutive nights.

1 values represent mean number per 100 emerged cercariae showing some signa of activity at the specified time following emergence. •• • 69

Age of No. Prey. Mean % Total Location of cercariae Mosq (%) Abundance Metacercariae Metacercariae (%) (hours) Exposed ±SE Head Thorax Abdomen

0- 1.9 240 74.3 1.84 ± 0.21 13.71 19.8 27.9 52.3 2- 3.9 240 86.9 2.46 ± 0.33 18.33 16.8 25.2 58.0 4- 5.9 240 91.5 3.84 ± 0.41 28.61 17.7 20.3 62.0 6- 7.9 240 90.7 2.11 ± 0.18 15.72 12.4 29.3 58.3 8- 9.9 240 82.5 1.47 ± 0.52 10.95 10.4 15.5 74.1 10- 11.9 240 66.7 0.91 ± 0.66 6.78 15.0 28.3 56.6 12+ 240 44.5 0.79 ± 0.89 5.89 6.3 48.4 45.3

Table 2.2. Prevalence, Mean Abundance, and Location of Metacercariae in fourth instar Aedes aegypti larvae exposed for 15 minutes to Plagiorchis elegans cercariae of different ages. Mosquito larvae were exposed individually for 15 minutes to 5 P. elegans cercariae ofdifferent ages at 30 minute intervals from 0.5-40h post emergence (6 replicates/cercarial age), and were examined for the presence of metacercariae 24h post exposure. • 70 LITERATURE CITED

Anderson, P.A., J.W. Nùwosi21ski, and N.A. CrolI. 1976. The emergence of cercariae of Trichobilharzia ocel1ata and its relationship to the activity ofits snail host Lymnaea stagnalis. Cano J. ZooI. 54: 1481-1487.

Anderson, RM. and P.J. Whitfield. 1975. Survival characteristics of the free-living cercarial population of the ectoparasitic digenean Transversotrema patialensis (8oparker, 1924). Parasitology 70: 295-310.

Anderson, RM., P.J. Whitfield, and C.A. Mills. 1977. An experimental study • of the population dynamics of an ectoparasitic digenean, Transversotrema patialense: the cercarial and adult stages. J. Anim. EcoI. 46: 555-580.

Asch, H.L. 1972. Rhythmic emergence of 8chistosoma mansoni cercariae from Biomphalaria glabrata: control by illumination. Exp. ParasitoI. 31: 350-355.

Bethel, W.M. and J.C. Holmes. 1974. Correlation of development of altered evasive behavior in Gammarus lacustris (Amphipoda) harboring cystacanths ofPolymorphus paradoxus (Acanthocephalal with the infectivity to the definitive hosto J. ParasitoI. 60: 272-274.

Blankespoor, H.D. 1974. Host-induced variation in Plagiorchis noblei Park, • 1936 (Plagiorchiidae: Trematodal. Am. Mid. Nat. 92: 415-433. 71 • Blankespoor, H.D. 1977. Notes on the biology of Plagiorchis noblei Park, 1936 (Trematoda: Plagiorchiidae). Proc. Helminthol. Soc. Wash. 44: 44-50.

Cable, RM. 1972. Behavior of digenetic trematodes. pp. 1-17 in Bt:havioural Aspects of Parasite Transmission. E.U. Canning and C.A. Wright eds. Academie Press, London.

Carney, W.P. 1969. Behavioural and morphological changes in carpenter ants harboring dicrocoeliid metacercariae. Am. Mid. Nat. 82: 605-611.

Curtis, LA 1987. Vertical distribution of an estuarine snail altered by a parasite. Science 235: 1509-1511.

Danien, D.L. and M.J. ffimer. 1964. Life cycle of Plagiorchis noblei Park, • 1936 (Trematoda: Plagiorchiidae). J. Parasitol. (Abstract). 50: 46.

de Jong-Brink, M. 1990. How trematode parasites interefere with reproduction of their intermediate hosts, freshwater snails. J. Med. and Appl. Malacol. 2: 101-133.

Dempster, S.J. and M.E. Rau. 1989. Plagiorchis noblei in Aedes aegypti: cercarial age and infectivit/. J. Am. Mosq. Contr. Assoc. 5: 261-263.

Dobson, A.P. 1988. The population biology ofparasite-induced changes in host behavior. Q. Rev. Biol. 63: 139-165.

Erasmus, D.A. 19'.'2. 'l'he Biology of Trematodes. E. Arnold, London. 312 • pp. 72 • Evans, N.A. and D.M. Gordon. 1983. Experimental studies on the transmission dynamics of cercariae ofEchinoparyphium recurvatum (Digenea: Echinostomatidae). Parasitology 87: 167-174.

Genov, T. and P. Samnaliev. 1984. Biology, morphology and taxonomy of Plagiorchis elegans (Rudolphi, 1802) (Plagiorchiidae) in Bulgaria. pp. 75-114 in lFauna, taksonomiyai ekologiya na khehninti po ptitsi. 1. Vasilev, ed. Bulgarian Academy of Science, Sofia Bulgaria.

Ginetsinskaya, T.A. 1968. Trematodes, Their Life Cycles, Biology, and Evolution. Nauka Publishers, Leningrad. [English version: 1988, Amerind Publishing Company. Pvt. Ltd., New Delhi], 559pp.

Holmes, J.C. and W.M. BetheI. 1972. Modification ofintermediate host behaviour by parasites. pp. 123-147 in Behaviour Modification by Parasites. E.U. Canning and C.A. Wright eds. Academie Press, London.

Hurd, H. 1990. Physiological and behavioural interactions between parasites and their hosts. Adv. ParasitoI. 29: 271-318.

Kavelaars, B.J. and T.K.R. Boums. 1968. Plagiorchis peterborensis sp. n. (Trematoda:Plagiorchiidae), a parasite ofLymnaea stagnalis appressa, reared in the labomtory mouse, Mus musculus. Cano J. ZooI. 46: 135-140.

Kendall, S.B. and F.S. McCullough. 1951. The emergence of cercariae of Fasciola hepatica from the snail Lymnaea truncrttula. J. Helminthol. 25: 77-92. 73 • Lester, R,J.G. 1971. The influence ofSchistocephalus plerocercoids on the respiration of Gasterosteus and a possible resulting effect on the behaviour of the fish. Cano J. ZooI. 49: 361-366.

Lewis, M.C., LG. Welsford, and G.L. Uglem. 1989. Cercarial emergence of Proterometra macrostoma and P. edneyi (Digenea: Azygiidae): contrasting responses to light:dark cycling. Parasitology 99: 215-223.

Lowenberger, C.A. and M.E. Rau. 1993. Plagiorchis elegans: requirements for metacercarial development to infectivity, and conditions required for excystment. J. HelminthoI. Soc. Wash. 60: 67-71.

Macy, R,W. 1960. The life cycle ofPlagiorchis vespertilionis parorchis, N. ssp. (Trematoda: Plagiorchiidae), and observations on the effects of • light on the emergence of the cercariae. J. ParasitoI. 46: 337-345. McBrien, H. and M. Mackauer. 1991. Decision to superparasitize based on larval survival: competition between aphid parasitoids Aphidius ervi and Aphidius smithi. EntomoI. Exp. AppI. 59: 145-150.

Moore, J. 1983. Altered behavior in cockroaches (Periplaneta americana) infected with an archiacanthocephalan, Moniliformis moniliformis. J. ParasitoI. 69: 1174-1176.

Olivier, L. 1951. The influence oflight on the emergence of Schistosoma douthitti cercariae from their snail hosto J. Parasitol. 37: 201-204.

Pages, J.R, and A. Théron. 1990. Analysis and composition of cercarial emergence rhythms of Schistosoma haematobium, S. intercalatum, S. bovis, and their hybrid progeny. Int. J. Parasitol. 20: 193-197. 74 • Pearre, S. 1979. Niche modification in Chaetognatha infected with larval trematodes (Digeneal. Int. Rev. des Hydrobiol. 64: 193-206.

Shoop, W.L. 1988. Trematode transmission patterns. J. ParasitoI. 74: 46-59.

Shostak, A.W. and G.W. Esch. 1990. Photocycle-dependent emergence by cercariae of Halipegus occidualis from Helisoma anceps, with special reference t.a cercarial emergence patterns as adaptations for transmission. J. Parasitol. 76: 790-795.

Smith Trail, D.R. 1980. Behavioral interactions between parasites and hosts; host suicide and the evolution of complex life cycles. Am. Nat. 116: 77-91.

Stirewalt, M.A. and W.A. Fregeau. 1968. Effect of selected experimental • conditions on penetration and maturation of cercariae of Schistosoma mansoni in mice. II. Parasite-related conditions. Exp. Parasitol. 22: 73-95.

Styczynska-Jurewicz, E. 1962. The life cycle of Plagiorchis elegans and the revision of the genus Plagiorchis Luhe, 1889. Acta Parasitol. Polon. 12: 419-445.

Swennen, C. 1969. Crawling-tracks of trematode infected Macoma balthica (L.l. Neth. J. Sea Res. 4: 376-379.

Théron. A. i984. Early and late shedding patterns of Schistosoma mansoni cercariae: Ecological significance in transmission ta human and murine hosts J. Parasitol. 70: 652-655. 75 • Théron, A. 1985. Polymorphisme du rythme d'émission des cercaires de Schistosoma mansoni et ses relations avec l'écologie de la transmission du parasite. Vie et Milieu 35: 23-31.

Théron, A. 1989. Hybrids between Schistosoma mansoni and S. rodhaini: characterization by cercarial emergence rhythms. Parasitoiogy 99: 225-228.

Théron, A. and C. Combes. 1983. Genetic analysis of cercarial emergence rhythms of Schistosoma mansoni. Behav. Genet. 18: 201-209.

Wagenbach, G.E. and A.L. Alldredge. 1974. Effect oflight on the emergence pattern ofPlagiorchis micracanthos cercariae from Stagnicola exilis. J. Parasitol. ':l0: 782-785.

• Webber, R.A., M.E. Rau, and D.J. Lewis. 1986. The effects ofvarious light regimens on the emergence ofPlagiorchis noblei cercariae from the molluscan intermediate host, Stagnicola elodes. J. Parasitol. 72: 703-705.

Williams, R.R. 1963. Life history studies on four digenetic trematodes that utilize Lymnaea (Stagnicola) refZexa (Say) as their first intermediate host in a temporary pond habitat. PhD thesis, Ohio State University. • 76 CONNECTING STATEMENT 1

The cercariae of Plagiorchis elegans represent a dispersal stage of the

parasite. It is their role to find and encyst as metacercariae within the

tissues of a suitable second intermediate hosto In Chapter 2 we showed that

the emergence of P. elegans cercariae coincides with a behavioural change in

the molluscan intermediate hosto Ultimately this behaviour, coupled with a

delay in reaching maximum infectivity, results in the dispersal of cercariae

over a larger area. This dispersal phase reduces the chance of superinfection

and parasite induced mortality of second intermediate hosts. Following

penetration of a suitable host the cercariae transform from the active, free

living stage into the relatively immobile metacercariae. The following study

addresses the activities and development of P. elegans metacercariae within

an insect second intermediate host, Aedes aegypti, and the conditions

required for excystment of infective metacercariae. • 77

CHAPTER3

PLAGIORCHIS ELEGANS: REQUIREMENTS FOR

METACERCARIAL DEVELOPMENT TO INFECTIVITY, AND

CONDITIONS REQUIRED FOR EXCYSTMEl\"'T.

• Carl A. Lowenberger and Manfred E. Rau.

lInstitute of Parasitology and 2Department of Entomology

Macdonald Campus of McGill University,

21,111 Lakeshore Rd, Ste-Anne-de-Bellevue

Quebec, Canada, H9X 3V9

J. Helminthol. Soc Wash. 60: 67-72 78 • ABSTRACT: The temperatures and times required for the development of Plagiorchis elegans (Digenea: ~JIagiorchiidae)

metacercariae from encystment to infectivity were studied in an

experimentai host, Aedes aegypti (Diptera: Culicidae). The ability of

mdacercariae to excyst was evaluated in vitro and was equated with

infectivity to the definit:ve hosto Development of metacercariae to

infectivity followed a sigmoidai curve at temperatures between 15 and

SOC. Rates of development increased significantly with temperature.

At 15C metacercariae first excysted 72h post infection (PI); at SOC

this occurred as early as 12h PI. Excystment reached 80% after IS2h

at 15e, and 60h at SOC. A minimum of 8h of contact with the host • was required for subsequent development to infectivity in vitro. Excystment ofinfective metacercariae was temperature dependent.

Less than 6% of metacercariae excysted at :'>SOC, whereas ~80%

excysted at ~37C. Such temperature requirements may explain, in

part, why adult P. elegans is principally a parasite of homeotherrnic

animaIs.

KEYWORDS: Plagiorchis elegans, Digenea, Trematoda, metacercaria,

Aedes aegypti, excystment, in vitro. 79 • INTRODUCTION Cercariae of Plagiorchis elegans (Rudolphi, 1802) actively penetrate their

insect intermediate hosts and then undergo morphological and physiological

changes from free swimming cercariae to encysted, relatively inactive

metacercariae (Lackie 1975). A variety of environmental factors may affect

the subsequent development of the parasite towards infectivity to the

vertebrate definitive hosto Metacercariae must be adapted to survive

intermediate host defense mechanisms as weIl as the digestive system of

the definitive hosto In the intestinal tract of the dùfinitive host,

metacercariae must respond to one or many of a wide range of stimuli,

excyst, and attach to the gut lining (Lackie 1975). This requires that the • parasite has reached a stage of development that allows it to receive and respond quickly to such stimuli.

The present study determined the temperature and time requirements for

development of P. elegans metaceruriae to infectivity in an experimental

insect host, Aedes aegypti (L.) and also determined the conditions and

temperature requirements for excystment of infective metacercariae in vitro.

Plagiorchis elegans

The behaviour and development of the various stages of P. elegans have • been described by Macy (1960), Blankespoor (1977), and Genov and 80 • Samnaliev (1984). Cercariae of the genus Plagiorchis are released from the molluscan first intermediate hosts, and penetrate a number of orders of

aquatic insects as weIl as crustaceans (Williams 1963). Cercariae attach to

the host cuticle and penetrate by means of a stylet and histolytic enzymes

(Bock 1984, Taft 1990), and then encyst as metacercariae in the haemocoel.

The time required for metacercariae to reach infectivity is reported as 4-5 d

for P. noblei (Blankespoor 1974) and 3 d for P. elegans (Genov and

Samnaliev 1984). Metacercariae excyst and transform into adults in the

intestine of the definitive hosto • MATERIALS AND METHOnS Groups of 200 fourth instar A. aegypti were maintained in plastic containers

containinr 300 ml tap water and fed ground Tetramin® fish fO'Jd ad

libitum. A 16:8 light:dark regime was maintained in each temperature­

regulated incubator. Infected snails, Stagnicola elodes (Say), were placed in

the dark to induce cercarial emergence. Eight hours later approximately

1000 cercariae were introduced to each container of mosquitoes which were

maintained at 15, 20, 25, 30, or 35C. Twenty minutes post exposure the

mosquitoes were transferred to containers of clean water to prevent

subsequent infection and maintained at the respective temperatures. 81 • At 12h intervals post exposure, 20 metacercariae were dissected from each group of larvae, placed in 20ml of an artificial excystment medium, MBEM

(0.015M NaHC03 , 0.015M NaCl, 0.5g/l Bile salts [50:50 Na cholate:Na

deoxycholate] (Sigma B-8756), pH 7.5), modified after Bock (1986), and

incubated at 37C. The number of successfully excysted metacercariae was

recorded 2h following incubation. This was repeated 5 times at each

temperature used to rear larvae. For the purpose of this study, a successful

excystment was considered to produce a mobile juvenile digenean. Those

partially emerged or still bound to the cyst wall were considered non-viable.

