Mermithid (Nematoda: Mermithidae) infections of black flies (Diptera: Simuliidae):

seasonal variation and developmental characteristics

by

Amy Sharp, B.Sc.

A thesis

submitted to the Department of Biological Sciences

in partial fulfillment of the requirements

for the degree of

Master of Science

April 2007

Brock University

St. Catharines, Ontario

® Amy Sharp 2007

Abstract

Mermithid nematodes (Nematoda: Mermithidae) parasitize larval, pupal and adult black flies (Diptera: Simuliidae), oftentimes resulting in partial or complete host feminization. This study was designed to characterize parasite-host seasonal variation and to estabUsh the developmental life stage at which feminization is initiated.

Data indicate that the total adult population of black flies collected from

Algonquin Provincial Park throughout the spring of 2004 was comprised of 31.8% female, 67.8% male and 0.4% intersex individuals. Of the total population, 0.6% was infected by mermithid nematodes (69.0% female, 3.5% male and 27.6% intersex).

Seasonal infection trends established over a 12-month period revealed that black flies

with different life histories host the same mermithid subfamilies, while black flies with

similar life histories host mermithids from different subfamilies. If a simuliid species simultaneously hosts two mermithid species, these parasites are from different subfamilies. Molecular mermithid identification revealed two mermithid subfamilies,

Me.somermithinae and Gastromermithinae, present in the simuliid hosts. Mermithid colour variation was not found to be a reliable species indicator.

The developmental stage at which feminization is initiated was determined by examining gonad morphology and meiotic chromosomal condition. Results indicate that mermithid-infected black flies exhibit feminization prior to larval histoblast formation.

Larvae can be morphologically male (testes present) or female (ovaries present), with morphological males exhibiting either male (achiasmate) or female (chiasmate) meiotic chromosomes; morphological females were only genetically female. Additionally, mermithid infection inhibits simuliid gonad development.

Acknowledgements

I owe heartfelt thanks to my sup)ervisor. Dr. Fiona F. Hunter, for allowing me the opportunity to conduct this study and guiding me through my development as a scientific researcher. Dr. Henry Cuevas offered assistance with statistical analyses. Dr. Youself

Haj-Ahmad, Dr. Jean Richardson and Dr. Miriam Richards offered direction and time as committee members. Dr. Brad Hyman (University of California Riverside) provided assistance and advice with nematode identification. Dr. Murray Colbo provided time and advice as the external examiner for this thesis. Cheers to my field assistant, Craig Ross, for sacrificing pints of blood in the name of science; to all Fly Lab hooligans for entertaining my endless questions and banter, especially to Curtis Russell for putting up with my .seemingly insane behaviour from time to time. Brad Baker and Claire Gamer assisted with sample processing. The Wildlife Research Station provided the stage for two extraordinary field seasons, with access to collection sites made possible by the

Ministry of Natural Resources and Algonquin Provincial Park. Support for this research was provided by Dr. Fiona F. Hunter through a grant from the Natural Sciences and

Engineering Research Council. I would also like to remember the late Mike Spironello, who.se contagious enthusiasm towards black flies and unparalleled fervour for life will be remembered by many.

Table of Contents

Page

Abstract 1

Acknowledgements 2

Table of Contents 3

List of Figures 6

List of Tables 8

1. Introduction 9

1.1. Black flies 9

1.1.1. Black fly history 9

1.1.2. Simuliid larval biology 10

1.1.3. Simuliid pupal biology 14

1.1.4. Simuliid adult biology 16

1.1.4.1. Simuliid adult eclosion 16

1.1.4.2. Simuliid sexual dimorphisms 16

1.1.5. Simuliid Mating 22

1.1.6. Oviposition 23

1.2. Mermithid nematodes 24

1.2.1. Mermithid biology 24

1.2.2. Mermithid development 25

1.3. Mermithid-simuliid interactions 26

1.3.1. Mermithid parasitism of black flies 26

1.3.2. Mermithid-simuliid life cycles 27

1.3.3. Host feminization 28

1.3.4. Evolutionary strategies 33

1.3.5. Trends in mermithid-simuliid populations 34

1 .4. Nematode identification 38

1.4.1. Traditional identification techniques 38

1.4.2. Molecular identification 39

1 .5. Sex determination of mermithid-infected simuliid larvae 40

1.6. Simuliid control measures 41

1.7. Purpose 42

2. Materials and Methods 43

2.1. Collections 43

2.1.1. Collection sites 43

2.1.2. Collection intervals 50

2.1.3. Larval and pupal simuliid collections 50

2.1.4. Adult simuliid collections 51

2.1.5. Mermithid collections 52

2.2. Specimen processing 55

2.2.1. Simuliid larval processing 55

2.2.2. Simuliid pupal processing 57

2.2.3. Simuliid adult processing 57

2.2.4. Mermithid processing 57

2.2.5. Specimen storage 58

2.3. Proportion of adult sexes from emergence trap collections 59

2.4. Monthly variation of parasite-host populations 59

2.4.1. CollecUon 59

2.4.2. Sample processing 60

2.5. Mermithid identification 60

2.6. Larval sex determination 61

2.6.1. Staining and dissection 61

2.6.2. Gonad morphology 62

2.6.3. Genetic sex 63

2.7. Statistical analyses 67

2.7.1. Monthly variation 67

2.7.2. Gonad morphologies 68

2.7.3. Gonad chromosomal conditions 68

3. Results 70

3.L Collections 70

3.2. Specimen processing 70

3.3. Adult sex proportions from emergence trap collections 71

3.4. Monthly variation of parasite-host populations 71

3.5. Mermithid Identification 76

3.6. Larval sex determination 80

3.6.1. Morphological sex determination 80

3.6.2. Genetic sex determination 84

4. Discussion 87

4.1. Collections 87

4.2. Specimen processing 87

4.3. Adult sex proportions from emergence trap collections 88

4.4. Monthly variation of parasite-host populations 89

4.5. Mermithid identification 90

4.6. Larval sex determination 94

4.6.1. Morphological sex determination 94

4.6.2. Genetic sex determination 94

5. Conclusions 97

6. Summary 102

Literature Cited 103

Appendices 112

Appendix 1 Recipes 112

Camoy's fixative 112

Feulgen stain 1 12

Sulphur dioxide water 1 12

50% Acetic acid 1 12

2% Aceto-carmine 112

Appendix II Total number of simuliid larvae and/or pupae collected from Costello and Mud Creeks from April 30 to August 19, 2005. No collections were made between June 29 and July 27, 2005. While 5. truncatum/venustum/rostratum numbers exceeded 250 during the summer months, the y-axis reaches a maximum of n=250 to highlight rarer species numbers 113

Appendix III Percent mermithid infection according to simuhid species collected from Algonquin Provincial Park from April 30 to August 18, 2005. No

collections were made between June 29 and July 27, 2005 1 14

Appendix FV Number of each mermithid colour variant that parasitized simuliid larvae, pupae and/or adults with respect to simuliid species. All samples were collected from Costello Creek and Mud Creek during the spring and summer of 2005 115

Appendix V Mean monthly air temperatures for Algonquin Provincial Park, obtained

from Environment Canada Climate Data Almanac 1 16

List of Figures

Figure 1 . Left lateral view of a Simulium venustum Say larva in the pharate pupal stage of development. (Obtained ft"om Adleret al. 2004) 13

Rgure 2. Left lateral view of a Simuliium vittatum Zetterstedt pupa enclosed in the pupal casing. (Obtained from Adler et al. 2(X)4) 15

Figure 3. Scanning electron micrographs of the heads of adult Simuliidae: male holoptic eyes (a) and female dioptic eyes (b). SEM images taken at Brock University by Glenda Hooper 19

Figure 4. Scanning electron micrographs of tarsal claws from male (a) and female (b)

black flies. All female simuliids lack the overhanging grapple of the male claws. Omithophilic females tend to exhibit a basal tooth on the claw (male SEM taken at Brock University by Glenda Hooper, female SEM obtained fromCrosskey 1990) 20

Figure 5. Left lateral views of simuliid adult male (a) and female (b) terminalia (adapted from Crosskey 1990). Externally, female simuliids exhibit characteristic cerci and anal lobes while males exhibit definite gonostyli 21

Figure 6. Scanning electron micrograph of an intersex adult black fly head exhibiting.