In order to test the in vivo contribution of the host to the development of • the parasite, mosquitoes were infected as above and maintained at 20, 25, and 30C. Metacercariae were dissected from mosquitoes at 2h intervals post

infection for the first 12h, and subsequently at 12h intervals. Groups of 20

such metacercariae were transferred to Dulbecco'e Phosphate-buffered

saline

Canada) and maintained at the same temperature as the mosquito host

from which they had been removed. The PBS was replaced daily. This

procedure was repeated 5 times at each temperature. At the time when in

lIivo incubation at the particular temperature would consistently provide

>80% excystment of metacercariae in vitro (as determined by the first

experiment) metacercariae were transferred from PBS to MBEM at 37C. 82 • AlI metacercariae maintained at the same temperature were excysted at the same age post infection. The number of rnetacercariae successfully ex::ysting

within 2h following the transfer was recorded.

To test the range of temperatures which will induce excystment of

metacercariae, mosquitoes were infected as above and the metacercariae

allowed 6 days at 20C to reach infectivity. Infective meta.::ercr.riae were

removed from the hosts and were placed in groups of 20, in MBE~.I at

temperatures ranging from 15-45C. The number of successfulexcystments

was recorded at 15 minute intervals for 2h. This was repeated 5 times at • each temperature. The effect of temperature alone on excystment was studied by placing

groups of 20 metacercariae in PBS or RPMI medium at 37C for 2h.

Unexcysted metacercariae were subsequently placed in MBEM at 20C for

2h. Those which still had not excysted were then incubated in MBEM at

37C for 2h.

Statistical analysis was done using SYSTAT 5.0 software using the Mann­

Whitney U-test, Tukey's multiple comparison tests, and Student's t-test.

The level of significance (x) was set at 0.05. S3 • RESULTS The effect of temperature on metacercarial development within the insect

host was significant. There was an inverse relationship between

temperature and time required to reach infectivity. The first successful

excystment at 15e occurred after 72h, compared to 36, 24, and 12h at 20,

25, and 30e, respectively. Levels of excystment reached SO % after 132, lOS,

60, and 60h at 15, 20, 25, and 30C, respectively. The development of

metacercariae at each temperature, as measured by the ability to excyst in

MBEM, followed a sigmoidal curve (Fig 3.1).

Metacercariae which received ~Sh of host contact showed no significant • diff~rence in their rate of development to infectivity compared with metacercariae maintained entirely in the insect hosts. However, regardless

of temperature, a minimum of Sh of host-parasite contact was required to

ensure development to SO% excystment levels (Fig 3.2). Sorne metacercariae

with fewer than Sh of host contact did excyst successfully, but levels of

excystment,were significantly lower (Mann-Whitney U-test, P<0.05). Host­

parasite contact in excess of Sh did not significantly increase levels of

excystment (Mann-Whitney U-test P>O.05).

The excystment ofinfective metacercariae was temperature dependent. Less

than 6 % of the metacercariae excysted at temperatures ~OC. There was no 84 • significant difference (Tukey HSD P>O.OS) between levels of excystment at temperatures ~37C (Fig 3.3). Levels of excystment at 3SC were intermediate

and significantly different from those at ::;30 and ~37C (Tukey HSD P

(Fig 3.3).

Elevated temperatures alone were insufficient in inducing excystment.

When infective metacercariae were placed in PBS or RPMI at temperatures

between 37 and 40C,

metacercariae failed to excyst when transferred to MBEM at 20C. However,

subsequent transfer to MBEM at 37C, resulted in >80% successful

excystment within 2h (Table 3.1). Significant differences (P

DISCUSSION

Whether metacercarial development is directly affected by temperature or is

mediated through the physiology of the insect host was not addressed in . this study. Reduced temperatures in the presence of an adequate food

supply slow the development of mosquito larvae (Christophers 1960), and

thus may also affect developing parasites. The excystment curves which

indicate development of metacercariae in mosquitoes were sigmoidal and 85 • paraIlel at the variou~ temperatures (Fig 3.1), differing only in the "Iag phase".

The contribution of the host per se to metacercarial development is

unknown. After only 8h within the host, metacerca.riae are able to develop

to infectivity in non-nutritive PB8. While c'verall development of

metacercariae is temperature dependent (Fig 3.1) there is a tempcrature

independent phase of metacercarial development; the first 8h of host contact

are requiréd by ail metacercariae independent of ambient temperature (Fig

3.2). There are several explanations for this, ail related to the uptake of

essential nm.aents from the host; 1) The metacercariae may have exhausted • reserves during penetration and encystment and may require 8h to restore nutrient levels required for subsequent development. 2) Uptake of specifie

compormde. may require 8h to be completed, or metacercariae may take up

nutrients only foIlowing the completion ofcyst wall formation. 3) The

deposition of cyst walllayers, of either host or parasite origin, may impede

the rapid influx ofnutrients which may impair parasite development. 4)

Encapsulation and melanization of the cyst by the insect host may interfere

with nutrient uptake. AlI of the above suggest a nutrient dependency on the

host during the first 8h foIlowing encystment. 86 Sorne successful excystment occurs when cysts are removed from tue insects

earlier than 8h PI. However, excystment of these metacercariae is

characteristicail.v "r.d sigllificantly less than thoSE! which have received >8h

of host contal~t Œ'ig 3.2). Smyth and Halton (1983) stat·) that metacer~ariae

can absorb nutrients during the process of growth and differentiation. What

nutrients cross the cyst wall, either actively or passively, is not known. The

completion of the 2-layered cyst wall may limit the influx of some

compoundc and reduce uptake (Bock 1988), and Taft (1990) suggests the

third cyst layer of hast origin may have a similar effect. The speed with

which these layers, parttcularly the third, l:ll"e deposited may vary with the

location of the cyst and the occurrence of other host ml'\diatecl factors (Taft • 1990). Where the deplJsition of this third layer of cyst wall is delayed or absent, metacercariae may obtain nutrients more effectively and may reach

infectivity with <8h ofhost contact.

Within the range of temperature~ used in these experiments, metacercariae

showed a distinct pattern of development, the most visible of which was an

increase in the size and optical density of the excretory vesicle. The cyst

walls ml'lY restrict the elimination of excretory products. Alternativdy, by

retaining these products, the parasite may avoid stimulating host defens6

mechanisms. The size and conspicuousness of this vesicle can be used as an • indicator of the age and infectivity of metacercariae. 87 • Excystment c: metacercariae may be passile or active, and parasites which are ingested by their hosts as inactive stages may play an active role in

their own excystment (Lackie 1975). The metacercariae of Plagiorchis excyst

intrinsically. Bock (1986, 1985)wund that metacercariae of Plagiorchis spec.

1 became very active when exposed to an artificial excystment medium. The

juveniles egested stored caecal fluid against the inner walls of the cyst and

;:1.~tively emerged through this area. This "explosive expulsion" ofjuveniles

has been reported by other authors (Howell 1970, Bass and LeFlore 1984).

The internaI pr'lssure of the metacercariae aids in rapid excystment and

allows early attachment by the parasite to the intestine (Bock 1989). Bile

and bile salts (a component ofMBEM) stimulate muscular activity in a • number of species (Lac!.cie 1975). This implies that the metacercariae are "mature", and are ready to contribute actively to their liberat.ion under

app~opriate conditions. 30me metacercariae excyst to produce immobile,

non-viable juveniles that differ morphologically from normal individudE.

This occurs most commonly at the time when metacercariae normally

approach infectivity, and is characterized by a poorly developed excretory

vesicle. Although such metacercariae may not have developed fully they

nevertheless respond to the excystment stimulus. Thus, the ability to excyst

may precede the ability to survive in a post-excystment environment.

Although we did not determine the infecêivity cf the excysted juveniles we • equated infectivity with the ability of metacercariae to excyst in vitro. 88 • In many digenean species excystment is temperature dependent and may be inhibited by incubation at inappropriate temperatures (Dixon 1966).

Similarly, P. elegans metacercariae showed essentially no excystment in

MBEM at temperatures <35C. However, when transferred ta MBEM at

37C, >80% of the wonns excysted within 2h. High temperature alone did

not induce excystment. There were significant differences in the levels of

excystment in those groups which included exposure ta MBEM at 37C and

aIl other groups (Table 3.1). A suitable temperature and medium must be

present simultaneously to ensure significant levels of excystment. Threshold

temperatures for excystment exist for many digeneans, and metacercariae

will not excyst in vitro unless the temperature approximates that of the • definitive host (Thompson and Halton 1982, Asanji and Williams 1975). Temperature differences of as little as 4C may influence excystment levels

(Fried and Huffman 1982).

Bock (1986) concluded that while pre-treatment by passage through the

stomach was not required for excystment, contact with gastric juices

enhanced the effects of bile. Since Plagiorchis spec. 1. excyst at

temperatures as low as 21C, Bock (1986) chose the speed of excystment as a

criterion of successful excystment. In contrast, P. elegans metacercariae did

not excyst at temperatures <30C. 89 • SUMMARY The present study defines the temperature-time relationship for the

development of metacercariae of P. elegans to infectivity within an insect

intermediate hosto We have shown a temperature- independent obligatory

period of host-parasite contact followed by a temperature-dependent

development period to obtain infective metacercariae. The physiological

interactions between intermediate host and parasite, including nutrient

uptake, remain unclear. Active excystment ofinfective metacercariae

requires both appropriate temperatures (~37C) and an activating stimulus. • ACKNOWLEDGEMENTS The authors thank K. Keller for assistance in preparation of the artificial

excystment medium (MBEM) and anonymous reviewers for helpful

comments on an earlier version of the manuscript. Funding was provided Ly

the Natural Sciences and Research Council of Canada (NSERC) (OGP

0007606) to MER and the Fonds pour la formation de Chercheurs et l'aide à

la recherche (FCAR) (ER 0129) to MER. Personal funding for CAL was

provided by a Walter M. Stewart Award and a Lynden Laird Lyster Award.

Research at the Institute of Parasitology of McGill University is supported

by NSERC and FCAR. • 90 Figure 3.1. In vitro excystment of Plagiorchis elegans metacercariae

removed from the insect host at various times post infection. Data are

presented as mean % excystment ± Standard Error.

•

• e • •

100 ~i-[] ~ 80 R t ~ ./ Q) El 60 ... L-Y- / ~ en e 15 C 1>- 1 : v 20 C a 40 / ...... 25 C H [] 30 C 1":1:1 /e [] ~ 10 /. o o 24 48 72 96 120 144 168 Time (hours) post infection • 92

Figure 3.2. Excystment of Plagiorchis elegans metacercariae removed from

the mosquito host at various times post infection and maintained in

Phosphate-buffered saline (PBS) until they reached the age required for

80% excystment as determined from Fig 1. Data are presented as mean %

excystment ± Standard Error. • • • •

100

~ 80 ~ Q) El 60 ~ v v 20C rn • 25C ~ 40 • 0 0 o SOC H P:.1 20 ~ O l , l , , , i ~ " , , o 2 4'.6 8 10 12"24 48 72 Tlme (hours PI) when transferred to PDS • 94

Figure 3.3. Excystment of infective Plagiorchis elegans metacercariae

fol1owing 2h incubation in excystment medium at various temperatures.

Data are presented as mean % excystment ± Standard Error.

•

• • 00000000 11001100110 .... 0110 .... (I4(14l1)lI)lI) ...... I>IJ~~.~.O

I>IJ~~.~.O

0 • ....(14 \ 1 1 • ..-.. rn \\ \ oG) ..... I.t .~ e\ ~ \ s:= Ilit 1 • 1 .l'''t • ::&1 '-" \\ 0 • 1 • 1 IDG) 8 \ \.IH \ ~e;.. ,,\ 1 e\ 0 ~~ li)

0 0 0 0 0 0 ....0 ID ID .... "(14 • . l ueWlS,(OX3 % e • • 96

Sequence of % Sequence of % Sequence of %

Conditions Excystment Conditions Excystment Conditions Excystment

PBS 37C 2.0 ± 1.2 RPMI37C 1.0 :!: 1.0 ,J, ,J,

MBEM 20C 2.0 ± 1.2 MBEM20C 1.6 ± 0.3 MBEM 20C 1.2 ± 0.2 ,J, ,J, ,J,

MBEM 37C 84.0 ± 2.9 MBEM37C 85.2 % 2.1 MBEM 37C 86.2 ± 4.1

Table 3.1. Excystment ofinfective metacercariae ofPlagiorchis elegans in various media and temperatures. Metacercariae were exposed to each medium for 2 h prior to transfer to the next medium in each sequence. Excystment recorded is the cumulative mean % excystment ± standard error. In each column (sequence) only combinations ofboth 37C and the artificial excystment medium (MBEM) yielded significant levels of excystment. Elevated temperatures alone or MBEM at low temperatures failed to elicit excystment. 97 • LITERATURE CITED

Asanji, M.F. and M.O. Williams. 1975. Studies on the excystment of trematode metacercariae in yitro. Z. Parasitenkd. 47: 151-163.

Bass, H.S. and W.B. LeFlore. 1984. In vitro excystment of the metacercariae ofAcanthoparyphium spinulosum (Trcmatoda: ·Echinostomatidae). Proc. Helminthol. Soc. Wash. 51: 149-153.

Blankespoor, H.D. 1974. Host-induced variation in Plagiorchis noblei Park, 1936 (Plagiorchiidae: Trematoda). Am. Mid. Nat. 92: 415-433.

Blankespoor, RD.. 1977. Notes on the biology of Plagiorchis noblei Park, 1936 (Trematoda: Plagiorchiidae). Proc. Helminthol. Soc. • Wash. 44: 44-50.

Bock, D. 1984. The life cycle of Plagiorchis spec. 1, a species of the Plagiorchi.5 elegans group (Trematoda, Plagiorchiidae). Z. Parasitenkd. 70: 359-373.

Bock, D. 1986. ln vitro excystment of the metacercariae of Plagiorchis species 1 (Trematoda, Plagiorchiidae). Int. J. ParasitoI. 16: 641­ 645.

Bock, D. 1988. Formation, histochemistry, and ultrastructure of the metacercarial cyst wall ofPlagiorchis species 1 (Trematoda, Plagiorchiidae). Int. J. Parasitol. 18: 379-388. 98 • Bock, D. 1989. Hatching mechanism of the metacercaria of Plagiorchis species 1 (Trematoda: Plagiorchiidae). J. Helmi-::thol. 63: 153-171.

Christophers, S.R. 1960. Aedes aegypti, The Yellow Fever Mosquito, its Life History, Bionomics, and Structure. Cambridge University Press, London. 739 pp.

Dixon, K.E. 1966. The physiology of excystment of the metacercariae of Fasciola hepatica L. Parasitology 56: 431-456.

Fried, B. and J.E. Huffman. 1982. Excystation and development in the chick and on the chick chorioallantois of the metacercaria of Sphaeridiotrema globulus (Trematoda). lnt. J. Parasito!. 12: • 427-431. Genov, T. and P. Samnaliev. 1984. Bi.ology, morphology and taxonomy of Plagiorchis elegans (Rudolphi, 1802) (Plagiorchidae) in Bulgaria. pp. 75-114 in Fauna, taksonomiya i ekologiya na khelminti poptitsi. 1. Vasilev, ed. Bulgarian Academy of Science, Sofia, Bulgaria.

Howell, M.J. 1970. Excystment of the metacercariae of Echinoparyphium serratum (Trematoda: Echinostomatidae). J. Helmintho!. 44: 35-56.

Lackie, A.M. 1975. The activation of infective stages of endoparasites of vertebrates. Bio!. Rev. 50: 285-323. 99 • Macy, RW. 1960. The life cycle of Plagiorchis vespertilionis parorchis, N. ssp. (Trematoda: Plagiorchiidae), and observations on the effects of light on the emergence of the cercariae. J. ParasitoI. 46: 337-345.

Smyth, J.D. and D.W. Halton. 1983. The Physiology of Trematodes. Cambridge University Press, Cambridge. 446 pp.

Taft, S.J. 1990. Cinephotomicrographic and histochemical observations on cercarial penetration and encystment by Plagiorchis sp. in larvae of Chaoborus sp. Trans. Am. Microsc. Soc. 109: 160-167.

Thompson, M. and D.W. Halton. 1982. Observations on excystment in vitro of Cotylurus variegatus metacercariae (Trematoda: • Strigeidae). Z. Parasitenkd. 68: 201-209.

Williams, RR 1963. Life history studies on four digenetic trematodes that utilize Lymnaea (Stagnicola) reflexa (Say) as their first intermediate host in a temporary pond habitat. PhD. Thesis, Ohio State University. 96 pp. • 100 CONNECTING STATEMENT 2

In Chapter 3 we showed that the overall development ofPlagiorchis elegans,

from cercariae to infective metacercariae, was temperature dependent.

However, there also was a temperature independent, 8 hour obligatory

period of parasite-host contact. We hypothesized in Chapter 3 that this 8h

period was one in which the young, recently encysted metacercariae

replenished reserves spent during the cercaria1 stage, or took up nutrients

from the insect host that were essentia1 for their ensuing development. In • the following study we address this issue by mea,;uring the metabolic activities of P. elegans metacercariae of different ages post infection as

defined by their ability to take up and incorporate the sugar and protein

precursors, 3H-glucosamine and 3H-leucine, respectively into parasite

tissues. In addition we eva1uate differential incorporation of these

compounds into parasite tissues and the distribution of3H-glucosamine into

parasite glycoproteins by metacercariae of different ages. • 101

CHAP~ER4

IN VITRO UPTAKE AND INCORPORATION OF 3H-GLUCOSAMINE

AND 3H-LEUCINE INTO METACERCARIAL PROTEINS BY

PLAGIORCHIS ELEGANS.

• Carl Lowenberger', Manfred Rau2, and Kris Chadee'

lInstitute of Parasitology and 2Department of Entomology

Macdonald Campus of McGill University,

21,111 Lakeshore Rd, Ste·Anne-de-Bellevue

Quebec, Canada, H9X 3V9

A revision of this chapter will be submitted to • The Journal of Parasitology 102 • AB8TRACT: Successful transmission of Plagiorchis elegans cercariae occurs when the active, free-living cercariae find and enter a suitable host

and transform into metacercariae. Such newly-formed rneta.:ercariae then

undergo physical and physiological changes to become infective to the

definitive host. This study examines the metabolic requirements of P.

elegans metacercariae at various times in their development by examining

their ability ta take up 3R-glucosamine and 3R-Ieucine in vitro, and to

incorporate these precursors into parasite proteins. Uptake of both

precursors was greater by young metacercariae (::;8h post infection (PI))

than old metacercariae (>8h Pl); approximately 73% of 3R-glucosamine and

70% of 3R-leucine were taken up by young metacercariae. There was a • marked difference in both the total uptake and pattern of uptake of these componnds. Virtually aIl 3R-glucosamine was incorporated into the juvenile

worms whereas 3R-Ieucine was partitioned in small amounts into both

juvenile worms and cyst walls. 3R-glucosamine was incorporated into a

variety of glycoproteins by 2h- and 5d- old metacercariae as determined by

SDS-PAGE and fluorography. There was evidence of deveIopmentally

regulated or stage specific expression of 14, 19.5 and 37kDa proteins by

older metacercariae. These results suggest a differential incorporation of

nutrients procured from the insect host inta parasite tissues by

metacercariae of different ages over the course of their development towards

infectivity. 103 • INTRODUCTION

The exploitation of a diversity of hosts and habitats has required distinct

adaptations to available food resources on the part of the Digenea (Ralton

1967). Metacercariae of many digeneans are able to absorb a wide range of

nutrients across their external surfaces (Uglem et al. 1985, Uglem and

Larsen 1987, Uglem et al. 1991). Such nutrient absorption may serve to

replenish reserves spent during the cercarial stage or to obtain specific

nutrients required for their development to infectivity and adulthood

(8myth and Ralton 1983).

• Cercariae of Plagiorchis elegans (Rudolphi) actively penetrate their insect intermediate hosts and then undergo morphological and physiological

changes from free-living, non-feeding cercariae to encysted, relatively

immobile metacercariae (Styczynska-Jurewicz 1962, Genov and Samnaliev

1984, Webber et al. 1986). Lowenberger and Rau (1993) have shown that

overall development ofP. elegans from the penetrating cercariac to infective

metacercariae in larvae of the experimental insect intermediate host Aedes

aegypti (L.) was temperature dependent. Rowever, there was also an initial

phase of metacercarial development that was temperature independent.

Metacercariae required a minimum of 8h in the insect host regardless of

temperature, following which development in vitro was directly temperature 104 • dependent. While little is known of the metabolic or nutritional requirements of P. elegans metacercariae, the authors suggested that the 8h

period of obligatory host-parasite contact may be used by the parasite to

procure essential nutrients for subsequent development or to rcplenish

nutrient reserves exhausted during the cercarial stage.

In order to test this hypothesis we evaluated the metabolic activity of P.

elegans metacercariae as defined by their ability to take up and incorporate

the sugar and protein precursors, 3H-glucosamine and 3H-Ieucine,

respectively. Differential uptake of the labels was compared between

metacercariae of different ages incubated with each label for the same • period, and between metacercariae of the same age incubated with each label for different periods. The site of label incorporation was assessed by

excysting metacercariae in vitro and determining proportional incorporation

of each label into the juvenile worm or cyst wall. DifferentiaI incorporation

of 3H-glucosamine into proteins by metacercariae of different ages was

determined by sodium dodecyl sulphate poly-acrylaiIDde gel electrophoresis

(SDS-PAGE). We have demonstrated that metacercariae of ail ages are

metabolically active, but that activity, as determined by 3H_ uptake and

incorporation, diminishes with age. While the majority of each 3H-Iabelled

compound was taken up by metacercariae ~h post infection (PI) the uptake • profile of the two 3H-Iabels was different, and the two compounds were 105 • partitioned distinctly into the juvenile worms and the cyst walls. 3H_ glucosamine was incorporated into a range of proteins in both 2h- and 5d­

old metacercariae and there was evidence of stage specific or

developmentally regulated protein expression by metacercariae of difTerent

ages. Metacercariae that were removed from the insect host 2h post

infection and incubated with 3H-glucosamine or 3H-Ieucine for 24h were 26h

old when evaluated for the uptake of the precursors. We have continued to

refer to the metacercariae by the age at which they were removed from the

insect host as this was the age when they were first exposed to the

precursors and could begin taking up and incorporating them into parasite

tissues. DifferentiaI uptake by metacercariae of different ages should reflect, • in large measure, the age at which they were exposed to the precursors.

MATERIALS AND METHOnS

3H-GLUCOSAMINE AND 3H-LEUCINE UPTAKE BY

METACERCARIAE

Infected snails

induce the emergence of P. elegans cercariae. At 07:00 groups of 200 fourth

instar A. aegypti mosquito larvae were exposed to batches of 1000 cercariae.

Following an exposure period of 15 minutes the larvae were isolated from • the cercariae, rinsed to remove adhering cercariae, and maintained at 27C. 106 • At intervals of 2, 4, 6, 8, 10, 12, 24, and 48h PI metacerca1Ïae were dissected free from larval tissues and washed in Dulbecco's Phosphate­

buffered saline

Ontario, Canada). Fifty metacercariae in l.0m! PBS were placed in each

weIl of a 24 weIl plate (Falcon, Becton Dickson, New Jersey, USA). One pCi

of either 3H-glucosamine (Specifie activity 40 Cilmmol; ICN Biomedicals

Inc, Irvine California) or 3H-Ieucine (Specifie activity 54 Cilmmol; ICN

Biomedicals Inc, Irvine California) was added to each weIl, and

metacercariae were incubated for 24h at 25C. Metacercariae fixed in 10%

glutaraldehyde (Sigma Chemical Co., St. Louis, MO, USA) were treated

similarly to assess non-specifie binding of 3H-labels. Following incubation,

metacercariae were washed three times with PBS, transferred to a

scintillation vial to which 5ml scintillation cocktail (ICN Biomedicals Inc,

Irvine California) were added, and counted in a beta counter. In a parallel

experiment, metacercariae of the same age PI were treated as above but

were incubated with either label for difI'erent periods from 2-12h, and the

uptake quantified as described above.

3H· DISTRmUTION IN METACERCARIAE

In order to determine the distribution of the label in the parasites, 5d-old

metacercariae were removed from the insect host and incubated for 24h

with either label. Following incubation, metacercariae were excysted in vitro 107 • for 2h at 37C in 20 m! of an artificial excystment medium (MBEM: 0.015 M NaHC03, 0.015 M NaCI, 0.5 g/l bile salts [50:50 Na cholate:Na deoxycholateJ, pH 7.5) modified after Bock (1986). Groups of 50 excysted

juvenile worms and their empty cysts were washed with PBS and evaluated

separately for the distribution of 3H-glucosamine and 3H-Ieucine to

determine the perc.entage of activity in each structure. These values were

compared with metacercariae not exposed to MBEM and metacercariae

which had been fixed for 1h in 10% glutaraldehyde to measure non-specifie

binding ta the cyst wall.

SDS-PAGE AND FLUOROGRAPHY • SDS-PAGE was used ta determine if metacercariae of different ages incorporated 3H-glucosamine differently into parasite proteins.

Metacercariae (2h and 5d PI) were removed from the larval tissues and

incubated with 3H-glucosamine for 24h as previously described. The

metacercariae were subsequently washed three times with PBS and boiled

for 5 minutes in sample buffer (4.0ml distilled water, l.0m! 0.5 M Tris (pH

6.8), 0.8m! glycerol, 1.6ml 10% SDS, OAml 2-~ mercaptoethanoD with

bromophenol blue ta mark the dye front. Proteins from 200 2h- and 5d-old

metacercariae, and equal aliqots of 3H- activity (CPM) from 2h- and 5d-old

metacercariae, were separated by (SDS-PAGE) with a 4% stacking gel and a

12% running gel under reducing conditions. Samples were electrophoresed 108 • at 20 mA/gel for 3.Sh, stained with Coomassie-blue, destained (200ml methanol, SOml acetone, 2S0ml distilled water), treated with fluorenhancer

(En3hance, Dupont, Boston Massachusetts), and exposed to X-OMAT AR

film (Kodak, Rochester, New York) for 28 days at -70C.

COLUMN CHROMATOGRAPHY

3H-glucosamine labelled metacercariae (2h and Sd PI) were incubated and

washed as described above. The metacercariae were solubilized in 8D8­

PAGE sample buffer for 10 minutes by agitation. Equal aliquots of 3H

activity (1S,OOO CPM) for each age of metacercariae were applied to a

8ephacryl 8-200 column (1.0 x 30 cm; Bio-Rad Laboratories, Richmond • California) previously equilibrated in O.OlM Tris-HCI buffer (pH 8.0). The column flow rate was 7 ml/h and O.S ml fractions were collected. Five ml of

scintillation fluid (lCN Biomedicals Inc.) were added to each fraction and

the 3H activity was determined for the whole elution profile. The column

was calibrated with blue dextran (BD,>2 x 1Q6 Mol.wt.), bovine serum

albumin (B8A; 67,000), chymotrypsinogen A (CTA; 2S,000) and ribonuclease

A (RA; 13,000) as molecular weight standards (Pharmacia, 8weden). 109 • RESULTS

UPTAKE OF sH-GLUCOSAMINE AND sH-LEUCINE BY P. ELEGANS

METACERCARIAE

The overall uptake ofSH-glucosamine was greater than that of3H-leucine.

There also were marked differences in the uptake profile between sH_

glucosamine and sH-leucine (Fig 4.1). The youngest metacercariae took up

the greatest amount ofSH-glucosamine followed by a steady decline in

uptake with age. In contrast, sH-Ieucine uptake was low in the youngest

metacercariae, peaked in metacercariae 6h old, and declined steadily

thereafter (Fig 4.1). Significantly more of each label was taken up by the • youngest metacercariae; 73.4% of the total SH-glucosamine uptake and 70.2% of the sH-leucine was taken up by metacercariae S;8h PI (Fig 4.2).

There was an age-dependent, differential uptake ofboth SH-glucosamine

and sH-Ieucine by metacercariae of the same age incubated with the labels

for different periods. The cumulative uptake of SH-glucosamine (Fig 4.3) was

greatest overall among the youngest (2-4h PI) metacercariae. Within these

groups, maximum uptake has occurred by 8-10h following incubation with

SH-glucosamine. While oIder metacercariae continued to take up sH_

glucosamine they did so in decreasing amounts, and the cumulative uptake

declined sooner and at lower levl1ls than that of younger metacercariae. The 110 • greatest amount of 3H-leucine was incorporated by Gh-old metacercariae incubated for G-8h with the label. Metacercariae incubated for <4h or >8h

showed similar levels and pattern of uptake of 3H-Ieucine (Fig 4.4).

PARTITIONING OF THE 3H-LABELS BY METACERCARIAE

3H-glucosamine and 3H-Ieucine were incorporated differently into juvenile

worms and cyst wans. 3H-glucosamine was found predominantly in the

juvenile with minimal incorporation into the cyst wans (Fig 4.5). Nonspecific

binding of the 3H-Iabels was negligible in metacercariae fixed with 10%

glutaraldehyde. The SUffi of3H-glucosamine found in cyst wans and juvenile

worms approximated levels found in non-excysted metacercariae, indicating

that almost all 3H-glucosamine taken up by metacercariae was incorporated

into the juvenile worms. In contrast, 3H·leucine was found in small amounts

in both the juvenile worms and the cyst wans. However, the combined

amounts found in the cyst wans and juvenile worms did not approach levels

found in non-excysted metacercariae (Fig 4.5).

SUBCELLULAR DISTRIBUTION OF 3H-GLUCOSAMINE INTO

GLYCOPROTEINS

Fluorography ofSnS-PAGE demonstrated the incorporation of3H­

glucosamine into a variety of proteins 12-200kDa by both 2h- and 5d-old

metacercariae. 3H-glucosamine was incorporated into specifie major proteins 111 • of 37, 19.5, and 14kDa by 5d-old metacercariae that were either absent or found in reduced amounts in 2h-old metacercariae (Fig 4.6). This

differential incorporation was shown when SDS-PAGE and fluorography

were used to compare similar numbers of each age of metacercariae or equal

aliquots of 3H_ activity (Fig 4.6). Quantification of the 3H-glycoproteins by

Sephacryl S-200 column chromatography (Fig 4.7) revealed major proteins

of 60 and 18.7kDa in 2h-old metacercariae, and a major protein of 14.5kDa

in 5d-old metacercariae.

DISCUSSION

• Plagiorchis elegans metacercariae, from 2-48h PI, were metabolically active and incorporated both 3H.glucosamine and 3H-leucine into parasite proteins.