The fly's right side is a holoptic male eye, while the left is a dioptic female eye. SEM taken at Brock University by Glenda Hooper 31

Figure 7. Interactions of the mermithid - simuliid life cycles (obtained from Crosskey 1990) 32

Figure 8. Map of Algonquin Provincial Park, Ontario, indicating Costello Creek (C) and Mud Creek (M) collection sites. Inset shows location of the park in Ontario (Canoe Routes of Algonquin Provincial Park) 45

Figure 9. Topographic map of Costello Creek. Circled area depicts sample site (Natural Resources Canada) 46

Figure 10. Topographic map of Mud Creek. Circled area depicts sample site (Natural Resources Canada) 47

Figure 11. View of downstream Costello Creek in mid-spring, 2004 48

Figure 1 2. View of upstream Mud Creek in mid-spring, 2004 49

Figure 13. A pan trap placed along the bank of Costello Creek, Algonquin Provincial Park. Photograph taken in June, 2005 53

Figure 14. An emergence trap suspended over trailing vegetation and sticks in Mud Creek, Algonquin Provincial Park. Photograph taken in May, 2004 54

Figure 15. Dorsal view of a Prosimulium mixtum larva with developing gill histoblasts marked. (Image obtained from Adler et al. 2004) 56

Figure 16. Morphology of larval simuliid gonads. Ovaries are elongated (a), while testes are round (b) 65

Rgure 17. Chromosomal condition of larval simuliid gonads during Meiosis I.

Chiasmate meiosis (a) is typical of female simuliids, while achiasmate meiosis

(b) is typical of male simuliids. Gonads without visible condensed chromosomes were "unscorable" (c) 66

Figure 18: Prevalence of mermithid-infection in mermithid-susceptible larvae collected from February 2005 to January 2006 in Algonquin Provincial Park. The

number of mermithid-infected larvae versus uninfected larvae is shown. The y-axes have different scales 73

Figure 19. Total number of mermithid infected Simuliidae collected from Algonquin Provincial Park between April 30 and August 18, 2005. No collections were made between June 29 and July 27, 2005 75

Figure 20. Preliminary distance tree based on COl sequences of mermithids recorded from black flies in Algonquin Provincial Park; data obtained from Dr. Brad Hyman (University of California at Riverside) 79

Figure 21. Frequencies of observed gonad morphologies in mermithid-infected and uninfected early instar P. fuscum/mixtum and 5. truncatum/venustum/ rostratum larvae. Morphologies were classified as having two ovaries (OO), one ovary (O), two testes (TT), one testis (T) or no gonads present (None).

Morphologies of infected versus uninfected specimens differed (F-F-H (4j = 34.99, P < 0.001). An asterisk (*) indicates a zero count 81

Rgure 22. Frequencies of gonad morphologies in mermithid-infected and uninfected P. fuscum/mixtum and 5. truncatum/venustum/rostratum pharate pupae. Morphologies were classified as two ovaries (OO), one ovary (O), two testes (TT), one testis (T) or no gonads present (None). Morphologies of infected

versus uninfected specimens differed (F-F-H [4] = 57.81, P < 0.001). An asterisk (*) indicates a zero count 82

Figure 23. Time line of interactions between simuliid and mermithid species in Algonquin Provincial Park. The presence of simuliid larvae (L), pupae (P) and adults (A) is indicated above the box depicting simuliid and mermithid presence. Mermithid species are paired with host simuliid species according to data obtained from molecular studies 95

List of Tables

Table 1. Two subfamilies of mermithid nematode were identified using DNA sequence

data from the COI, 1 8S and 28S genes, with three species branching out within each subfamily. Mermithid colour and simuUid species association are listed (clear = C, white = W, green = G, pink = P, yellow = Y), with each letter indicating the presence of that particular mermithid colour morph within the respective simuHid host species 77

Table 2. Number of mermithid nematodes of five colour categories found in different simuliid host species in Algonquin Provincial Park in 2005. The number of mermithid nematodes sent for barcoding to Dr. Brad Hyman (University of California at Riverside) is shown in parentheses 78

Table 3: Comparison of the number of gonads of each morphology present in P. fuscum/mixtum and S. rostratum/venustum/truncatum early larvae versus pharate pupae. Separate tests were done for each infection status group. Fisher- Freeman-Halton exact tests found no significant difference exists between the two developmental stages, in both uninfected and mermithid-infected specimens 83

Table 4. Influence of infection status on black fly larval gonad chromosomal conditions. Developmental stages were kept as independent variables. Fisher-Freeman- Halton exact tests found significant differences exist between uninfected and mermithid-infected simuliid larvae of both morphological sexes in early larvae and pharate pupae 85

Table 5. Influence of developmental stage on chromosomal conditions in the gonads of simuliid larvae. Infection statuses were kept as independent variables. Fisher- Freeman-Halton exact tests found no significant variation exists between developmental stages with respect to chromosomal condition, regardless of sex or infection status 86

1. Introduction

1.1. Black flies

1.1.1. Black fly history

Black flies (Diptera: Simuliidae) exhibit a global distribution, bridging both aquatic and terrestrial environments. As aquatic organisms, they are beneficial by filtering dissolved organic matter fi-om the water and filling a role in the food web as prey for other macroinvertebrates and fish. As terrestrial organisms they are pests and transmitters of disease resulting from their conspicuous blood feeding behaviour

(Clubber 1998), a food source for birds, possible pollinators of plants and thieves of nectar (Hunter et al. 20(X)).

First appearing in the fossil record 1 20 million years ago (Currie and Walker

1992), Simuliidae have remained virtually unchanged ever since. There are more than

1 809 recognized simuliid species known to date ( 1 798 extant and 1 1 fossil) (Adler et al.

2004, Crosskey and Howard 2004), a number that continues to grow in concert with the advent of new and improved species identification technologies. Simuliid larval structure has adapted to a filter-feeding lifestyle, with developing larvae and pupae occupying the substrate of highly oxygenated riffles of flowing freshwater. Primarily haematophagous,

female adult Simuliidae are biting (>ests. Not only are the characteristic swarming and blood feeding behaviours of simuliid females a nuisance, but black flies can also act as vectors of vertebrate pathogens (Clubber 1998). Simuliids carry filarial Onchocerca sp. to humans and livestock; in humans this causes onchocercia.sis, or "river blindness", a

debilitating disease that renders victims blind and physically incapacitated (Grillet et al.

10

2001). Simuliidae also transmit filarial Omithofilaria and the protozoan Leucocytozoon

to birds (Werner and Pont 2003, Mondet and Bernard 1981). Black flies reduce yields from livestock in terms of dairy and meat production (Crosskey 1990), while the irritating haematophagous behaviours have a negative impact on tourism in several areas of the world, including the Canadian Shield.

1 . 1 .2. Simuliid larval biology

Larval black flies are easily identifiable, aggregating in highly oxygenated, unpolluted, flowing freshwater habitats, and forming a large portion of the benthic

biomass (Werner and Pont 2003). Larvae can be found on virtually all available

submerged solid substrates, including trailing vegetation, wood, and rocks (Merritt et al.

1982). Simuliid larvae exhibit characteristic labral fans arising from the anterior portion of a heavily sclerotized head capsule, which attaches to a club-shaped body with

prothoracic and posterior prolegs. Each proleg has a circlet of hooks on its apical end used to secure the larva to a silk pad secreted onto the substrate by the mouthparts, as well as for locomotion. These appendages are present throughout larval development,

from hatch to pupation (Adler et al. 2004).

Upon completion of embryogenesis, which ranges from four days to eight months

depending on the species and if the egg has undergone diapause (Currie 1986), first instar larval simuliids utilize an egg burster on the cephalic apotome to assist in egg eclosion

(Alencar et al. 2001, Ross and Merritt 1978). Simuliid larvae then undergo a moulting process between each of approximately six developmental instars, increasing in size with each moult (Currie 1986, Currie and Craig 1987). Growth rates increase with each larval

11

instar, with over 81% of growth occurring during the final two larval instars; larval

development is influenced by nutrient availability (dissolved organic content) in the

streams, temperature and parasitism (Bernardo et al. 1986, Merritt et al. 1982, Colbo

1982). Mature larvae measure up to 15 mm long in the largest simuliid species, and only

4 mm long in the smallest, with an average size of approximately 9 mm in most species

(Crosskey 1990).