It is likely that nutrient uptake in the manner shown here supplies young

metacercariae with essential nutrients required for the transformation from

non-feeding cercariae to infective metacercariae. Metacercariae can be

induced to encyst in vitro (Lowenberger, unpublished data) but will not

develop to infectivity in non-nutritive media. While metacercariae of all ages

were active metabolically, younger metacercariae (:58h Pl) took up the

greatest amount of both 3H-glucosamine and 3H-leucine (Fig 4.2). This

supports the hypothesis ofLowenberger and Rau (1993) that the 8h period

of obligatory host-parasite contact was one of nutrient uptake from the host, 112 • although the mechanism of tlùs uptake has not been studied. Metacercariae of other Digenea take up nutrients through either facilitated difib.sion or

active transport (Uglem and Larsen 1987, Uglem et al.1985). The

mechanism of uptake by different species may be related to the niche tha:t

the adult fluke inhabits (Whitfield 1979). While facilitated diffusion is

common in adult parasites that inhabit the gut of the host (such as P.

elegans) and active transport is more common in tissue dwelling species

(Whitfield 1979, Uglem et al. 1985), some species such as Clinostomum

marginatum may employ both facilitated diffusion and active transport

(Uglem and Larsen 1987). Tlùs correlation between niche and mode of

transport also applies ta larval forms, and transport mechanisms may • change in different habitats. Active transport in Proterometra macrostoma is expressed by the intra-molluscan rediae while facilitated diffusion is

expressed by the cercariae (Uglem 1980).

The observation that more 3H-glucosamine than 3H-leucine was taken up by

metacercariae of aIl ages may represent a hierarchy of nutrient uptake;

compounds may be taken up only as required for development. 3H·

glucosamine may be readily used as an energy source by the developing

worm, hence its minimal incorporation into the cyst wall. Alternatively

some compounds may present more difficulties ta cross the cyst wall, or may • he required in minimal amounts. The majority of3H-glucosamine was found 113 • in juvenile worms which may indicate a requirement for 3H-glucosamine and its rapid incorporation into glycoproteins during the period of

transformation and development ofmetacercariae. The 3H-leucine profile,

however, was not as clear. While there was incorporation into the cyst wall

and juvenile worms, much of the label was not bound to parasite tissues.

Whereas the amount of 3H-glucosamine found in the cyst wall and juveniles

equalled the amounts found in non-excysted metacercariae this was not the

case with 3H-leucine.

Infective metacercariae are characterized by a large excretory vesicle

containing dark, globular inclusions. The size and conspicuousness of the • vesicle may be used as an indicator of the developmental stage (infectivity) of the parasite. Immediately follov.ing in vitro excystment, once the juvenile

has completely extricated itselffrom the cyst wall, the contents of the.·

excretory vesicle are voided. If3H-leucine had been free between the

juvenile worm and cyst wall, or incorporated into the contents of the

excretory vesicle, this would have been lost promptly following excystment.

A loss of3H-leucine in this manner would explain the differential recovery

of 3H-leucine between excysted and non-excysted metacercariae. Little is

known of the activities and function of the excretory vesicle. It may play a

role in osmoregulation, nutrient absorption, excretion (LeFlore 1978) and

phosphorylated transfer mechanisms (Halton 1967). The structure is 114 • metabolically active and has strong intrinsic dehydrogenase activity (LeFlore 1978). The excretion of 3H-leucine would also explain the

differential uptake of 3H-leucine compared to that of 3H-glucosamine; there

is no need to take up significant amounts ofa compound destined for

excretion. Perhaps 3H-Ieucine was taken up in abnormally high amounts not

because it was in great demand, but because it was the only compound

available to the metacercariae. Unlike 3H-glucosamine, 3H-leucine was not

readily metabolized as a food source and the excess was excreted.

Alternatively, the differential uptake of3H-glucosamine and 3H-leucine may

have been due to difficulties associated with the transport of compounds

across the cyst wall. The cyst wall of P. elegans metacercariae is deposited

in two layers. The completion of the second layer of parasite origin may

limit the influx of some compounds or reduce their uptake (Bock 1988).

Similarly, Taft (1990) suggests that the third cyst layer which is deposited

by the host may have a similar effect. The reaction of many insects to

invasion by parasites is to encapsulate and/ormelanize the invader .

(Christensen et al. 1984, Getz and Boman 1985, Getz 1986, Christensen

1986, Gupta 1986, Nappi and Christensen 1987, Christensen and Tracy

1989). Melanization may be apparent as early as 10 minutes following

invasion (personal observation). Metacercariae which are completely

surrounded by a melanin layer will not excyst in vitro (Lowenberger, • unpublished data). A rapid deposition of a melanin layer may retard 115 • metacercarial development by impeding the transport of essential nutrients across the cyst walls. The melanotic capsule also may reduce the ability of

the metacercariae to receive and respond to the stimuli for excystment.

The fluorographs of SDS-PAGE (Fig 4.6) showed an incorporation of :IH_

glucosamine into a range of proteins by both 2h- and 5d-old metacercariae,

indicating that, although already infective, the older metacercariae are still

active metabolically. Ofinterest was the incorporation of 3H-glucosamine

into specifie proteins by the older metacercariae that were not present in

younger metacercariae based on the assays used. This may be indicative of

the ongoing transformation from young to infective metacercariae and • developmentally regulated or age specifie proteins that are produced at particular stages in the process of differentiation. This pattern of agl'­

specifie incorporation of 3H-glucosamine into parasite glycoproteins was

demonst.rated when gel electrophoresis was used to compare similar

numbers ofmetacercariae or equal aliquots of 3H- activity from young and

old metacercariae. A similar distribution was resolved by gel filtration

chromatography under non-reducing conditions. The major 14kDa protein

found in 5d-olà metacercariae was c1early evident as compared to the 2h-old

metacercariae. However, due to the range of molecular weights (l3-25kDa)

found within fractions 38-41, it is difficult to distinguish clearly if the

changes in activity reflect the differential expression of the 14kDa protein. 116 • The data suggest that P. elegans metacercariae of different ages utilize compounds obtained from the host differently. Young metacercariae may

concentrate on the production of the cyst wall and initial differentiation

from the cercarial stage to the developing metacercariae. During

development towards infectivity, metacercariae may incorporate compounds

inta food reserves or structural components. The metacercariae of P. elegans

excyst intrinsically. Bock (1986, 1989) found that during the process of

excystment, the juveniles egested stored caecal fluid against the inner walls

of the cyst and actively emerged through this weakened area. These caecal

fluids may contain enzymes into which 3H-glucosamine and 3H-Ieucine had

been incorporated. Stage-specific proteins also could be incorporated inta • developing organ systems, into defense mechanisms involved in protection against host immunity, or inta specific parasite organs or tissues found only

in adults. While the specific location and function of the 14kDa protein was

not determined, it appears to be developmentally regulated at sorne stage

between 2h and 5d postinfection.

SUMMARY

Plagiorchis elegans metacercariae of aIl ages were metabolically active and

took up 3H-glucosamine and 3H-Ieucine in vitro. The former was taken up in

greatest amounts by the youn!lest metacercariae and was incorporated in • high proportions into the juvenile worms with minimal incorporation into 117 • the cyst wall. The latter was taken up in greatest amounts by metacercariae 6h PI and was incorporated into both the juvenile and the cyst wall. SDS·

PAGE showed differential incorporation of 3H.glucosamine into proteins by

2h· and 5d·old metacercariae whic1-.. may be stage specifie or

developmentally regulated.

ACKNOWLEDGEMENTS

Thê authors thank Kathy Keller for expert technical assistance. Funding

was provided by the Natural Sciences and Research Council of Canada • (NSERC) (OGP CÙ07606) to MER and (OGPIN 030) to KC and by the Fonds pour la formation de Chercheurs et l'aide à la recherche (FCAR) (ER 0129)

to MER. Personal funding for CAL was provided by a Walter M. Stewart

Award and a Lynden Laird Lyster Award. Research at the Institute of

Parasitology of McGill University is supported by NSERC and FCAR. • 118

Figure 4.1. 24 hour in vitro uptake of 3H-glucosamine and 3H-leucine

(+ 1 standard error) by Plagiorchis elegans metacercariae of different

ages post infection.

• • Q) CD -pot ... El= Ils Q) en r:I 0 _pot ..."l () () ::s ::s ."...... -t Q) ..c. ~ ...-t ,..."l~ 1 1 (1) 0 .0)=0)= .- L. ,...0 0 0 I~ L. (1) 0 CD 0 o+J (1) E CD \t-a

~ ... «Cl

e· o ~ 0 ~ 0 ~ 0 tt) C'I C'I -- (~O ~X) ~dO • 120 Figure 4.2. Proportional uptake of3H-glucosamine and 3H-leucine by

young (~8h) and old (>8h) Plagiorchis elegans metacercariae fol1owing

24h incubation.

• • 0) .-c: E o 0) rn c: 0._ o 0 :::J :::J ~ -0) CO .c 0)_ /\ '-" 1 1 ::c::c CI) ~C':) e .-L- e () I~ L- CI) () • e CO ...... CI) VI ~

'+- 0

CI) «Cl

o o o o o ....o CD ID .... • 122 Figure 4.3. Cumulative uptake of 3H-glucosamine by Plagiorchis

elegans metacercariae of different ages post infection incubated for

different periods. •

.2000 2h .2000 4h

1500 1800

1000 1000

500 500 0 .2 ... 8 8 10 1.2 0 .2 ... 8 8 10 1.2 .2000 eh .2000 8h

1500 1100 ::If e 114 U 1000 1000

500' 100 0 2 ... 8 8 10 1.2 0 2 ... 8 8 10 1.2 .2000 , 10h ' .2000 12h

1500 1100

1000 1000

100 100 0 .2 ... ,8 8 10 12 0 .2 ... 8 1 10 1.2 e Time of incubation (hours) • 124 Figure 4.4. Cumulative uptake of 3H-leucine by Plagiorchis elegans

metacercariae ofdifferent ages post infection incubated for different

periods.

• •

.2500 2h .2500 .2000 .2000 4h 1500 1500 1000 1000 500 500 0 0 0 .2 4 8 8 10 1.2 0 .2 4 e 8 10 1.2 .2500 .2500 eh 8h .2000 .2000 ::111 I:l.. 1500 1500 • t) 1000 1000 500 500 0 0 0 2 4 ft 8. 10 1.2 0 .2 4 8 8 10 1.2 .2500 .2500 10h 12h .2000 .2000 1500 1500 1000 1000 500 500 0 0 0 .2 4 e 8 10 1.2 0 .2 4 8 8 10 1.2 Time of incubation (hours) • 126 Figure 4.5. DifferentiaI incorporation of 3H-glucosamine and 3H_

leucine into Plagiorchis elegans juvenile worms and cyst walls. Five

day old P. elegans metacercariae were incubated for 24h with each

label, and then excysted in vitro. The proportion of 3H- incorporation

into juvenile worms, empty cysts, and glutaraldehyde-fixed

metacercariae were compared with total uptake (100%) by non­

excysted metacercariae. Virtually all the 3H-glucosamine (95.6%) was

incorporated into the juvenile worms whereas oruy 14.9% of 3H_

leucine was found injuvenile worms. More than 50% of the 3H_

leucine Îound in non-excysted metacercariae was not accounted for

following excystment and quantification of 3H- levels in cyst walls and

juvenile worms. • • •

Non- Excysted Metacercariae

Juvenile worms

Cyst Walls

Fixed Metacercariae ..­ 3H-glucosamine ~- 3H-leucine

o 10 20 30 .40 &0 80 70 80 80 100 ~ of Total Uptake 128 • Figure 4.6. Fluorograph ofSDS-PAGE (4% stacking gel; 12% running gel) of 2h- and 5d-old Plagiorchis elegans metacercarial glycoproteins

labelled with 3H-glucosamine for 24h in vitro. 6A: 200 2h- and 5d-old

metacercariae per lane, 6B: equal aliquots (15,000 CPM) of solubilized

2h- and 5d-old metacercariae. The arrowhead indicates the border of

the stacking gel. M,. standards used were Myosin (200,000),

Escherichia coli ~-galactosidase (116,250), rabbit muscle

phosphorylase b (97,400), bovine serum albumin (66,200), hen egg

white ovalbumin (42,699), bovine carbonic anhydrase (31,000),

soybean trypsin inhibitor (21,500), and hen egg white lysozyme • (14,400) (Pharmacia). • 6A 6B

Mol.-Wt 2h 5d Mol. Wt 2h 5d 200-- 200-

66.2- 66.2- 42.7- 42.7- -37.5 31-

31- -37.5 21.5- 21.5- -19.5 ""-...... 14- -14 12- -12 14- -14 • 12- ·1 -12

• • 130 Figure 4.7. Fractionation of glycoproteins from 2h- and 5d old

Plagiorchis elegans metacercariae incubated with 3H-glucosamine for

24h in vitro using 8ephacryl 8-200 column chromatography. 15,000

CPM of soluble metacercarial proteins were added to the column and

the proteins eluted with O.OlM TRIS-HCI buffer. Fractions (1.0ml)

were collected and the 3H_ activity for each fraction was determined

as describei1 in Materials and Methods. The M,. standards used were

6 blue dextran (BD; >2 x 10 ), bovine serum albumin (B8A; 67,000),

chymotrypsinogen A (CTA; 25,000), and ribonuclease A (RA; 13,000) • (Pharmacia).

.. . •

o•

1 1 , 1 1 o 1 " ID 1 " " ...' " " « ....------::::::::::::::::::::::':':':---.----- ct: - ,,--- ::: « .t'_':a-;;::'~ o 1-_ "- . ... o ---__ ~ --- .t'__-, ,)

«cn_ ..------~ m ------­ "---...\ ,-.--~ ..._---­------.. ...o

1

L.._...... I...... _--L__...L__....._~ 0 ID ... o • 132 • LITERATURE CITED

Bock, D. 1986. In vitro excystment of the metacercariae ofPlagiorchis species 1 (Trematoda, Plagiorchiidae). Int. J. ParasitoI. 16: 641-645.

Bock, D. 1988. Formation, histochemistry, and ultastructure of the metacercarial cyst wall of Plagiorchis species 1 (Trematoda,Plagiorchiidae). Int. J. ParasitoI. 18: 379-388.

Bock, D. 1989. Hatching mechanism of the metacercariae ofPlagiorchis species 1 (Trematoda: Plagiorchiidae). J. HelminthoI. 63: 153-171.

Christensen, B.M. 1986. Immune mechanisms and mosquito-filarial worm relationships. Symp. ZooI. Soc. Lond. 56: 145-160.

Christensen, B.M. and J.W. Tracy. 1989. Arthropod-transmitted parasites: Mechanisms ofimmune interaction. Amer. ZooI. 29: 387-398.

Christensen, B.M., D.R. Sutherland, and L.N. Gleason. 1984. Defense reactions of mosquitoes to filarial worms: comparative studies on the response of three different mosquitoes to inoculated Brugia pahangi and Dirofilaria immitis microfilariae. J. Invert. PathoI. 44: 267-274.

Genov, T. and P. Samnaliev. 1984. Biology, morphology and taxonomy of Plagiorchis elegans (Rudolphi, 1802) (Plagiorchiidae) in Bulgaria. pp. 75-114 in Fauna, taksonomiyai ekologiya na khelminti po ptitsi. I. Vasilev, ed. Bulgarian Academy of Science, Sofia Bulgaria.

Giitz, P. 1986. Mechanisms of encapsulation in dipteran hosts. Symp. ZooI. Soc. Lond. 56: 1-19. 133 • Gotz, P. and H.G. Boman. 1985. Insect Immunity. pp. 453-485 in Comprehensive Insect Physiology, Biochemistry and Pharmacology, G.A. Kerkut and L.I. Gilbert (eds.). Pergamon Press, New York.

Gupta, A.P. 1986. Hemocytic and Humoral Immunity in Arthropods. John Wiley and Sons, New York. 535pp.