Internally, simuliid larvae are composed of alimentary, silk-producing, respiratory, muscular, nervous, and reproductive systems. The alimentary system consists

of two main comf)onents: i) the digestive tract, and ii) the excretory Malpighian tubules.

A separate silk-producing system consists of two long salivary glands running along the

ventral side of the body cavity, connecting to silk ducts. The silk produced is used for substrate attachment and cocoon spinning.

Larval black flies have no specialized organs for the acquisition of "free" air

(Crosskey 1990), since they remain submerged in an aquatic environment, only surfacing upon adult eclosion. The haemolymph and fat body of larval Simuliidae reflect the overall physiological condition of the individual, acting as an area for metabolism through nutrient storage, hydrolysis, transformation and transport (Rubstov 1989). The muscular and nervous systems of larval simuliids permit the larvae to perform a wide

range of activities, ranging from locomotion to filter feeding (Adler et al. 2004, Rubstov

1989).

The simuliid larval reproductive .sy.stem consists of two gonads that foreshadow

ftilly developed testes and ovaries of adult black flies (Alencar et al. 2001 , Crosskey

12

1990). These gonads form a pair of small organs located near the dorsal body wall of the sixth abdominal segment, just posterior to the Malpighian tubules. Female gonads are elongated "sausage-shaped" ovaries, while male gonads are rounded "pear-shaped" testes

(Adler et al 2004, Alencar et al. 2001, Crosskey 1990, Rothfels and Dunbar 1953). In

ovaries, chromosomal divisions during Meiosis 1 are chiasmate, exhibiting genetic crossover, while testes divisions are achiasmate, not exhibiting genetic crossover

(Rothfels 1979, Moens 1978). Increased rapid development of the gonads occurs during the final two larval instars (Rubstov 1989).

The final larval instar stage is referred to as the pharate pupal stage. The term

"pharate" is used to describe the stage in which a new cuticle of any instar has developed and an old cuticle from the previous instar has not yet been shed (Crosskey 1990).

Pharate pupae possess gill histoblasts present laterally on the thorax region of the larval

body (Figure 1). Gill histoblasts ultimately form the respiratory organs used throughout the pupal stage.

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14

1.1.3. Simuliid pupal biology

The pharate pupa spins a cocoon, known as the pupal casing, from salivary silk secreted by salivary glands just prior to pupation. Morphological characteristics of the pupal casing are species specific, ranging from a tightly woven housing to a loosely woven pad upon which the pupa sits, partially or completely enclosing the developing pupa. The pupal casing also acts as an anchor, securing the pupa to the substrate (Adler et

al. 2004, Rubstov 1989). The length of the pupal stage depends on both environmental conditions and the simuliid species, lasting from four days to two weeks (Currie 1986).

The pupal black fly is incapable of locomotion, with any movement confined to rotation of the body as a whole within the cocoon. During pupation the organism does not feed, relying on the pupal gills, derived from the histoblasts of the pharate pupae, which branch out beyond the pupal casing into the surrounding environment as respiratory organs

(Currie 1986) (Figure 2). Once the simuliid has undergone transition from a pharate pupa to an actual pupa, the internal organs and tissues become a liquid-like mass held within

the shell of the pupal body. It is during this stage that the intricate transformation from larva to adult occurs. Gradually, adult features develop, such as the head, eyes, antennae,

mouthparts, thorax, spiracles, legs, abdomen and terminalia (Figure 2).

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16

1 . 1 .4. Simuliid adult biology

1.1.4.1. Simuliid adult eclosion

Once pupation is complete and the simuliid is prepared to emerge as an adult, the

pharate adult releases air from its respiratory system, essentially blowing a self- enveloping air bubble. This action also causes the pupal cuticle to crack open from increased gas pressure, followed by the pharate adult both exiting the exuvium and floating to the surface in an air bubble (Currie 1986). In some species, such as Cnephia dacotensis (Dyar & Shannon) and the Simulium decorum Walker and Simulium vittatum

Zetterstedt complexes, if the pupal casing is attached to a plant stalk or some form of support that leads to the water surface, the adult crawls out of the pupal exuvium and follows the stalk to the surface (Crosskey 1990). Once at the surface, the emerged adult

commences the aerial stage of its lifecycle, composed primarily of feeding and mating.

Adult Simuliidae exhibit protandry, wherein males start to emerge prior to females

(Rubstov 1989).

1.1.4.2. Simuliid sexual dimorphisms

Adult black flies display distinct sexual dimorphisms in the eyes, tarsal claws and terminalia, as well a.s distinct sex-specific behaviours (Crosskey 1990). For vision and light perception, black flies rely entirely on compound eyes composed of many individual ommatidia, or facets (O'Grady and Mclver 1987). Female adult black flies maintain dioptic eyes, separated by a distinct sclerite on the upper vertex of the head know as a frons. Facets of the female eyes are uniform in size (Peterson 1981 , Davies 1978, Torre-

Bueno 1985) (Figure 3). Adult male black flies utilize holoptic eyes, lacking a frons and

17

having dorsal facets much larger than the ventral facets, specialized to detect females flying above the swarms (Wenk 1987, Peterson 1981, Davies and Peterson 1956) (Figure

3).

Adult male black flies have a large hood-like grapple overhanging the base of the claw, whereas tarsal claws of female black flies lack a grapple (Crosskey 1990). The

grapple is used to grasp the female abdomen by the male black fly during copulation

(Craig and Craig 1986) (Figure 4). Adult female simuliid claw structure reflects host type. Some species of adult female black flies have adapted a basal tooth on the tarsal claw, creating a bifid claw used to cling to a host while blood feeding (Malmqvist et al.

2004, Crosskey 1990) (Figure 4). Mammalophilic species tend to have simple claws,

lacking basal teeth, while omithophilic species have bifid claws. It is hypothesised that the bifid claw design facilitates movement through host feathers (Malmqvist et al. 2004,

Crosskey 1990). Simulium rostratum and Simulium truncatum are examples of

mammalophilic black fly species with simple claw morphologies (Malmqvist et al. 2004).

Cnephia omithophilia is an omithophilic species with bifid claw morphology.

Adult simuliid abdomens consist of nine segments, commonly referred to as tergites, with the tenth segment modified into sexually dimorphic terminalia utilized in

mating and oviposition (Figure 5). Male simuliid genitalia are composed of the gonopods

and the aedeagus with associated parameres (Figure 5). The gonopods, also referred to as claspers, are a pair of two-.segmented appendages comprising a large basal subconical gonocoxite and a distal longer, slender gonostylus, u.sed to grasp the female between the ninth and tenth abdominal segments (Wood et al. 1963). The aedeagus, also referred to as the ventral plate, is a tubular muscular structure with two or three sclerites associated

18

with its walls (the ventral, median and dorsal plates). The aedeagus has a role in gonopod articulation and support during mating. The parameres, one on either side of the aedeagal

base, articulate with the aedeagus and gonocoxite (Adler et al. 2004).

Female black fly terminalia begin on the eighth abdominal tergite, with a posterior hypogynial valve present as the functional ovipositor. This is followed by the well developed ninth abdominal segment, connecting the lateral arms of the Y-shaped genital

fork. The next abdominal segment, the tenth tergite, is small with a sternum composed of

two pairs of cerci and anal lobes (Figure 5). Anal lobes are utilized during mating, coupling with the male claspers and involved with spermatophore transfer. Setae-covered cerci lie just posterior to the anal lobes, leading to the internal spermatheca used to store

sperm. Simuliidae possess a single spermatheca (Adler et al. 2004).

19

a)

— dorsal facets

ventral facets

b)

frons

Figure 3. Scanning electron micrograplis of tlie tieads of adult Simuliidae: male holoptic eyes (a) and female dioptic eyes (b). SEM images taken at Brock University by Glenda Hooper.

20

grapple

b)

basal tooth

Figure 4. Scanning electron micrograplis of tarsal claws from male (a) and female (b) black flies. All female simuliids lack the overhanging grapple of the male claws. Omithophilic females tend to exhibit a basal tooth on the claw (male SKM taken at Brock University by Glenda Hooper, female SEM obtained from Crosskey 1990).

21

a)

paramere

gonostylus

gonocoxite

b)

spermatheca

cercus

anal lobe

genital fork

hypogynial valve

Figure 5. Left lateral views of simuliid adult male (a) and female (b) terminalia (adapted from Crosskey 1990). Externally, female simuliids exhibit characteristic cerci and anal lobes while males exhibit definite gonostyli.

22

1 . 1 .5. Simuiiid Mating

Male Simuliidae eclose into adulthood ready to mate, with seminal vesicles full of sperm; in some species females also emerge ready to mate, with the ovaries full of ova.

However, in most simuiiid species, the female emerges with partially developed eggs, requiring a sugar meal to facilitate the post-pupal phase of ova development. Adult black flies of both sexes feed on honeydew produced from aphid excretions, flower nectar or

occasionally sap as energy sources (Burgin and Hunter 1997, Rubstov 1989, Davies et al.

1962). In most simuiiid species, females often disperse from their emergence site in search of a blood meal to finalize ova development, sometimes flying several kilometres

(Crosskey 1990). Some female simuiiid species, such as Simuliium vittatum, do not

require a blood meal to mature the ova (Bernardo et al. 1986). SimuUid females that do not require blood meals are referred to as autogeneous, carrying nutrients over from the larval stage of development to support a single gonotrophic cycle. Such females usually emerge with mature ova and mate close to the stream from which they emerged, and oviposit shortly thereafter (Malmqvist et al. 2004, Davies and Peterson 1956). Some species have anautogeneous females, requiring a blood meal for ova maturation

(Malmqvist et al. 2004), while other simuiiid species eclose into autogeneous adults, but have subsequent gonotrophic cycles that require anautogeneous behaviour.

Mating generally occurs in massive swarms, especially in northern temperate regions where harsh winters constrain development and mating time (Davies and

Peterson 1956). To increa.se the chances of successful mating in these regions, simuiiid adult males have evolved swarming behaviours and morphologies to support such practices. Unlike in temperate regions, simuiiid mating and development in warmer

23

equatorial regions are not restricted by environmental demands, and the flies have

therefore not evolved swarming behaviours to support rushed mating (Wenk 1987). In

such regions, it is more common to see ground mating, that is, with no swarm formation

occurring (Crosskey 1990). In northern regions, Cnephia dacotensis is one of the only

species to mate on the ground without swarming (Adler et al. 2004), although such terrestrial mating does involve thousands of individuals in one spot, which technically could be considered "terrestrial swarming". Aerial swarms are most often formed at a height of between two to five meters above ground level, depending on the size of the swarm itself. Swarming occurs during daylight hours, with peak numbers occurring just before dusk (Crosskey 1990). Females that fly over a swarm are sighted by the males, chased and mated with (Davies 1978, Peterson 1962). Male simuliids that undergo characteristic ground mating without swarming, such as C. dacotensis (Davies and

Peterson 1956), do not require the massive holoptic eyes as in swarming species, thus displaying dioptic eyes with uniform facets (Blenkhom 1998, Davies 1978).

1.1.6. Oviposition

It is believed that female black flies mate only once during their lifetime, fertilizing all ova with a single insemination, even though the oviposition might occur in

separate batches (Crosskey 1990). In P. mixtum, fecundity of an individual female is

positively related to adult body size as well as larval nutrition (Malmqvist et al. 2004,

Colbo 1982, Chutter 1970).

After mating and ova maturation, gravid females return upstream to oviposit fertilized eggs (Hunter and Jain 2000). h has been suggested that simuliid females utilize

24

visual and anaemotactical (air sensing) cues to locate suitable upstream oviposition sites

(Hunter and Jain 2(X)0, Crosskey 1990, Grenier 1949 in Colbo and Moorhouse 1979).

One signal is the hue of the site itself; green and yellow substrates resemble healthy

vegetation, and are thus chosen by black flies (Golini and Davies 1975). Ovipositional

swarming occurs by gravid female black flies at such ideal oviposition sites above

flowing bodies of water, since simuliid ova must be in close proximity to water to prevent

desiccation. The tendency for gravid females to return upstream is to compensate for larval drift downstream in the natal current (Davies and Peterson 1956, Crosskey 1990).

Gravid females oviposit either directly on the surface of the water or onto substrate that lie beneath the water's surface, such as trailing vegetation, sticks or rocks (Golini and

Davies 1987, Imhof and Smith 1979, Davies and Peterson 1956); such behaviours are species specific.

1 .2. Mermithid nematodes

1.2.1. Mermithid biology

Nematodes play a significant role in the energy cycle of the biosphere. They are found globally and are the most numerous of all invertebrate groups with species numbers estimated between 100,000 and one million (Malakhov 1994). Mermithid nematodes (Nematoda: Mermithidae) are primarily parasites of insects, yet they also infect spiders, crustaceans, earthworms, leeches and molluscs (Vandergast and Roderick

2003. Poinar 1979). The mermithids in this study are aquatic and obligate parasites.

These mermithid nematodes require aquatic hosts, and inhabit the .same aquatic

25

environments during their pre- and postparasitic stages of development as their aquatic

hosts (Rubstov 1989). These endoparasitic mermithids tend to exhibit host species

specificity, with host kiUing almost always being the ultimate outcome of infection

(Poinar 1979).

1 .2.2. Mermithid development

Mermithids have three stages of development: preparasitic, parasitic and postparasitic. The preparasitic phase of life includes both ova and juvenile nematodes. As aquatic organisms, mermithids commence their life cycle as ova in the substrate of a flowing body of water that is co-inhabited by a healthy host population (Malakhov 1994).

Once fully developed, juvenile mermithids eclose from the ova and enter the stream water from the substrate. The preparasitic juvenile is extremely small, less than 300 pm long (Mondet and Bernard 1981), and exhibits nondirectional swimming behaviour, eventually making physical contact with a suitable host (Molloy and Jamnback 1975).

Once this association is made the preparasitic juvenile attaches to the early instar host and uses a stylet to penetrate the cuticle. The mermithid then enters the host haemocoel within

a matter of minutes, during which time the host is temporarily paralyzed (Bailey et al.

1977, Molloy and Jamnback 1975). Once in the host haemocoel, the mermithid is now considered parasitic.

Parasitic mermithids have long, slender bodies with permeable cuticles used to absorb nutrients from the host haemocoel, exchange waste products and maintain an osmotic balance. Mermithids in this parasitic life stage moult four times, with the most intensive growth periods occurring in the third and fourth developmental stages. New

26

growth is accompanied by a significant alteration in body size, mass and proportion. Most

animal-parasitic nematode species exhibit a 50 to 80 percent increase in body mass

during the fifth and final growth stage following the fourth moult (Malakhov 1994). After

the fifth growth stage, mermithids complete the parasitic phase of life cycle by exiting the

host directly through the host cuticle.

Now aquatic post-parasites, the mermithids return to the stream substrate and

moult twice prior to sexual maturity. Post-parasitic mermithids do not require food,

unlike the parasitic life stage that exhibits tremendous metabolic activity. The post-

parasites instead rely on nutrient reserves obtained during the parasitic stage of life. Once

sexually mature, the postparasitic mermithids mate and oviposit directly into the stream

substrate (Gordon 1981, Bailey et al. 1977). Mature adult mermithids are sexually dimorphic, requiring internal fertilization (Malakhov 1994), and are semelparous, mating only once in a lifetime and dying post-copulation (males) or post-oviposition (females).

In dry areas mermithid ova can remain quiescent in the desiccated substrate for

con.secutive seasons (Mondet et al. 1979).

1 .3. Mermithid-simuliid interactions

1 .3. 1 . Mermithid parasitism of black flies

Mermithid para.sitism of simuliid larvae was first noted by Siebold in 1848 and in adult black flies in 1913 by Swinton (Siebold 1848 and Swinton 1913, in Crosskey 1990).