Halton, D.W. 1967. Observations on the nutrition of digenetic trematodes. Parasitology 57: 639-660.

LeFlore, W.B. 1978. Plagiorchis elegans: histochemicallocalization of dehydrogenases in the cercarial stage. Exp. ParasitoI. 46: 83-91.

Lowenberger, C.A. and M.E. Rau. 1993. Plagiorchis elegans: requirements • for metacercarial development to infectivity, and conditions required for excystment. J. HelminthoI. Soc. Wash. 60: 67-71.

Nappi, A.J. and B.M. Chrïstensen. 1987. Insect immunity and mechanisms ofresistance by nematodes. pp. 285·291 in J.A. Veetch and D.W. Dickson (eds.), Vistas on Nematology.'Society of Nematologfsts, Hyattsville, Maryland.

Smyth, J.D. and D.W. Halton. 1983. The Physiology of Trematodes. Cambridge University Press. Cambridge. 446pp.

Styczynska-Jurewicz, E. 1962. The life cycle of Plagiorchis elegans and the revision of the genus Plagiorchis Luhe, 1889. Acta ParasitoI. Polon. 12: 419-445. 134 • Taft, S.J. 1990. Cinephotomicrographic and histochemical observations on cercarial penetration and encystment by Plagiorchis sp. in larvae of Chaoborus sp. Trans. Am. Microsc. Soc. 109: 160-167.

Uglem, G.L. 1980. Sugar transport by lar-:al and adult Proterometra macrostoma (Digenea) in relation to environmental factors. J. ParasitoI. 5: 748-758.

Uglem, G.L. and O. R. Larson 1987. Facilitated diffusion and active transport systems for glucose in metacercariae ofClinostomum marginatum. Int. J. ParasitoI. 17: 847-850.

Uglem, G.L., M. C. Lewis, and O. R. Larson. 1985. Niche segregation and sugar transport capacity of the tegument in digenean flukes. • Pa:-Ùitology 91: 121-127. Uglem, G.L., O.R. Larson, J.M. Aho, and K.J. Lee. 1991. Fine structure and sugar transport functions of the tegument in Clinostomum rlwrginatum (Digenea: Clinostomatidae): environmental effects on the adult phenotype. J. ParasitoI. 77: 658-662.

Webber, RA, M.E. Rau, and D.J. Lewis. 1986. The effects ofvarious light regimens on the emergence ofPlagiorchis noblei cercllliae from the molluscan intermediate host, Stagnicola elodes. J. ParasitoI. 72: 703-705.

Whitfield, P.J. 1979.,. The Biology of Parasitism: an Introduction to the Study ofAssociating Organism@. University Park Press, Baltimore Md. 277pp. 135 • CONNECTING STATEMENT 3

In Chapters 3 and 4 we determined the developmental requirements and

metabolic activities of Plagiorchis elegans metacercariae within the insect

hosto Previous studies have shown that infections with P. elegans

metacercariae can kill mosquito larvae. In Chapter 1 we discussed the

notion of incorporating organisms such as Plagiorchis elegans into biological

control programmes for the control of mosquito larvae. For successful

control, the parasite and target species must overlap spatially and

temporally. Natural selection would favour mosquito species that avoid

ovipositing in sites deleterious to larval development, and kin selection

theory predicts that parasitized larvae should communicate the

unsuitability oflarval habitat to ovipositing adults. Mosquitoes do oviposit

selectively in sites devoid of predators. The following study evaluates the

oviposition response ofgravid adults to the presence of the molluscan first

intermediate host (uninfected or shedding P. elegans cercariae), P. elegans

cercariae in the absence of the snai! host, or in the presence of

unparasitized or P. elegans parasW.zed mosquito larvae. The study also

assesses oviposition in waters which had previously contained larvae to

determine ifgravid adults recognize and discriminate betwGen waters that

had previously held parasitized or unparasitized larvae. • 136 CHAPTER5

EVIDENCE FOR THE PRODUCTION OF AN OVIPOSITION

DETERRENT OR REPELLENT COMPOUND BY AEDES AEGYPTI

LARVAE PARASITIZED BY THE DIGENEAN PLAGIORCHIS

ELEGANS.

• Carl A. Lowenberger1 and Manfred E. Rau2

lInstitute of Parasitol0lD' and 2Department of Entomology

Macdonald Campus of McGill University,

21,111 Lakeshore Rd, Ste-Anne-de·Bellevue

Québec, Canada, H9X 3V9

A Revision of this Chapter will be

submitted for publication to

Oecologia 137 • AB8TRACT: Gravid Aedes aegypti oviposit preferentially on waters containing conspecific immatures due to attractive pheromones produced by

larvae and pupae. We have investigated the effects of the digenean parasite

ofmosquito larvae, Plagiorchis elegans, on this oviposition behaviour.

Natural selection should favour species that avoid ovipositing in sites

deleterious ta larval development. Waters containing parasitized larvae

received significantly fewer eggs than waters containing unparasitized

larvae. There were no significant effects of larval density on oviposition.

Waters that had previously contained unparasitized larvae (LHWUP)

received significantly more eggs than either waters that had previously

contained no larvae (LHW 0) or parasitized larvae (LHWPP), and LHW 0 • received significantly more eggs than LHWPP. When waters were boiled, treated with penicillin and streptamycin ta curb bacterial growth, diluted, or

filtered sterilized, LHWPP continued to receive significantly fewer eggs than

LHWUP. The behaviour ofmosquitoes in the presence ofthese waters was

recorded. Mosquitoes landed as frequently on both waters, and laid similar

numbers of eggs/minute spent on the surface. However, mosquitoes

remained for significantly shorter periods on LHWPP and consequently laid

significantly fewer eggs there. We propose that parasitized A. aegypti larvae

produce an oviposition deterrent compound that repels adults and deters

oviposition in sites unsuitable for larval development. While the production 138 • of such compounds is common in phytophagous insects, this is the first evidence for their use by haematophagous Diptera.

• 139 • INTRODUCTION

In studies on the life histories of mosquitoes, much recent research has

focused on the endogenous regulation of mosquito reproductive behaviour,

and on the exogenous factors which gravid fl'males use to discriminate

between potential oviposiUon sites (Klowden 1989, Klowden 1990). Many of

the complex stereotyped behaviours involved in host finding, blood feeding,

pre-oviposition and oviposition are genetically programmed into the central

nervous system and are released by exogenous and endogenous stimuli

(Klowden 1990). The selection of a suitable, indeed an optimal, oviposition

site is an integral part of the life history of mosquito species that has

repercussions on subsequent generations (Bentley and Day 1989). Sites

containing few resources and high larval populations produce smaller, less

fit pupae and adults (Christophers 1960, Moore and Fisher 1969, Ikeshoji

and Mulla 1970a). DifferentiaI oviposition between potential sites has been

attributed to visual cues and odours (Wood 1961, McIver 1982, Strickman

1982), the presence ofmicroflora and their metabolites (Gjullin and Johnsen

1965, Ikeshoji et al. 1979, Benzon and Apperson 1988), and the presence

and density oflarvae in the waters (Ikeshoji and Mulla 1970a, Chadee et al.

.1990). • 140 • Most oviposition studies have focused on the attractiveness of specifie sites with the aim of determining which factor(s) contribute to site selection.

Gravid females display a marked preference for sites containing larvae of

the same species (Maire 1984, 1985, Ikeshoji and Mulla 1970b, Bentley et

al. 1981, Osgood 1971), or larvae of different species within the same genus

(Ikeshoji and Mulla 1970b, Maire 1984, 1985, Maire and Langis 1985). The

presence of larvae of other genera may be either repellent or attractant

(Ikeshoji and Mulla 1970b). The preference for ovipositing in sites

containing conspecifics has been attributed to oviposition attractant

pheromones produced by larvae and pupae (Davis 1976, Bruno and

Laurence 1979, Maire 1984). Bacteria and their metabolites also have been • implicated in attracting gravid adults (Benzon and Apperson 1988). However, Maire (1985) reported that even axenically raised Aedes

atropalpus larvae enhanced oviposition in sterile water and concluded that

aIl larval stages produced an attractant pheromone. Several of these

attractant compounds have been isolated and synthesized (Ikeshoji et al.

1979, Klowden 1982, Hwang et al. 1987, Dawson et al. 1989, Knight and

Corbet 1991, Dawson et al. 1990) and sorne have been evaluated in field

trials (Otieno et al. 1988).

The majority cf these studies have assessed the oviposition response of • adults to the attractive stimulus(i) produced by la~oratory reared-Iarvae. 141 • Very little is known about the effects oflarval predators, parasites, and pathogens on the ability of mosquito larvae to produce semiochemicals and

communicate with adults. With the increasing emphasis on biological

control programmes employing natural enemies to control mosquito larvae,

it is imperative that we understand the implications of the infection status

of larvae and pupae on the oviposition site selection by gravid mosquitoes.

Natural selection should strongly favour adult behaviours that reduce

oviposition in [!iI;~s where larvae are likely to suffer high mortality

(Petranka and Fakhoury 1991). Ovipositing females should maximize both

their individual and inclusive fitness (Hamilton 1964) by judging and • discriminating between waters containing healthy or parasitized larvae as iI'.dicators of site quality. Kin selection theory (Smith Trail 1980) suggests

that parasitized larvE.e should communicate suboptimal conditions for

development to ovipositing females. Appropriate responses by the adults

would produce a reduction in oviposition and diminished recruitment of

cor.specifics into conditions unsuitable for larval development.

The present stuày tests these hypotheses by comparing the oviposition

response ofA. aegypti to waters containing, or which had pleviously

contained, unparasitized larvae or larvsG parasitized by the digenean

Plagiorchis elegans to determine if mosquitoes recognize and discriminate 142 • between potential oviposition sites based on the health status of the larvae these contain.

Plagiorchis elegans

The biology of the various stages of P. elegans has been described (Macy

1960, Styczynska-Jurewicz 1962, Blankespoor 1974, 1977, Genov and

Samnaliev 1984). Plagiorchis elegans cercariae emerge nocturnally from the

molluscan first i.ntermediate host and penetrate a wide range ofinsect

orders that serve as second intermediate hosts. The cercariae attach to the

host cuticle and penetrate by means of a stylet and histolytic enzymes (Bock

1984, Taft 1990), and then encyst as metacercariae in the haemocoel. • Overall development of metacercariae within the insect is primarily temperature dependent (Lowenberger and Rau 1993). Metacercariae excyst

and transform into adults in the intestine of the vertebrate definitive host

under conditions ofhigh temperature, alkaline pH, and the presence ofbile

salts (Bock 1986, 1989, Lowenberger and Rau 1993). High intensities of

infection with P. elegans metacercariae can kil! mosquito larvae or inhibit

ecdysis (Dempster and Rau 1990, Dempster and Rau 1991, Dempster et al.

1986), while adults which survive infections show reduced longevity and

fecundity (Kimoro 1991). • 143 • MATERIALS AND METHOnS

A colony ofA. aegypti has been maintained in our laboratory for more than

6 years. Adults were kept under a 14L:lOD photocycle at 27 ± 2C, provided

with a 10% sucrose solution ad lib., and blood-fed twice weekly. Larvae

were reared under similar temperature and light conditions and were fed

finely ground Tetramin® fish food ad lib.

A colony offield collected and laboratory infected Stagnicola elodes

(Mollusca: Pulmonata) has been maintained for more than 3 years in the

laboratory. Snails were maintained at 20 ± 4C under a 16L:SD photocycle • and fed lettuce and Tetramin® fish food ad lib.. Plagiorchis elegans cercariae emerge nocturnally, and were collected by placing 10 infected

snails in 100ml of aerated tap water prior to the scotophase. Water that had

contained UIÙnfected snails was termed snail water. Cercariae were used for

experimental infections approximately Sh following emergence.

The following experiments assessed the oviposition response ofA. aegypti in

the presence ofinfected or uninfected snails, cercariae or snail water, and

parasitized or unparasitized mosquito larvae. The experiments were carried

out in cages (75 x 39 x 32cm) at 27 ± 2C under a 14L:10D photocycle, with a

10% sucrose solution available ad lib.. Recently emerged mosquitoes (100 il' 144 • + 100 ~) were introduced to each cage and were blood-fed every 48h. A centrally located oviposition dish '.vas used to determine the time of initial

oviposition. Four days later this dish was removed and replaced by two

experimental oviposition dishes placed 40cm apart. These were 450ml

round, clear plastic containers lined with white nlter paper, and filled with

400ml aerated tap water. Oviposition was monitored over 60 day periods (3

replicatesiexperiment). Eggs were collected, adults were blood-fed, and the

relative position of the two dishes (UR) was randomized every 48h.

EXPERIMENT 1 • Oviposition was assessed in the presence of the snail first intermediate hosto Oviposition was compared when dish A contained an infected snail

shedding approximately 500 cercariae/day and dish B contained an

uninfected snail. Twenty ml of snail water were added ta each container

every 48h to replace water lost ta evaporation.

EXPERIMENT 2

In order ta determine ifthe presence of cercariae in the absence of the snail

host would affect oviposition, dish A received 400 8h-old P. elegans cercariae

in 20ml of snail water every 48h. Dish B received 20ml snaii water without

cercariae every 48h. • '- ,-"' ',' 145 • EXPERIMENT 3 Oviposition was evaluated in the presence of parasitized or unparasitized

1" i"vae. Twenty first instar A. aegypti larvae were introduced to both dishes

every 48h. Each day, dish A also received 400 8h·old P. elegans cercariae in

20ml snail water while dish B received 20ml snail water as a control. Pupae

were removed and estimations oflarval densities were made daily by

counting larvae at the periphery of the oviposition dish. The adults were

allowed to em"erge and were examined to determine the prevalence and

intensity of infection with P. elegans metacercariae. Two visibly moribund

fourth instar larvae were removed every 48h to determine levels of • infection. EXPEUIMENT 4: LARVAL HOLDING WATER EXPERITUENTS

The following experiments were done to determine if, in the absence of

larvae, female A. aegypti would oviposit differentially on waters which had

previously contained parasitized or unparasitized larvae. Cages (26 x' 22 x

23cm) were established containing 20 male and 20 female l'ecently emerged

A. aegypti, maintained under a 14L:10D photoperiod at 27 ± 2C with a 10%

sucrose solution available ad lib. Mosquitoes were blood·fed daily. Four days

following the onset of oviposition the central oviposition dish was removed

and replaced by 2 experimental round, glass oviposition dishes (60 x 15mm)

placed 20cm apart. Each dish contained 25ml of larval holding waters 146 • (LHW). For 7 consecutive days the dishes were removed, the eggs counted, and the dishes washed, dried, and replaced in the enclosure with 25ml fresh

LHW. The relative position (UR) of the dishes was randomized daily. A

minimum of five replicates were included in each trial and the same adult

mosquitoes were used for one trial OIÙy. LHW were prepared by incubating

a known number of washed third and fourth instar A. aegypti larvae for 72h

at 27C in a 21 flask containing 11 distilled Wf'Ler and 0.25g finely ground

Tetramin® fish food. Following incubation the contents of the flask were

filtered through 2 Melita® coffee filters and stored at 4C. LHW prepared

with parasitized larvae were prepared similarly using larvae which haà·

been parasitized 24h prior ta the 72h incubation period, and were • designated as havingbeen prepared with lightly «5 metacercariae/larva) or heavily (>5<10 met1cercariae/l~.rva) parasitized larvae.