Most knowledge of mermithids has .stemmed from the greater interest of the potential use

27

of these nematodes as biological control agents against black fly populations (Molloy

1981, Mondet and Bernard 1981, Ezenwa 1974a). Based upon morphological

identification of the mature adults, 67 species of Mermithidae parasitize black flies

(Poinar 1981). Mermithid infection rates vary considerably between habitats and seasons

due to localized, discontinuous distributions (Vandergast & Roderick 2003, Welch and

Rubstov 1965, Phelps and DeFoliart 1964 cited in Vandergast and Roderick 2003). At times mermithid populations attain levels of parasitism sufficient to reduce host

populations considerably, regulating natural populations of Simuliidae (Gordon et al.

1973).

1.3.2. Mermithid-simuliid life cycles

The mermithid life cycle is closely interrelated with the simuliid life cycle.

Mermithid and simuliid eggs eclose at roughly the same time (Ebsary and Bennett 1973)

and mermithids infect early (first or second) instar simuliid larvae almost immediately

(Colbo and Porter 1980) by entering the simuliid haemocoel directly through the simuliid

larval cuticle. Some researchers hypothesize that mermithids can enter the host either via

the head if caught by the simuliid labral fans or via the abdomen if the mermithids cling to the host (Mondet and Bernard 1981). The mermithid nematodes parasitize the simuliid

larvae, occasionally remaining in the host pupae and adult flies. Multiple mermithids may

infect a single simuliid host, with the maximum number of mermithids found in a

simuliid larva being eight (Mondet et al. 1976). A 1998 survey of mermithid infection in

Costello Creek, Algonquin Provincial Park, found that C. dacotensis are capable of

harbouring up to five mermithids at once, while S. venustum/verecundutn larvae only

28

harbour up to a maximum of three mermithids at any given time. The larvae of both species, however, were infected primarily by single mermithid nematodes (Phillips 1998).

Mermithid nematodes grow to near-adult size within the host, obtaining all nutritional requirements to complete the parasitic stage of the life cycle (Colbo 1990).

Mermithid parasites decrease levels of protein, glycogen and nonglycogen carbohydrates in the simuliid haemolymph, potentially altering host metabolism and causing deleterious effects on host tissue (Rutherford and Webster 1978, Gordon et al. 1978, 1973, Welch

1965, Nickle 1972). Stickland (191 1) was the first to record mermithids having pathogenic effects on the host simuliid (as cited in Gordon et al. 1973). Mermithid parasitism may also suppress larval gill histoblast development and lead to abnormally large host simuUid larvae having undergone supernumerary moults (Phillips 1998).

After fulfilling the parasitic requirements of development, the mermithid parasites rupture and emerge through the host cuticle, killing the host in the process. Mermithids

tend to fulfill the requirements for parasitic development prior to the completion of host larval development; otherwise remaining in the host through pupation into the adult simuliid life stage (Rubstov 1964 in Crosskey 1990, Colbo 1978, Peterson 1960).

1.3.3. Host feminization

Mermithid infections carrying through to the host adult phase frequently result in

morphological and/or behavioural host feminization (Adler et al. 2004, Crosskey 1990,

WUlker 1975). Sexually dimorphic morphological traits in adult Simuliidae are the eyes, tarsal claws and terminalia (Crosskey 1990). Feminization can either be complete or partial, the former exhibiting all female sexually dimorphic features, and the latter being a

29

sexual mosaic, combining male and female traits in one individual. Phenotypic feminization has been observed in numerous black fly species (Davies 1989, Gordon

1981, Nickle 1972, Freeden 1970), as well as in other insect families such as mosquitoes

(Diptera: Culicidae) (Brust 1966) and mayflies (Ephemeroptera) (Vance 1996).

Sexually mosaic individuals can be classified as either gynandromorphs or

intersexes; it is not easy, however, to distinguish these two forms of sexual mosaics

(Crosskey 1990). A gynandromorph is defined as "an abnormal individual exhibiting characters of both sexes in various parts of the body, a sexual mosaic", while an intersex

is defined as an individual "intermediate in sexual characters between a typical male and a typical female" (Merriam-Webster Online Dictionary 2006). Some entomological sources do not distinguish between intersexes and gynandromorphs. Torre-Buenno

(1985), for example, defines gynandromorph as "any insect combining the secondary male and female characters in the same individual, not necessarily an hermaphrodite, although sometimes such", yet fails to offer a definition for intersex.

Simuliid literature defines sexual mosaics in more detail. An individual black fly exhibiting gynandromorphy has a combination of male and female cells. These cells are expressed phenotypically as recognizably male and female body parts. Bilateral gynandromorphy occurs most often in simuliids, homologizing male and female structures with the left and right sides of the body being asymmetrically of the opposite sex (Adler et al. 2004, Brust 1966), as is seen in Figure 6. Unlike a gynandromorph, an intersex posses.ses cells of only a single genotype. While usually bilaterally symmetrical, intersexes typically exhibit a progressive transformation into the opposite sex in an anterior-to-posterior direction, with sexually mosaic features appearing as inconspicuous

30

transformations rather than definite segregations as with a gynandromorph (Adler et al.

2004, Brust 1966). Due to the ambiguity in distinguishing the difference between a

gynandromorph and intersex, feminized individuals and all sexual mosaics in this thesis

are commonly referred to as intersexes.

Adult mermithid-infected male, female and intersex black flies have been reported

to exhibit not only feminized morphologies, but also characteristic female behaviours.

Some researchers believe mermithid-infected adult black flies are not haematophagous

(Colbo and Porter 1980), although observed cases of blood feeding mermithid-infected

adult Simuliidae have been reported (Mondet et al. 1979; F. F. Hunter, personal communication). Mermithid-infected adult simuliids also exhibit characteristic uninfected gravid female oviposition behaviours, travelling to oviposition sites and essentially ovipositing the parasites into the upstream waters (Phelps and Defoliart 1964 cited in

Vandergast and Roderick 2003, personal observations). Figure 7 illustrates the life cycles of the mermithid and simuliid populations, and dynamic interactions between the parasite and host.

31

Figure 6. Scanning electron micrograph of an intersex adult black fly head exhibiting. The fly's right side is a holoptic male eye, while the left is a dioptic female eye. SEM taken at Brock University by Glenda Hooper.

32

BLOOOSUCKIMO

OVIPOSITION BCMAVIOUR

SEDIMENT RtVC

Figure 7. Interactions of the mermithid - simuliid life cycles (obtained from Crosskey 1990).

33

1.3.4. Evolutionary strategies

It is in the best interest of the mermithid nematodes not to kill the simuliid hosts

or interfere seriously with survival capabilities (Colbo 1982), since development and

distribution of the mermithid nematodes themselves are dependent upon the host living at

least long enough for the mermithid to complete the parasitic stage of its life cycle.

Mermithid preparasites do not kill the host upon entry, since the overall size of the

preparasite is extremely small relative to the simuliid host (Mondet and Bernard 1981). If the mermithid passes through to the adult host, the simuliid must have both well-

developed flight muscles to ensure the nematode is deposited back in the water, as well as

the ability to digest nectar or honeydew to fuel flight (Colbo 1982). Thus, mermithids are dependent upon healthy, well-nourished simuliids. Malnourished hosts cannot support

large parasite populations, and any fluctuations in nutrient availability should keep the

mermithid population at low levels (Colbo 1982). Also, nutrient-deficient larval simuliid

populations tend to exhibit prolonged emergence periods when compared to well-

nourished counterparts (Colbo 1982). Balanced nutrient reserves within a habitat provide

a suitable environment for the sustainability of mermithid infections (Bailey et al. 1977).

If mermithid infections are only present in the larval simuliid population and do

not pass over to the adult host pha.se, then parasitism occurs primarily downstream since

mermithid-infected larvae drift downstream as time passes, without returning the

mermithids back upstream (Colbo 1979). Such tendencies could hamper dispersal of the

mermithid population. However, if mermithid infection passes into the adult host, then

parasitism is ob.served upstream (Colbo and Porter 1980). Likewise, during late spring the

number of parasitized simuliids is very high downstream, yet in earlier spring it is high

34

upstream (Colbo and Porter 1980). Mermithid-infected adult black flies are important

both for maintaining the parasite population in the stream by reintroducing mermithids to

the head waters and for dispersal from the natal stream (Colbo and Porter 1980). It is

speculated that mermithid infection in mayflies results in similar behavioural alterations,

with upstream female dispersal and oviposition behaviour, uncharacteristic of uninfected

mayflies (Vance 1996). Male mermithids are smaller than female mermithids (Poinar and

Takaoka 1981), and some studies have found that multiple mermithid infections in a single simuliid host, referred to as superparasitism, result in male mermithids, while infection by a single parasite results in female adult mermithids. Such trends influence the sex ratio of mermithid ]X)pulations (Phillips 1998, Mondet and Bernard 1981). Male worms must be brought up stream by adult flies with multiple infections to balance out sex ratios.

1.3.5. Trends in mermithid-simuliid populations

Evidence exists that mermithid-infected black fly populations have stable distribution patterns within the natal stream, and are not fleeting occurrences. While such parasite-host interactions might experience annual variation, they tend be constant over long periods of time (Colbo 1990). Interspecies infection rates vary considerably. The percentage of parasitism in most black fly populations is less than one percent, with some exceptions having up to 70 percent infection (Gordon et al. 1973). Mokry and Finney

(1977) found in collections from Newfoundland that 1 1.7% of adult female Prosimulium mixtum Syme and Davies, and 22.2% of adult female Simulium venustum Say had mermithid infections, while no adult male simuliids were parasitized. Pupal collections from this same study, however, found five percent of male S. venustum pupae were

35

parasitized, and only eight percent of females were parasitized (Mokry and Finney 1977).

It has also been noted that simuliid species with similar life histories, that is, occupying the same spatial and temporal distributions within a stream, tended not to be parasitized by mermithids at the same time (Colbo and Porter 1980).

Previous studies of mermithid parasitism of black fly populations in Algonquin

Provincial Parte found that P. fuscum/mixtum, St. mutata, S. vittatum s.l, C. dacotensis and

S. truncatum/venustum/rostratum are susceptible to mermithid infection (Blenkhom

1998, Phillips 1998). P. fuscum/mixtum and St. mutata are univoltine species,

overwintering in the creeks as larvae; S. vittatum s.l. is a multivoltine species, with an overwintering generation and one or more generations throughout the spring and summer;

C. dacotensis is a univoltine species having one generation in May; the S.

truncatumAenustum/rostratum complex is composed of three consecutive but overlapping species in Costello Creek and Mud Creek from late spring to summer. These

species overwinter as eggs and are known to be S. truncatum, S. venustum and 5.

rostratum (Hunter 1990). S. rostratum is then multivoltine thereafter until the season ends, with 5. truncatum and 5. venustum being univoltine.

Prevalence of black flies and mermithid nematodes is strongly influenced by stream temperatures, with 7 °C being the maximum temperature at which Prosimulium and Stegoptema ova and the corresponding mermithid ova eclose; 3 °C is the minimum temperature at which hatching occurs in these species (Colbo 1979, Ebsary and Bennett

1974). ALso, each black fly species has its own degree day requirements for development; such factors could play a role in mermithid development and/or which simuliid species the parasite infects. P. fuscum/mixtum (Syme and Davies) requires approximately 240

36

days above °C to complete larval development, referred to as degree days (D°C); St. mutata Malloch development is similar to that of P. fuscum/mixtum, with approximately

250 D°C to 275 D°C above °C. St. mutata tend to pupate later than P. fuscum/mixtum, indicating that the former might be better adapted to develop at higher temperatures than

the latter. C. dacotensis requires approximately 475 D°C above °C to complete larval development, thus occurring later in the season than the former two species. Such delayed development could be due to the autogeneous behaviour of adult female C. dacotensis, thus requiring a longer development time for egg maturation. S. venustum undergoes larval development later in the season at approximately 230 D°C to 260 D°C above 10 °C

(Ross and Merritt 1978).

Trends in mermithid and simuliid populations have been characterized within a habitat in relation to those black flies that exhibit multiple annual generations

(multivoltine) and those with single annual generations (univoltine). As the larvae of a univoltine host species overwinter, the mermithids also partly or completely overwinter

as parasites in the host rather than as ova (Ezenwa 1974b). P. fuscum/mixtum and St.

mutata are univoltine, overwintering as larvae (Colbo and Porter 1980), S. vittatum s.l. also overwinter as larvae; however, they are multivoltine with two or sometimes three generations per annum (Bernardo et al. 1986).

Studies of mermithid-infected black fly populations in Newfoundland have found that mermithid nematodes also have multi- and univoltine species. While both multivoltine and univoltine simuliid sjjecies exhibit susceptibility to the univoltine mermithid species {Neomesomermis flumenalis, Gastromermis viridis and Isomermis

wisconsinensis), all exhibit unique parasite-host infection periods (Ezenwa 1974a).

37

Parasites of a multivoltine host species tend to prevail as multivoltine parasites in the host population throughout the entire season (Ezenwa 1974b), that is, until there are no longer potential host larvae in the water (Ebsary and Bennett 1975). Likewise, mermithids infecting univoltine species tend to be univoltine parasites. Another study of

Newfoundland black fly populations found that there were two groups of mermithid species, with one group overwintering in Prosimulium larval hosts, while the remaining group of mermithids underwent parasitic development in summer-developing Simulium

larval hosts (Bailey et al. 1977).

Previously, mermithids of P. mixtum and the Simulium venustum/verecundum complex were considered the same species, N.flumenalis (Ebsary and Bennett 1974).

However, such identifications were questioned by Colbo and Porter (1980). It was found that although ova of mermithids infecting early season Simulium can develop in

laboratory conditions in time to infect Prosimulium larvae (Bailey et al. 1977), this does not occur in nature (Colbo and Porter 1980). In natural settings, simuliid hatch occurs

synchronously with the mermithids that will parasitize them. Though P. mixtum and St. mutata are closely associated in terms of life history traits by overwintering in the same

stream, P. mixtum was found to be infected by different mermithids than St. mutata

(Colbo 1978). P. mixtum also eclose from the ova earlier than do the St. mutata, suggesting that the mermithids that infect P. mixtum hatch out at the same time as the P. mixtum larvae, but would not survive in the streams as preparasitic mermithids long

enough to also infect early in.star St. mutata. It has been hypothesised that the failure of

mermithids to infect simuliid species with similar life histories is not due to physiological limitations or differences in distribution within the stream (Colbo and Porter 1980).

38

1 .4. Nematode identification

1 .4. 1 . Traditional identification techniques

Numerous studies have attempted to identify mermithid parasites of black flies to the species level using traditional morphological taxonomy. In one study, mermithids recovered from P. mixtum were Neomesomermis flumenalis, Isomermis sp. and

Gastromermis viridis, while mermithids from 5. venustum were Hydromermis sp., G.

viridis and N.flumenalis (Mokry and Finney 1977). G. viridis and /. wisconsinensis have

been reported in 5. vittatum s.l. in Wisconsin, and Mesomermis flumenalis from 5.

venustum in Ontario (Welch 1 962). Four species from the genera Neomesomermis,

Gastromermis and Isomermis were all found at the same breeding site in West Africa

(Mondet & Bernard 1981). N.flumenalis was found to be the most abundant mermithid in

Newfoundland populations (Ezenwa 1974a). Mermithid identification has always been

plagued with inconsistency in taxonomic methods. Morphological taxonomy is not always reliable for mermithid specimens that are not fully mature, and access to mature

adult specimens, a necessity for proper morphology-based taxonomy, is not always

available (Blaxter 2004, Colbo 1990, Mondet and Bernard 1981, Bailey et al. 1977,

Gordon et al. 1973, Welch and Rubstov 1965). Nematodes are extremely difficult to identify to the species level using conventional morphological methods, including scanning electron microscopy and live video analysis (Blaxter 2004).