Oviposition was compared between dishes containing LHW prepared with 0,

100, :300, and 600 unparasitized (U) larvae/l. lOOU and GOOU were then

compared with waters prepared with 100 lightly (100L) or 100 h'~avily

(100R) parasitized larvae. Further comparisons of oviposition.'Nere made

between 100U anlhv':lters prepared by concurrent or sequential incubations

of parasitized and unparasitized larvae. • 147 • Comparisons of oviposition were made between 100H and lOOU when both waters had been i) boiled for 10 minutes, ii) treated with 10,000 units of

penicillin G, sodiwn (Œbco BRL #860-1830MJ) and streptomycin sulphate

(Œbco BRL #860-1860IM) (100 units/ml ofLHW) daily during the

incubation and storage periods, iii) filter sterilized (Acrocap 0.2jlM, Gelman

Sciences, Ann Arbor, Mi, USA), or iv) diluted to 10% of stock,·oiution .

The bacteria present in lOOU, 100H, lOOU with penicillin-streptomycin,

lOOH with penicillin-streptomycin, LHW 0, and LHW 0 with penicillin­

streptomycin were identified and enumerated. Sampies were diluted serially

to 1:10.000 in sterile distilled water (2 replicates). Each dilution was spread

plated (O.lml/dilution) onto plate COtilit agar aXd nutrient agar

supplemented with yeast extract (Difco; and glucose. Plates were incubated

at 27C for one week and were observed daily for growth. Bacterial colonies

from the antibiotic treated lOOU and 100R were plated on brain heart

infusion agar and identified using the biochemical API 20E identification

strips for Gram negative bacteria. One bacterial colony that was found only

in 100R was cultured for further studies. Colonies of this bacteria were

counted on the original plates and grown on chocolate agar supplemented

with 3% yeast extract (Difco), 0.5 mM CaCI2, and 5.0ml/l IsovitaleX® as per

Gerhardt et al. (1981). The bacteria were streaked onto a plate of

supplemented chocolate agar and incubated at 27C for 7 days to develop 148 • abundant growth. Bacteria were washed from the plate using brain heart infusion broth (BHIE) and diluted to approximately 1 x lOG/ml in sterile

distilled water at 27C. This dilution was then used in ovipo~ition trials and

was compared with 100H and LHW 0, and 100H and LHW 0 to which 7.0ml

of BHIE had been added.

EXPERIMENT 5: MOSQUITO BEHAVICUtR

The behaviours of mosquitoes within the cages were recorded on videotape

to determine how mosquitoes responded to 100U a'ld 100H. Two round,

glass oviposition dishes (60 x 15mm) containing 25ml of 100U and 100H

respectfully were introduced lOcm apart at 11:45 each morning. A Hitachi • CVCC camera with an automatic, light sensitive diaphragm was used to record mosquito activities from noon to midnight (8h light:4h dark) for 5

consecutive days. Recordings were made when the cages contained: i) 20

males, ii) 20 unmated females not blood-fed, lii) 20 mated females not blood­

fed, or iv) 20 mated, blond-fed females. Subsequent analysis measured

diiferential mosquito behaviour and the numbers of eggs laid in each dish.

ANALYSIS

Statistical analysis was done on SYSTAT 5.0 software, using Stu~l.ent's t­

test for diiferences between numbers of eggs laid in the presence of snails • and/or cercariae. The numbers of eggs laid on LHW were transformed 149 • (Arcsine square root) and compared using Studeni's t-test for pairprl samples (Sokal e;'ld"Rohlf1981). The effect of the relative position (UR) of

the oviposition dishes was compared using the KruskalWallis test. The

Mrnn·Whitney U- test was used to compare the number oflandings, the

duration of contact, the number of eggs laid per unit time and per landing

on the LHW. The level of significance (a) was set at 0.05 in al! tests.

RESUI,TS

EXPERIMENTS 1·3

There was no significant differen_~e m :;hênumber of eggs laid id dishes • containing snails, either uninfected or infected and shedding P. elegans cercariae (t-tesi; P=0.853) (Fig 5.1). In Experiment 2, there was no

significant difference in the numbers of eggs laid in dishes that received

'~ercariae or snail water controls (t-test P=0.329) (Fig 5.1). In experiment 3

there was a significant reduction in oviposition in the dish containing

cercariae and larvae (=parasitized larvac,) (t-test, P<0.001) (Fig 5.1). The

relative position (UR) of the oviposition dishes did not influence oviposition

in any ofthese experimelJts (Kruskal Wallis P>0.05). The prevalence of

infection with P. elegans metacercariae in pupae and emerged adults was

99.1%, and the mean intensity ofinfection in emerging adults was 4.97

(Table 5.1). Larval densities were reduced due to parasite induced host 150 • mortality. Larvil.i density was higher in the control (estimated minimum 260, maximum 511) larvaell) than in the dish containing parasitized larvae

(esti~ated minimum 126, maximum 328 larvaelD.

EXPERIMENT 4: LARVAL HOLDING WATER EXPERIMENTS

While waters prepared with 100, 300, or 600 unparasitized larvae/l received

significantly more eggs than waters prepared with 0 larvae (LHW 0)

(P

oviposition between these solutions (600 vs 100, P=0.111; 600 vs 300,

P=0.487; 300 vs 100, P=0.666) (Fig 5.3). When identical solutions (LHW 0)

were placed in both dishes there was no significant difference in the • numbers of eggs laid in each location (P=0.726) (Fig 5.3), and the relative position (IJR) of dishes did not influence oviposition in any of the trials

(Kruskal Wallis P>0.05).

There was no significant difference in the number of eggs laid on 100L or

100H (P=0.283) (Fig 5.4). However, dishes containing 100L or 100H received

significantly fewer eggs than dishes containing either 100U or 600U (100U

vs 100L, P

100H, P

than waters prepared by concurrent incubation of 100 unparasitized and

100 parasitized larvae (P

Whereas LHW 0 received significantly fewer eggs than lOOU (P

5.2), LHW 0 received significantly more eggs than either 100L (P

100H (P

treated with antibiotics throughout the incubation and storage periods,

filter sterilized, or when 100H was diluted to 10% of stock solution, 100H

continued to receive significantly fewer eggs than did 100U (P

cases) (Fig 5.7).

• Total bacterial counts of the larval holding waters showed no obvious differences between lOOU and 100H, or between lOOU with antibiotics and

100H with antibiotics (Table 5.2). Ten major bacterial colonies were found

on the culture plates, one ofwhich was not present in both 100H and lOOU.

This colony was identified from the API 20E strips as a FlaUflhacterium sp.,

possibly F. aquatile. In oviposition trials, waters containing suspensions of

this bacteria received significantly more eggs than 100H (P

BHill (P

LHW 0 + BHill ((P::0.324) (Fig 5.8). 152 • EXPERIMENT 5: MOSQUITO BEHAVIOUR Analysis of mosquito behaviour showed that only mated, blood-fed females

visited either dish with any frequency. Male mosquitoes landed only 6 times

on lOOU and 7 times on 100H over the 60 hours recorded (Table 5.3). While

the males remained on lOOU for almost twice as long as on 100H, the

landings were too few to allow statistical analysis. Similarly, females which

were neither blood fed nor mated rarely landed in either dish: 8 times on

100U and 5 times on 100H (Table 5.3). Two of these females remained for 4

h:22min:39sec and 28min:41sec respectively which skewed the mean

resting timellanding. Again the low number of landings makes statistical

comparisons difficult. Females that were mated but not blood-fed landed 19 • and 20 times on lOOU and lOOH respectively with a mean of 61.9 seconds/landing on lOOU and 30.1 secondsllanding on 100H (Table 5.3). The

number oflandings and duration of stay on the surface were not

significantly different bctween lOOH and lOOU (Mann-Whitney lI-test

P>0.05).

Blood-fed, mated mosquitoes landed more frequently on lOOU than 100H,

although this difference was not significant (Mann-Whitney U-test, P=0.076)

(Table 5.3). The total time spent on the surface, the mean time spent on the

surface per landing, the total number of eggs laid, and the mean number of • eggs laid per landing by mated, blood-fed mosquitoes were aIl significantly 153 • higher on loOU than on 100H (Table 5.3). However, there was no significant difference in the number of eggs laid per minute on the surface (Table 5.3).

DISCUSSION

Neither the presence of snails (uninfected or infected and shedding

cercariae) nor cercariae in the absence of snails affected oviposition by

mosquitoes. However, oviposition was significantly reduced in dishf:s

containing parasitized larvae. The density of larvae in this dish was

significant1y and visibly reduced due to parasite induced mortality, and

larval density is known to affect oviposition. Culex tritaeniorhynchus • preferred to oviposit in "uncrowded conditions" (Reisen and Siddiqui 1978) and larval holding waters prepared with high densities (>900-1000 larvae/l)

repelled gravid A. atropalpus (Maire 1985). We have not used larval

densities approaching repellent levels and accordingly the differential

oviposition we saw in experiment 3 cannot be explained solely by differences

in larval density. Larvae parasitized by more than 3 metacercariae are

reported to behave abnormally (Webber et al. 1986) and the pres~!.(;e of

fewer, abnormally behaving larvae may have affected oviposition site

selection. The presence of sorne visibly moribund or dead larvae also may

have affected oviposition, although A. aegypti prefer to uviposit on waters

containing deadlarvae than in distilled water (Soman and Reuben 1970). 154 • In the absence oflarvae, oviposition was significantly higher in waters that had previously contained unparasitized larvae when compared with waters

that had held no larvae. This is consistent with other studies (Osgood 1971,

Bentley et al. 1981, Maire 1984, 1985, Bentley and Day 1989) which have

shown that mosquitoes oviposit preferentially in waters that had contained

conspecifics. There was no significant difference in oviposition over the

range oflarval densities tested (l00-600 larvae/l) indicating that we did not

approach densities at which oviposition would be reduced.

There was, however, a significant reduction in oviposition in dishes

containing waters that had held parasitized larvae, regardless oflarval • densities used to prepare the waters. This pattern was maintained when 100U was compared with waters prepared by concurrent or sequential

incubations of parasitized and unparasitized larvae. As long as waters had

contained parasitized larvae at sorne time, they received significantly fewer

eggs than waters prepared exc1usively with unparasitized larvae. These

data suggest that larvae release a semiochemical that deters oviposition in

sites containing parasitized larvae.

Parasites of mosquito larvae may affect the metabolism, growth, and

development of their hosts (Galloway and Brust 1985, Giblin and Platzer • 1985). IfP. elegans metacercariae had inhibited or reduced the production of 155 • the larval attractant pheromone we would have expected similar oviposition levels in 100U and concurrent or sequential incubations ofparasitized and

unparasitized larvae. Similarly, we would have expected similar oviposition

levels in LHW 0 and 100L or 100H. However, not only is oviposition

significantly reduced in waters prepared by concurrent or sequential

incubations of parasitized and unparasitized larvae when compared with

100U, oviposition was significantly reduced in lOOL and 100H when

compared to waters which had never contained larvae (Fig 5.6). These data

suggest that not only do LHWPP fail to at.tract gravid females, they may

repel them or deter them from ovipositing.

• When 100H was diluted to 10% of stock solutions, or when loOU and 100H were boiled for 10 minutes, treated with antibiotics to curb bacterial growth,

or filter sterilized, 100H continued to receive significantly fewer eggs than

lOOU. T}1is suggests that the factor r~sponsible for the differential

oviposition is heat stable and is active even at low concentrations. Benzon

and Apperson (1988) suggested that differential oviposition was caused by

bacteria and their metabolites. While bacteria would have been removed

during filtration, bacterial metabolites may have passed through the 0.2 /lM

filter. As waters prepared with suspensions of Flavobacterium sp. received

significantly more eggs than 100H or LHW 0, the reduced oviposition in • 100H, was not due to the presence of this bacterium. 156 • When lOOU and 100H were filter sterilized we found a total of 10 eggs laid on 100H. Proportional1y, this is about 10 fold fewer eggs than we found in

earlier comparisons of unfiltered 100H and lOOU. The process of filtration

may have removed bacteria and sorne metabolites which normal1y would

have been attractive to ovipositing mosquitoes. The removal of these

aj;>~<'actants may have released the full potential of the oviposition deterrent

or repel1ent factor. Therefore, the effects of!,HWPP, as shown in earlier

comparisons of oviposition in LHWPP and LHWUP, may have significantly

underestimated the potential effects of this compound(s) in reducing

oviposition in waters containing parasitized larvae.

• Analysis of mosquito behaviour within the cages showed that only blood-fed, mated females spent significant t'eriods on potential oviposition sites. This

is to be expected since only gravid, mated females initiate pre-oviposition

behaviours: gravid'virgins or mated, non-gravid adults do not respond to

oviposition stimuli (Klowden 1990). The initiation of preoviposition

behaviours requires that the female has received substances from the male

accessory reproductive glands during copulation. These compounds are also

responsible for inducing monogamy (Craig 1967, Ramalingham and Craig

1976), autogeny (O'Meara and Evans 1976), altered circadian flight activity,

and for inactivating an endogenous oviposition inhibitor which prevents • oviposition prior to copulation (Fuchs and Kang 1978). 157 • Gravid females landed more often, spent significantly more time pel' landing, laid significantly more eggs, and more eggs pel' landing on 100U

than on 100R. Rowever the number of eggs laid pel' minute on the surface

of the waters was not significantly different. Mosquitoes were deterred from

landing on 100R, and if they did land there, they q;.;ickly left. While the

number of eggs laid/minute on the surface was not significantly different

between lOOU and IOOR, the results may have been affected by the small

size of the oviposition dishes and the relatively large number of mosquitoes.

Many collisions were seen between mosquitoes 'landing and those already

sitting on the waters, causing aIl participants to fly away. Larger

oviposition dishes, or fewer mosquitoes might have further reduced the / • number of landings and oviposition on 10OR.

The reduced oviposition in waters prepared with parasitized larvae may be

due to the excessive production of excretory compounds or El. crowding

metabolite produced by parasitized larvae to which the ovipositing adults

respond. If this were the cause of the diffe:rential oviposition, then the

production of such compounds must have been at least 100 fold greater than

that ofunparasitized larvae, since we used only 100 larvae/l, and

subsequently diluted the 100R by 10 fold, with no loss of the deterrent

factor. • 158 • The reduced oviposition, number of landings, and duration of stay on LHWPP are consistent with the production of an ovipositi~n deterrent or

repellent compound by parasitized larvae that is released into the aquatic

environment. Approaching adults may be repelled from landing by volatile

fractions of compounds detected on the blunt-tipped sensilla of the antennae

(Davis 1976). V/hile mosquitoes landed fewer times on LHWPP than

LHWUP, this difference was not significant. Compounds oflow volatility are

detected primarily on the labella of the mosquito following landing (lkeshoji

1968) and may deter oviposition and reduce the duration of the resting

period on the waters. While attractant pheromones can become repellent in

situations of excessive larval crowding (Maire 1985, Reisen and Siddiqui • 1978) our experiments did not approach such densities.

Mechanisms of oviposition inhibition may have evolved to reduce oviposition

in unsuitable or crowded sites and act to prevent excessive competition for

limited resources (Bentley and Day 1989). This would be highly adaptive

since nutrient stress has pronounced effects on developmental rates of

mosquito larvae (Ikeshoji and Mulla 1970a, Haramis 1985). Excessive larval

densities may affect the size, fecundity, longevity, vectorial capacity and

overall fitness of emerging adults (Christophers 1960, Clements 1963,

Ikeshoji and Mulla 1970a, Haramis 1985, Hawley 1985, Service 1989). • Reduced egg laying due to oviposition deterrent. pheromones is common in 159 • terrestrial insects. Oviposition deterrent or epideictic pheromones are produced by at least 35 species of phytophagous insects and serve to reduce

oviposition in hosts already occupied by conspecifics (prokopy 1981, Prokopy

et al. 1982, Roitberg and Prokopy 1987, Pittara and Katsoyannos 1990,

Papaj et al. 1992). While the majority of these compounds are produced by

adults, oviposition may be deterred by compounds associated with

previously laid eggs (Klijnstra 1986) or by compounds excreted in the frass

of larvae (Renwick and Radke 1980). This pheromone-mediated

communication acts to disperse adults over the range of the hosts and

reduces lethal competition between conspecifics which are often genetically

related (Prokopy 1981). These pheromones benefit both the producer and • recipient. Mosquito larvae, however, have no offspring to protect and cannot easily disperse to new environments. Nor do mosquito larvae benefit directly

from the production of such compounds. Another rationale is required to

explain the production of oviposition deterrent compounds by parasitized

mosquito larvae. This may be explained by kin selection theory.