Rather than using morphological taxonomy, some studies assign mermithid nematodes from multiple simuliid hosts found across North America into one species

classification (Harkrider 1988); by doing .so, intricate host-parasite relationships are

39

masked. Also, mermithid colour variation has been used by some researchers as a character in species recognition (Blenkhom 1998, Phillips 1998); however, there is no concrete evidence that mermithid colour is actually indicative of species. Mermithid

taxonomy is uncertain (Gordon et al. 1973), and there exists a need for improved mermithid identification techniques. To gain a better understanding of correlations that might exist between the simuliid-mermithid populations, in particular seasonal host- parasite species variations, accurate mermithid species identification is necessary.

1.4.2. Molecular identification

Molecular taxonomy offers an effective alternative to moiphological identification (Szalanski et al. 2001), especially in a situation when morphological

characteristics are not fiilly developed (Blaxter 2004). Molecular taxonomy, also referred to as genetic barcoding, or deoxyribonucleic acid (DNA) taxonomy, proves quite useful

as a taxonomic aid (Hebert et al. 2003, Blaxter 2004).

Some genomic regions are highly conserved, such as the mitochondrial genome, thus permitting the use of universal oligonucleotide primer sets for polymerase chain reaction (PCR) amplification, but also contain informative sequence differences that are,

oftentimes, species-specific (Blaxter 2004). Sequence-based molecular taxonomy is a

u.seful taxonomic tool, using the following common sequences: the nuclear small subunit ribosomal DNA (rDNA) gene (SSU, or 16S in prokaryotes and 18S in eukaryotes), the

nuclear large-subunit rDNA gene (LSU, or 23S and 28S), the internal-transcribed spacer

(ITS) region of the rDNA separated by the 5.8S rDNA gene into ITSl and ITS2 regions.

40

and finally, the mitochondrial cytochrome c oxidase 1 gene (COl) (Blaxter 2004).

Perhaps molecular taxonomy can assist with identification procedures.

1 .5. Sex determination of mermithid-infected simuliid larvae

The developmental stage at which feminization is initiated in mermithid-infected

black flies remains unknown. It could occur at any point in development, from the

moment the mermithid first enters the simuliid host to the point at which morphological

sexual features of the adult fly form during the late pupal stage. Also, if feminization is initiated during larval development, could mermithid-infected black fly larvae exhibit feminized characteristics similar to the feminized mermithid-infected adult black flies?

The exact qualities of the observed feminization also remain a mystery; while it is known

that this sexual alteration is morphological and behavioural, it remains unknown as to whether or not the feminization occurs at a genetic level as well. Feminization of black

flies by mermithid nematodes is often identified as feminization of genetic males

(Crosskey 1990); however, no research currently exists to support such claims. Perhaps

these alterations are not feminization (males converting into females) at all, but actually masculinization (females converting into males).

Row cytometry was used in a study by Vance (1996) to determine the genetic sex of mermithid-infected mayflies that were morphologically female, yet could not be distinguished as being genetically male or female. Genome sizes of uninfected and

mermithid-infected mayflies were compared. Results indicated that there is a difference in genome size between uninfected male and female mayflies, while parasitized

41

morphological females were indistinguishable from either unparasitized male or female

flies, and parasitized intersexes were indistinguishable from unparasitized male mayflies

(Vance 1996), supporting the theory that the parasitized individuals are actually feminized males.

Differences in genetic cross-over exist between male and female reproductive cells of the same species (Moens 1978). In larval black flies, males have achiasmate meiosis, while females have chiasmate meiosis (Rothfels and Mason 1975). Chiasmata occur during meiotic prophase, when two homologous chromatids crossover, exchanging genetic information. Achiasmate meiosis occurs in animals and largely in insects, but only in the heterogametic sex (White 1973). Male larval Simuliidae, as well as many other Dipterans, undergo achiasmate meiosis (Rothfels and Mason 1975, Moens 1978).

1 .6. Simuliid control measures

Potential biological control methods against black flies are always under investigation. Black flies are susceptible to viruses, microsporidia, fungi and bacteria

(Lacey and Undeen 1986), making any of these simuliid invaders potential biological control agents. Currently simuliid control depends on larvicidal chemicals and biological

agents such a.s bacteria (Werner and Pont 2(X)3). The bacterium Bacillus thuringiensis serovar israelensis (Bti) was introduced in the 1980s as an economically feasible, highly specific insecticide against black flies and mosquitoes with low activity on non-target organisms (Merritt et al. 1989, Colbo and Undeen 1980). Since then Bti has been commonly used to as a primary control agent against black fly populations. Intensive

42

research has been focused on Bti formulations with improved potency, stability, ease of application and residual activity (Araiijo-Coutinho et al. 2005).

Mermithid nematodes were once thought of as promising biological control agents against black flies (Mondet and Bernard 1981, Bailey et al. 1977, 1974, Ebsary and Bennett 1973) since some simuliid populations retain high mermithid infection rates

(Gordon 1984 in Colbo 1990, Bailey et al. 1977), which ultimately alter host

reproduction and regulate natural black fly populations (Gordon et al. 1973).

Implementation of mermithid nematodes as a large-scale biological control measure against Simuliidae hosts was never achieved since the procedures for mass collection and rearing of mermithid nematodes are extremely labour intensive and economically

unfeasible (Finney 1981, 1976, Ebsary and Bennett 1973, Bailey et al. 1977, 1974).

Large quantities of mermithid nematodes would be required to initiate effective control programs (3.6x10^ mermithids per mile of stream), and costs to produce such numbers was thought to be unreasonable (Molloy and Jamnback 1977).

1.7. Purpose

This thesis examines the complex relationship between mermithid nematodes and

simuliid hosts. Specifically, it investigates i) influence of mermithid infection on

proportions of adult black fly morphological sexes within a population, ii) variation in the seasonal ecology and species as.sociations of the mermithid and simuliid populations

through population monitoring and modem taxonomic approaches, and iii) a detailed examination of the development and genetic sex of mermithid-infected black fly larvae.

43

2. Materials and Methods

2.1. Collections

2.1.1. Collection sites

For this study, two sites in Algonquin Provincial Park, Ontario (Figure 8) were

used: Costello Creek (45°35'57"N, 78°19'49"W) (Figures 9 and 1 1) and Mud Creek

(45°32'25"N, 78°15'54"W) (Figures 10 and 12). The park is a 7,725 acre area in a transition zone between southern deciduous forests and northern coniferous forests; flora and fauna characteristic of both forest types thrive within the park boundaries.

Costello Creek is an outflow of spring-fed Costello Lake, originating at a beaver dam on the northwest end of the lake. Approximately ICX) metres long and varying between one and 10 metres wide, the sample site at Costello Creek is composed primarily of a rocky substrate. The site terminates when the substrate changes from rock to peat and the current slows, the creek then meanders through a bog for approximately 10 kilometres before emptying into Lake Opeongo.

Originating from West Smith Lake, Mud Creek merges with Headstone Creek approximately 2(X) metres upstream from the sample site. The site begins at a beaver dam built around the support beams of an old wooden bridge on Highway 60, where the water pours over/through the dam and flows downstream approximately 100 metres through a portion of the creek that ranges from one to eight metres wide. TTie sample site terminates where the current drops off and the creek then proceeds to meander through a bog,

44

eventually emptying into Clarke Lake. The substrate of Mud Creek is sandy with smaller, pebble-sized rocks.

For sampling purposes, both Costello and Mud Creeks were divided into

"upstream" and "downstream" zones; upstream at both sites comprised the upper half of the site, originating at the beaver dams and travelling to approximately half way through the area of flowing water, downstream included the final half of the area over which oxygenated water flowed.

45

tmimm0tmt. n»

Figure 8. Map of Algonquin Provincial Park, Ontario, indicating Costello Creek (C) and Mud Creek (M) collection sites. Inset shows location of the park in Ontario (Canoe Routes of Algonquin Provincial Park).

46

Figure 9. Topographic map of Costello Creeic Circled area depicts sample site (Natural Resources Canada).

47

Figure 10. Topographic map of Mud Creek. Circled area depicts sample site (Natural Resources Canada).

48

Figure 1 1. View of downstream Costello Creelt in mid-spring, 2004.

49

Figure 12. View of upstream Mud Creek in mid-spring, 2004.