Kin selection theory in parasite-host interactions proposes that parasitized

hosts can increase their inclusive fitness by lowering the risk of parasitic

infection in their kin. The theory requires that the parasit" is more likely ta

parasitize the host's kin than non-kin, and that the reproductive success of • kin will be increased due to a lower risk of parasitism (Smith Trail 1980). 160 • Bince A. aegypti females are monogamous (Craig 1967), prefer to oviposit in sites containing eggs of conspecifics rather than their own from previous

gonotrophic cycles (Chadee et al. 1990), and have a normal flight range as

little as 25-30m from their site of emergence (Soper 1935), the probability of

genetically related females using the same oviposition sites is high.

Moribund larvae, having their individual fitness reduced to almost zero by

the parasite, may increase their inclusive fitness by reducing oviposition,

and therefore death of kin, in sites harbouring parasites such as P. elegans.

Mosquitoes already use and respond to semiochemicals to avoid

superoviposition and the subsequent crowding, competition, (Chadee et al.

1990) and opportunistic cannibalism of younger instars (Mogi 1978). The • employment of oviposition deterrent compounds would act similarly to prevent losses ofaquatic stages to parasites, predators and, in general, to

conditions unsuitable for the development of larvae. Benzon and Apperson

(1988) suggested that the use of an oviposition deterrent or repellent

compound is more plausible than that of an attractant compound, especially

under conditions of stress and resource depletion. Maire (1985) suggested

that Aedes larvae might produce an inhibition factor under conditions of

intense crowding. Our data suggest that larvae indeed produce oviposition

deterrent compounds that have a volatile fraction which repels adults prior .. to landing on the water, and possibly a fraction of low volatility perceived 161 • after landing which deters oviposition and encourages rapid departure from waters containing these compounds.

These results raise important issues. The rapid depletion of mosquito larvae

foIlowing introductions of control agents such as the predatory mosquito

fish, Gambusia sp. have been directly attributed to the introduced agent

(Murdoch and Dence 1987). Our data suggest that target species such as

mosquitoes may oviposit less frequently in sites deleterious to larval

development in direct response to an introduced agent. Rerluced numbers of

the target species may reflect a shift of that species to nearby habitats

devoid of the control agent. Chesson (1984) showed that Culex pipiens • quinquefasciatw:: oviposited less in sites containing notonectid predators. Kats and Sih (1992) showed that Ambystoma barbouri laid fewer eggs in

pools containing fish predators. Selective oviposition by adults may be an

indirect effect of the presence of predators and parasites that has

repercussions on larval density, distribution, aÏJ.d the structure of aquatic

communities (Petranka and Fakhoury 1991). Biological control programmes

for mosquito larvae must address these indirect effects ofintroduced control

agents as weIl as the more easily measured direct effects. • 162 • SUMMARY Whereas oviposition by Aedes aegypti was not affected by the presence of

snails or Plagiorchis elegans cercariae, oviposition was reduced in waters

which contained or had previously contained larvae parasitized by the

digenean P. elegans. We have shown that neither larval densities nor

bacterial contaminants were factors influencing this differential oviposition.

Our results are consistent with the production of oviposition deterrent

and/or repellent compounds by parasiti.zed mosquito larvae. This

compound(:;) would deter oviposition and recruitment of conspecifics into

environments unsuitable for larval development due to the parasite. W'nile

oviposition deterrent compounds have been shown in adult phytophagous • Diptera, this is the first evidence for the production ofcompounds which influence the oviposition behaviour of terrestrial adults by parasitized,

aquaiic, larval Diptera.

ACKNOWLEDGEMEN'l'8

We thank Dr. Alain Maire of the Université de Québec à Trois Rivières for

discussions on our initial results and comments and suggestions on. the

larval holding water studies. Joan Kearvell cultivated, identified, and

enumerated the bacteria from LHW and prepared the suspensions of • Flavobacterium sp. Funding was provided by the Natural Sciences and 163 • Research Council of Canada (NSERC) (OGP 0007606) to MER and the Fonds pour la formation de Chercheurs et l'aide à la recherche (FCAR) (ER

0129) to MER. Personal funding for CAL was provided by a Walter M.

Stewart Award and a Lynden Laird Lyster Award. Research at the Institute

of Parasitology of McGill University is supported by NSERC and FCAR.

•

• • 164

Figure 5.1. Comparison of oviposition by Aedes aegypti in dishes containing

400 ml of aerated tap water to which snails, cercariae, or larvae were

introduced. Expt 1: A received a snail shedding - 500 Plagiorchis elegans

cercariae/day while B received an uninfected sti..lil. Expt 2: A received 400

Bh-old P. elegans cercariae in 20ml snail water daily while B received 20ml

snail water daily. Expt. 3: A and B both received 20 first instar larvae

every 4Bh. A also received 400 Bh-old P. elegans cercariae in 20 ml snail

water daily while B received 20 ml snail water. Eggs were removed every

4Bh and egg numbers were compared using Student's t-test for paired • samples. Significantly different levels of oviposition within pairs (P

• •

....• 0­ X * I.LJ

....• 0­ < X • I.LJ

....• 0­ X I.LJ

1 1 1 1 0 0 0 0 0 0 0 0 0 CIO CO ~ ~ SJlI sôôe UDe~ • (:3S+ ) -~ ~ et/ 166 • Figure 5.2. Comparison of oviposition by Aedes aegypti on larval holding waters (LHW) prepared with 0, 100, 300, or 600 unparasitized A. aegypti

larvael!. Eggs were removed daily for 7 days and egg numberR transformed

(Al'csine square root) and compared using Student's t-test for paired

samples. Significantly different levels of oviposition within pairs CP

are indicated by *". LHW were prepared by incubating a lmown number of

washed third and fourth instar larvae for 72h at 27C in a 2L fh:.sk

containing IL distilled water and 0.25g finely ground Tetramin® fish food.

Following incubation the contents of the flask were filtered through 2

Melita® coffee filters and the resulting LHW stored at 4C. •

• • ~ ~ o 0 o 0 * toi) CO * 1 1 ~~

o ~ :1: 0 :I: 0 ..J ,- 1 1 * • DI *

*

o o o o o o 10 o 10 o 10 e· CIt CIt ...... (35+) ,(DP/4S 1P/8559 uDe~ 168 • Figure 5.3. Oviposition by Aedes aegypti on larval holding waters (LHW) prepared with 0, 100, 300, or 600 unparasitized Aedes aegypti larvaell. Eggs

were removed daily for 7 days and egg numbers transformed (Arc:;i:lè

square root) and compared using Student's t-test for paired samples. No

significant diffcrences in oviposition were found bet\veen LHW prepared

with different densities of unparasitized larvae CP>O.OS). LHW were

prepared as described in Fig 2.

•

• • :;):;) 00 00 l'()CO 1 1 ~~

o :;) ;=0 ::I:O ..J1- 1 • DI

o o o o • ' ....10 ....o 10 . - (35+) ,(DP/4S !P/s55e uDew 170 • Figure 5.4. Comparison of oviposition by Aedes aegypti on larval holding waters (LHW) prepared with 100 (lOOU) or GOO (GOOU) unparasitized

larvae!l and 100 lightly (lOOL) or 100 heavily (lOOH) Plagiorchis elegans

parasitized larvae. Eggs were removed daily for 7 days and egg numbers

were transformed (Arcsine square root) and compared using Student's t-test

for paired samples. Significantly different levels of oviposition within pairs

(P<0.01) are indicated by **. LHW were prepared as described in Fig 2.

LHW prepared with parasitized larvae were prepared similarly using larvae

which had been parasitized 24h prior to the 72h incubation period. These

waters were designated as having been prepared with lightly «5

metacercariae!larva) or heavily (>5<10 metacercariae!larva) parasitized • larvae.

• • -I::I: 00 ,...00,... 1 1

ml i'-'* ::::>::::> 00 ,...co00 1 1 * • I~ *

* .....

*

o 0 0 0 0 0 a 0 a 0 a .' CIil CIil ...... (3S+) ,(DP/LfslP/s668 UD8Vi 172 • Figure 5.5. Comparison of oviposition by Aedes aegypti on larval holding waters (LHW) prepared with 100 unparasitized larvae/l (lOOU) and waters

prepared by either concurrent (lOOM) or sequential (lOOSL and 100HL)

incubations of parasitized and unparasitized larvae. Eggs were removed

daily for 7 days and egg numbers were transfonned (Arcsine square root)

and compared using Student's t-test for paired samples. Significantly

different levels of oviposition within pairs CP

LHW were prepared as described in Fig 2 and 4. 100M was prepared by

incubating 100 unparasitized larvae with 100 heavily parasitized larvae for

the 72h incubation period. lOOSL and 100HL were prepared by incubating

100 lightly or 100 heavily parasitized larvae respectfully in loOU for 72h at • 27C.

• • ...1% en(/) 00 '00 * --1 1 * O]~

~::I 00 00 --1 1 ID * • *

* *

, , o o o o o o o o 10 o 10 o 10 .' te') ~ ~ ..- ..- (3S+) ,(np/481P/8668 UD8" 174 • Figure 5.6. Comparison of oviposition by Aedes aegypti on larval hOlding waters (LHW) prepared with 100 unparasitized (lOOU), 100 lightly

parasitized (100L) or 100 heavily parasitized (lOOH) A. aegypti larvae, or

without larvae (LHW 0). Eggs were removed daily for 7 days and egg

numbers were transformed (Arcsine square root) and compared using

Student's t-test for paired samples. Significantly differont levels of

oviposition compared within pairs œ

prepared as described in Figs 2 and 4. •

• • ..1% '00 00 --1 1 []JI * 0 ::» ~O %0 -! - DI1 1 • *

*

o o o o o o o o 10 o 10 o 10 .' If) C\4 C\4 ...... (3S+) ,{DP/48!P/8668 Il UD8" 176 • Figure 5.7. Comparison of oviposition by Aedes aegypti on larval holding water" (LHW) prepared with 100 unparasitized (100U) or 100 heavily

(100H) parasitized Ae.!es aegypti larvae. Prior to being used in oviposition

trials the solutions were subjected to one of the fol1owing treatments: il

boiled for 10 minutes, ii) treated with 10,000 units ofpeniciIlin G, sodium

and streptomycin su1phate (100 units/ml of LHW) during the incubatioi'. and

storage periods, iii) a dilution of 100H to 10% of stock solution, or iv) filter

sterilized (Acrocap 0.2J.lM, Gelman Sciences, Ann Arbor, Mi, USA). Eggs

were removed daily for 7 days and egg numbers were transformed (Arcsine

square root) and compared using Student's t-test for paired samples.

Significantly different levels of oviposition compared within pairs (P

"C" CD~ L..::t ...... CD('l * =0 * l.&.. '-' J: 0 ,...0 c: 0 .-..... ::::t ~ .-C

0) • () .-...... -0 * ::J .Q * 0 .-..... 0 c: ,... <

"C CD 1 .-0 al

o o o o o o 10 o 10 o 10 C"l C"l ...... • (3S+) AOp /4S !p/s55s uosVi 178 • Figure 5.8. Comparison of oviposition by Aedes aegypti on larval holding waters (LHW) prepared without larvae (LHW 0), with 100l-jeavily

parasitized A. aegypti larvae (100H), LHW 0 + brain heart infusion broUl

(BHm), 10llH + BHm, suspensions ofFlavobacterium sp. (l x lOB/ml). Eggs

were removed daily for 5 days and egg numbers were transformed (Arcsine

square root) and compared using Student's t-test for paired samples.

Significantly different levels of oviposition compared within pairs (P

are indicated by **. LHW were prepared as described in Figs 2 and 4. LHW

0+ BHm and 100H + BHm received 7.0 ml BHm following filtration and

prior to storage. •

• •

al al ~ ::J: :I:- al c:: al 0 + .-œ + c:: 0 1) :I: C. 0 3= œ 0 :I: :::J ..... -1 œ E :::J -.L- • lm ....I) () 10 0 ..Q 0 :I: > 0 3= 10 0 :I: ...... -1 -LL. ~~I

o 10 o ,...10 o o -10 -C"'l -o 10 • (35+) ,{op/4S IP /SÔÔ9 U09~ '. • • 180

Status Estimated Pupae Emerged d':f/. Prey , Mean Intensity of Infection Larval (%) with Plagiorchis elegans Densityl1 Adults Metacercariae

Min Max Total Dead Pupae Adults Larvae

Parasitized 126 328 238 66 172 1.05 99.1 11.1 4.9 14.7

Control 260 510 394 8 386 1.01 0 0 0 0 .

Table 5.1. Summary of pupal and adult production, prevalence and intensity of infection, and estimated larval densities from oviposition sites containing Aedes aegypti larvae in the presence and absence of Plagiorchis elegans cercariae. • 181

LHW Total bacterial counts (SE)/ml of LHW at various timei> following l preparation .

Day 1 Day 7

100U 6.7 (1.J.) x 107 8.5 (2.4) x 106 100R 4.1 (0.8) x 106 1.6 (0.2) x 106

7 7 100U +P/S * 1.7 (0.4) X 10 2.4 (0.6) x 10 7 7 100R + PIS * 2.2 (1.2) x 10 1.2 (0.8) X 10 LHWO --- 2.7 (0.03) x 107 LHWO +P/S * --- 1.9 (0.05) x 107 • ,

Table 5.2. Microbial analysis of Larval Holding Waters (LHW) prepared with 100 unparasitized (100U), 100 heavily parasitized (lOOR), or no (LHW 0) fourth instar Aedes aegypti larvae.

* 10,000 units ofpenicillin G, sodium and streptomycin sulphate (100 unitslml of LHW solutions) were added daily to solutions during the incubation and storage periods.

1 Standard error was corrected using the Gurland and Tripathi correction factor in Sokal and Rohlf (1981) for small sample sizes.

• •• • 182

Males (20) Females (20) Females (20) Females (20) unmated mated mated, not blood-fed not blood-fed blood-fed 100U 100H 100U 100H 100U 100H 100U 100H # Landings 6 7 8 5 19 20 218 119 Total Time spent 80 184 17841 85 1177@ 602 22223 @ 6444 on surface (seconds) Mean time! 13.3 26.3 2230 17 61.9 @ 30.1 175.14 @ 52.87 landing (4.6) (7.1) (1943) (6.5) (25.6) (7.1) (82.9) (13.5)

(seconds)(SE) . Total eggs ------1163 11 295 laid Mean # Eggs/ ------6.41 @ 2.17 landing (SE) (1.5) (0.7)

Mean # Eggs! ------2.92 2.51 minute on (0.46) (0.42) surface (SE)

Table 5.3. Activities of adult Aedes aegypti mosquitoes on oviposition sites containing larval holding waters (LHW) prepared ,vith 100 unparasitized larvae (100U) or 100 larvae heavily parasitized by Plagiorchis elegans (100H). The numbers represent total activities ofmosquitoes over 5 days recorded on videotape.

@ Mann-Whitney U-test, P<0.05 # Egg numbers were transformed (Arcsine square root), followed by Student's t-test on paired samples, P

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Ikeshoji, T. and M. Mulla. 1970a. Overcr:owding factors of mosquito larvae. J. Econ. Entomo!. 63: 90-96.

Ikeshoji, T. and M. Mulla. 1970b. Oviposition attractants for four species of mosquitoes in natural breeding waters. Ann. • Entomo!. Soc. Am. 63: 1322-1327.

Ikeshoji, T., 1. Ichimoto, J. Konishi, Y. Naoshima, and H. Ueda. 1979. 7,1l-dimethyloctadecane: an ovipositional attractant for Aedes aegypti by Pseudomonas aeruginosa on capric acid substrate. J. Pesticide Sci. 4: 187-194.

Kats, L.B. and A. Sih. 1992. Oviposition site selection and avoidance of fish by streamside salamanders (Ambystoma barbouri). Copeia 1992: 468-473.

Kimoro, C.O. 1990. The effects ofPlagiorchis noblei, Park 1936 on the reproductive success and behaviour of adult Aedes aegypti. • MSc. thesis, McGill University. 188 • Klijnstra, J.W. 1986. The effect of an oviposition deterrent pheromone on egglaying in Pieris brassicae. EntomoI. Exp. AppI. 41: 139­ 146.

Klowden, M.L. 1982. Nonspecific effects oflarge doses of 20-hydroxyecdysone on the behaviour of Aedes aegypti. Mosq. News 42: 184-189.

Klowden, M.J. 1989. Influence of the ovaries and fat body on the initiation and termination of pre-oviposition behavior in the mosquito, Aedes aegypti. J. Insect PhysioI. 35: 567-570.

Klowden, M.J. 1990. The endogenous regulation of mosquito reproductive behavior. Experentia 46: 660-670.

• Knight, J.C. and S.A. Corbet. 1991. Compounds affecting mosquito oviposition: structure-activity relationships and concentration effects. J. Am. Mosq. Contr. Assoc. 7: 37-41.

Lowenberger, C.A. and M.E. Rau. 1993. Plagiorchis elegans: requirements for metacercarial development to infectivity, and conditions required for excystment. J. Helmintho!. Soc. Wash. 60: 67-71.

Macy, R.W. 1960. The life cycle of Plagiorchis vespertilionis parorchis, N. ssp. (Trematoda: Plagiorchiidael, and observations on the effects oflight on the emergence of the cercariae. J. Parasito!. • 46: 337-345. 189 • Maire, A. 1984. An analysis of the ovipositional response ofAedes atropalpus to experimental oviposition waters. Mosq. News 44: 325-329.

Maire, A. 1985. Effect ofaxenic larvae on the oviposition site selection by Aedes atropalpus. J. Am. Mosq. Control Assoc. 1: 320-323.

Maire, A., and R. Langis. 1985. Oviposition responses ofAedes (Ochlerotatus) communis (Diptera: Culicidae) to larval holding water. J. Med. Entomoi. 22: 111-112.

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Murdoch, W.W. and J. Bence. 1987. General predators and unstable prey populations. pp. 17-30 in Predation: Direct and Indirect Impacts on Aquatic Communities. C. Kerfoot and A. Sih, eds. University Press of England, London.

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Otieno, W.A., T.O. Onyango, M.M. Pile, B.R Laurence, G.W. Dawson, L.J. Wadhams, and JA Pickett. 1988. A field trial of the synthetic oviposition pheromone with Culex quinquefasciatus Say (Diptera: Culicidae) in Kenya. Bull. Ent. Res. 78: 463-478.

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CHAPTER6

GENERAL DISCUSSION AND

CONTRIBUTIONS TO ORIGINAL KNOWLEDGE

GENERAL DISCUSSION

• The central objective of this thesis was to examine various aspects of the transmission ofPlagiorchis elegans from the snai! first intermediate host to

the insect second intermediate host, and its consequences on subsequent

generations of conspecific larvae. More specifically 1 wanted to address

mechanisms utilized by the parasite ta increase the rate of transmission to

insects and the developmental requirements of the metacercariae within the

insect hosto Thus in Chapter 2, the emergence ofP. elegans cercariae was

. shown to cause a behavioural change in the molluscan hosto Infected

Stagnicola elodes moved ta the surface, and remained there for 2-3 hours,

during which time approximately 80% of aIl cercariae emerged. Since the

snails were relatively immobile, the cercariae were shed in a dense cloud • around the snail from which they were passively dispersed. One might 194 • tlrink that a dense cloud ofinfective cercariae would be maladaptivl' to transmission since insects entering the cloud would be so heavily

parasitized that rapid death would ensue. However, cercariae had a low

level ofinfectivity upon emergence. Under field conditions cercariae would

probably be dispersed prior to attaining maximum infectivity, hence

reducing the probability of superinfection and parasite induced mortality of

. hosts. The basis of the behavioural modification of the snail hosts has not

been studied. In tact, there are very few reports of trematode induced

behavioural alterations in any molluscan first intermediate host (Curtis

1987, de Jong-Brink 1990). While the benefit to the parasite is apparent,

one wonders what the effect is on predation of infected snails. Are immobile • snails at the water surface easier prey? Ifso, is this effect neutralized by the fact that cercariae emerge from the snail under low light conditions

whl;n inactive snails may be difficult to see? Direct observations of cercariae

emerging from the snaH (unpublished data) suggest that cercariae exit the

snail through the lung cavity via the pneumostome, and cercarial emergence

is accompanied by heavy mucous secretion by the snail. Damage ta the lung

cavity by thousands of emerging cercariae may affect air exchange, causing

the snail ta move ta the air-water interface. These are questions that should

he addressed ta further understand the interactions between digenean • parasites and their hosts during the process of cercarial emergence. 195 • l have shown in Chapter 2 that cercariae emerge en masse, and that cercarial infectivity changes over time. Of the thousands of cercariae

produc~d. each night, it is probable that few successfully find, penetrate, and

encyst in suitable insect hosts. The literature is vague on the developmental

processes and times required for metacercariae to i'each infectivity

(Williams 1963, Daniel and Ulmer 1964, Blankespoor 1977), and on the

specific environmental conditions under which metacercariai development

was measured. In Chapter 8) looked at the developmental rates ofP.

elegans metacercariae within an insect host in relation ta temperature. In

this study infectivity was equated with the ability of metacercariae ta excyst

in vitro in an artificial excystment medium modified after Bock (1986). The • overall deveJopment from the penetrating cercariae to an infective metacercariae was temperature dependent. Similarly, excystment of

infective metacercariae was temperature dependent; temperatures ~37C

were required in the presence of bile salts to elicit excystment. A range of

compounds and conditions were used to prepare earlier versions of the in

vitro excystment medium (unpublished data). In all combinations of media

tested, bile salts and high temperatures were required. As P. elegans is

essentially a parasite of homeotherms, response ta the two triggers,

appropriate temperature in the presence of bile salts, has probably arisen ta

elicit excystment only in environments likely to support the development of

adults. However, the means by which metacercariae receive the cue ta • excyst is not known. How do the juvenile worms perceive the bile salts? 196 • What mechanisms exist to aUow the stimuli to cross the cyst wall? Metacercariae that have been melanized by the host did not excyst in vitro

(unpublished data). Was this due to poorly developed parasites within

melanized cysts or did the juvenile worms not receive the stimulus to

excyst? Why were aU metacercariae not melanized? Melanization rates in

experimental infections normaUy ranged from 2-5%, although there were

occasions when 100% of the metacercariae were melanized. Was this due to

cohorts of mosquito larvae with weil developed defence mechanisms or.

cohorts of cercariae that were compromised? Other mosquito parasites can

inactivate the phenoloxidase cascade responsible for melanin production.

Did unmelanized metacercariae inactivate the hosts defences or were the • majority cf metacercariae not recognized as non-self? The recognition of non-selfin insect-parasite systems has received extensive investigation, yet

we do not fully understand the processes involved. These questions have

been raised by the results of Chapter 3, and should be addressed.

ln Chapter 3 1 also showed that there "las an 8 hour temperature

independent period of obligatory parasite-host contact required for

subsequent development of metacercariae in vitro. 1 hypothesized that this

period was one of high metabolic activity in which the parasite procured

essential nutrients from the host, or replaced reserves exhausted during the

cercarial stage. In Chapter 4 the metabolic activities of P. elegans • metacercariae, as determined by their ability ta take up and incorporate the 197 • sugar and protein precursors 3H-glucosamine and 3H-leucine, respectively, were fxamined. The resuJts showed that indeed, young metacercariae

absorbed more of these compounds than did old metacercariae, thus

supporting the hypothesis in Chapter 3. The majority of 3H-glucosamine

was incorporated into glycoproteins within the juvenile wormtl. The data

presented showed a dependency of P. elegans metacercariae on the host for

the nutrients required for development, without which, presu-TIlably, the

metacercariae would die. The mechanism of nutrient transfer across the

cyst wall remains to be studied. Other species use facilitated diffusion or

active transport. Despite the physiological dependency on the mosquito for

development of metacercariae, as few as 3 metacercariae!larva can kill the • hosto The factors that are responsible for the death of P. elegans-infected A. aegypti larvae are not known. Loss of haemolymph during cercarial

penetration, damage to larval tissues during cercarial migration within the

insect, or the introduction ofprotozoan parasites with penetrating cercariae

may aIl play a role. While sorne parasites routinely kill their hosts te

complete their life cycle (Petersen 1984, 1985), it is highly maladaptive for

P. elegans to do so. Death of the larva results in death of metacercariae, and

no transmission to a definitive hosto

ln Chapter 1 1 cited several studies suggesting that digeDeans such as P.

elegans be incorporated into biological control programmes for the control of • mosquitoes. Successful control programmes require that mosquito larvae 198 • and the controlling agent overlap spatially and temporally. Natural selection would tend to favour species that avoid ovipositing in sites

containing parasites, predators and pathogens. One question addressed in

Chapter 5 was whether mosquitoes would oviposit selectively in the

presence ofparasitizet1Jr unparasitized larvae, or in waters which had

previously held these larvae. Mosqcitoes landed fewer times (although this

was not significant1y different from controls), remained for shorter periods,

and laid fewer eggs on waters that had previously contained parasitized

larvae. Did mosquito larvae produce oviposition deterrent or repellent

compounds that resulted in reduced oviposition? Chesson (1984) reported

that mosquitoes laid fewer eggs in sites containing notonectid predators, • although whether this was due to the presence of the predator, dead larvae, or chemicals produced by larvae was not addressed. The identification of the

oviposition deterrent compound and determination of the mechanisms

leading to its production and release by the parasitized insect will allow us

ta understand in more detail the tactics used by mosquitoes to maximize

their fitness. The implications of these results can be extrapolated to other

biological control systems. Are reduct.ions in target species due to introduced

biological control agents, or due to selective oviposition by the target species

in response to the introduced control agent? Researchers in this field are • faced with the task of determining the relative weights of both factors. 199 • In conclusion, this thesis has explored aspects of the transmission of Plagiorchis elegans from the molluscan first intermediate host ta the insect

second intermediate host, and the requirements for parasite development.

In addition it has addressed a possible mechanism by which mosquitoes

may avoid ovipositing in waters in which their larvae would be parasitized

and killed by the parasite. Further studies should address the mechanism

by which cercariae kill the host, and the identification and synthesis of the

oviposition deterrent or repellent compound produced by parasitized larvae. •

• 200 • CONl'RmUTIONS TO ORIGINAL KNOWLEDGE

1. Parasite induced host behavioural changes were demonstrated in

snails (Stagnicola elodes) during Plagiorchis elegans emergence.

During cercarial emergence, infected snails moved to the top of the

water column and remained there, relatively motionless, for 2-3

hours.

2. The position of cercariae within the water column and the rate of

settling was shown for P. elegans. Cercariae emerged at the top of the • water column, and settled within 2-4 hours following emergence. 3. The infectivity of P. elegans cercariae was characterized. Infectivity

was low upon emergence, peaked 4-6h post emergence, and declined

to <5% by 24h.

4. The temperature-time relationship for the development of P. elegans

metacercariae within Aedes aegypti larvae was determined: overall

development was temperature dependent.

5. While overaIl development of P. elegans metacercariae was

temperature dependent, an 8 hour temperature-independent period of • obligatory parasite-host contact was demonstrated. 201 • 6. The presence of bile salts and high temperatures (~37C) were shown to be required for in vitro excystment of infective P. elegans

metacercariae.

7. The metabolic activity of P. elegans metacercariae, as measured by

the uptake of 3H-glucosamine and 3H-Ieucine, was quantified. Both

compounds were taken up by metacercariae removed from the insect

host 2-48h post infection. Proportionally, more of each precursor was

taken up by younger (~8h post infection) metacercariae.

8. The distribution of 3H-glucosamine and 3H-Ieucine into juvenile • worms and empty cyst walls was determined. The former was incorporated almost exclusively inta juvenile worms while the latter

was incorporated into both the juvenile and the cyst wall.

9. 3H-glucosamine incorporation inta metacercarial glycoproteins was

shown to be developmentally regulated or stage specifie, as

determined using SnS-PAGE.

10. The oviposition response by adult A. aegypti was unaffected by the

presence of snails (uninfected, or infected and shedding cercariae), or

P. elegans cercariae in the absence of the snail first intermediate • hosto • W2 11. Oviposition by adult Aedes aegypti was significantly reduced in

waters containing P. elegans-parasitized larvae, compared with

waters containing Wlparasitized larvae.

12. Oviposition by adult A. aegypti was significant1y reduced in waters

which had previously contained P. elegans-infected A. aegypti larvae.

13. Oviposition by adult A. aegypti was significantly reduced in larval

holding waters which had been boiled, filter sterilized, or treated with

antibiotics to curb bacterial growth.

• 14. The behaviour of adult A. aegypti in the presence oflarval holding waters was quantified. Gravid females did not land significant1y

fewer times on waters which had contained parasitized larvae.

However, they remained there for significantly less time and laid

significant1y fewer eggs on waters prepared with parasitized larvae.

• 203 • LITERATURE CITED

Blankespoor, H.D. 1977. Notes on the biology of Plagiorchis noblei Park, 1936 (Trematoda: Plagiorchüdae). Proc. Helminthol. Soc. Wash. 44: 44-50.

Bock, D. 1986. ln vitro excystment ofthe metacercariae of Plagiorchis species 1 (Trematoda, Plagiorchiidae). Int. J. Parasitol. 16: 641-645.

Chesson, J. 1984. Effect of notonectids CHemiptera: Notonectidae) on mosquitoes (Diptera: Culicidae): predation or selective oviposition? Environ. Entomol. 13: 531-538.

Curtis, LA 1987. Vertical distribution of an estuarine snail altered by a • parasite. Science 235: 1509-1511. Daniell, D.L. and M.J. Œmer. 1964. Life cycle of Plagiorchis noblei Park, 1936 (Trematoda: Plagiorchüdae). J. Parasitol. (Abstract). 50: 46.

de Jong-Brink, M. 1990. How trematode paras!tes interfere with reproduction of their intermediate hosts, freshwater snails. J. Med. and Appl. Malacol. 2: 101-133.

Petersen, J.J. 1984. Nematode parasites ofmosquitoes. pp. 797-847 in Plant and Insect Nematodes. W.R. Nickle ed. Marcel Dekker Inc. New York.

Petersen, J.J. 1985. Nematodes as biological control agents: Part I. • Mermithidae. Adv. Parasitol. 24: 307-345. 204 • Williams, R.R. 1963. Life history studies on four digenetic trematodes that utilize Lymnaea (StagnicolaJ reflexa (Say) as their first intennediate host in a temporary pond habitat. PhD thesis, Ohio State University.

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