50

2. 1 .2. Collection intervals

Field collections were made every 48 hours from early May to late August, 2004 and from late April to late August, 2005. These "spring and summer" collections

conmienced earlier in 2005, because it was observed that mermithid infection was already present upon arrival at the study sites in May 2004.

Monthly collections were also made from February 2005 to January 2006 on approximately the same date each month from the same locations at Costello and Mud

Creeks as per the spring and summer collections.

2.1.3. Larval and pupal simuliid collections

On each collection date, quantitative larval and/or pupal collections were made at both Mud and Costello Creeks. Approximately 100 individual larvae and/or pupae were collected from upstream and downstream at each creek for a total of approximately 400 specimens per collection date. When simuliid larval and/or pupal presence was low, as many individuals as possible were obtained. Pupae were not consistentiy collected in

2004.

Developing simuliid larvae and pupae were collected off rocks, sticks and trailing vegetation in the streams using fine forceps and transferred into re-sealable plastic containers half filled with water obtained directly from the creek. Samples were transferred to the field laboratory at the Wildlife Research Station in a cooler with an ice pack, where they were then kept in a refrigerator until processed. Feeding was not neces.sary during this storage time, since stream water contained sufficient dissolved

51

organic nutrients (Gordon et al. 1973), and all specimens were processed within 24 hours of collection.

2. 1 .4. Adult simuliid collections

Adult black flies were collected using pan traps (Figure 13), emergence traps

(Figure 14), sweep netting and manual aspiration. The yellow plastic pan traps were approximately 30 cm^ in diameter and 10 cm deep. They were filled one quarter full with water directly from the stream with a few drops of biodegradable dish soap added to break the surface tension. Traps were left on the river bank as close to the water level as possible for 48 hours, after which they were emptied into re-sealable plastic containers and transported to the field laboratory at the Wildlife Research Station.

Emergence traps were constructed from fine netting glued into a cone shape, which was then glued to a ring of 12 mm PVC tubing, making a base approximately 30 cm in diameter. A collecting chamber was constructed from a 500 mL Nalgene bottle with a 40 mm hole cut into the base, and a piece of 40 mm PVC tubing approximately 10 cm long was inserted into this hole and affixed with silicone sealant, as well as attached to the apex of the cone-shaped netting using a metal pipe clamp. A piece of string attached to both the Nalgene bottle and an overhead L-shaped wooden support suspended the emergence trap directly over a developing simuliid community in the stream. The collecting chamber was partially filled with stream water with a drop of biodegradable soap added. Contents of the emergence trap collecting chambers were emptied into re- sealable plastic containers every 48 hours and transported back to the Wildlife Research

Station field laboratory for processing.

52

Sweep netting in a figure eight pattern was done along and just above the

vegetation near the headwater of the creeks. A manual aspirator was used to transfer any

adults caught in the sweep net into a 20 mL scintillation vial containing 95% ethanol.

Adult simuliids were also aspirated directly from the researchers when possible and

transferred into the same scintillation vials as with the sweep collections. These samples

were then transported back to the Wildlife Research Station for further analysis.

2.1.5. Mermithid collections

Mermithid nematodes were not collected directly from the creeks, but rather dissected from the simuliid host. Any free-swimming mermithids that had emerged from the larval, pupal or adult host in the sample containers during transportation were also processed.

53

pan trap

Figure 13. A pan trap placed along the bank of Costello Creek, Algonquin Provincial Park. Photograph taken in June, 2005.

54

wooden brace

collecting chamber

pipe clamp

netting

plastic tubing

Figure 14. An emergence trap suspended over trailing vegetation and sticks in Mud Creelc, Algonquin Provincial Park. Photograph taken in May, 2004.

55

2.2. Specimen processing

2.2.1. Simuliid larval processing

Larvae were individually assessed for mermithid infection status by examining the larval body through a Leica Wild M3B stereo microscope. Mermithid(s) were generally visible through the cuticle of the host larva. Occasionally, however, mermithid presence was questionable in a simuliid larval specimen. In such an event the host was dissected along the ventral midline using fine forceps to confirm infection status.

All larvae exhibiting a mermithid infection were identified to species level using the identification key from Adler et al. (2004), and when necessary Currie (1986) and

Wood et al. (1963). Since P.fuscum and P. mixtum larvae are extremely difficult to separate, they were grouped together as the P. fuscum/mixtum complex (Ross and Merritt

1978, Ebsary and Bennett 1974). Likewise, members of the 5. venustum/verecundum

group are difficult to differentiate. Three members of the S. venustum/verecundum

complex are present in Costello and Mud Creeks: S. truncatum, S. venustum and S. rostratum (Hunter 1990), thus reference is made to 5. truncatum/venustum/rostratum in this thesis.

To distinguish larval developmental stages, gill histoblast development was noted

using the following categories: 1 ) none (no indication of gill histoblast development), 2) clear (gill histoblasts just beginning to form), 3) white, 4) grey or 5) black (fully formed

gill histoblasts) (Figures 1 and 15).

56

*4i'il£

head capsule length

head capsule width

gill histoblast

larval length

Figure 15. Dorsal view of a Prosimulium mixtum larva with developing gill hliitoblasts marked. (Image obtained from Adier et al. 2004).

57

2.2.2. Simuliid pupal processing

To assess individual mermithid infection status of a simuliid pupa, the pupal body

was first removed from the pupal casing using fine forceps. Upon examination under a

Leica Wild M3B stereo microscope, mermithid infection was generally visible through the pupal cuticle. Occasionally, as with the host larvae, mermithid specimens were difficult to distinguish through the host cuticle. In such an event the host was dissected using fine forceps to confirm infection status. Mermithid-infected pupae collected in

2005 (n=247) were identified to species level using the keys of Adler et al. (2004), and when necessary Currie (1986) and Wood et al. (1963).

2.2.3. SimuUid adult processing

Adult black flies from the pan trap, emergence trap, sweet net and aspiration samples were transferred into 5 mL Sarstedt© vials filled with 95% ethanol. Adult black flies were classified as being male, female or intersex based upon morphologies of the

sexually dimorphic eyes (Figure 3), tarsal claws (Figure 4) and genitalia (Figure 5).

Intersexes are those individuals exhibiting combined male and female sexually dimorphic

traits.

2.2.4. Mermithid proce.ssing

Mermithid nematodes were dissected out of approximately half of all samples collected. Mermithids were not removed from larvae to be dissected for gonad examination, so as to not disturb the internal contents of the larval haemocoel. Nematodes that were found free-swimming in the samples were assumed to have emerged from the

S8

hosts; these mermithids were stored in separate vials and labelled as being "free- swimming".

Mermithid nematodes exhibit dramatic colour variation, and detailed observations

of such variation were made since it was unknown whether or not mermithid colour variation reflects species variation. Such variations in colour have been abridged into five colour morph categories: clear (C), white (W), green (G), pink (P) and yellow (Y). Each of these five categories contains variations of that colour morph. The "clear" category contains only clear mermithids. The "white" category includes the following colour morphs: white, whitish-clear, whitish-brown, opaque greyish-white, greyish-clear, clear- white-brown, brownish-white, white with brown flecks, and white with grey patches. The

"green" category includes green, greenish-clear, greenish-white, greenish-clear-white, pale green, and blue-green mermithids. "Pink" mermithids include pink, pinkish-clear, white-pale pink, pinkish-white, and pinkish-white-brown individuals. The "yellow" category contains yellow and yellowish-white mermithids.

2.2.5. Specimen storage

Individual specimens were stored in 95% ethanol in individual 5 mL Sarstedt© vials. On occasion mennithid(s) were dissected from the simuliid host during sample processing; in such cases the mermithid(s) were .stored in the same vial as the simuliid host. Mermithids found free-swimming in the samples were stored in vials according to colour variation, thus multiple mermithids of one colour could be stored in a single vial.

Vials were labelled using a fine-tip permanent marker and a small piece of cardstock paper with the label information written in pencil was placed within the vial.

59

2.3. Proportion of adult sexes from emergence trap collections

Proportions of adult simuliid sexes were established by determining the morphological sexes (as per section 2.2.3) of uninfected and mermithid-infected adult black flies collected from emergence traps suspended over Costello Creek throughout the spring and summer of 2004. While adults were collected from pan traps as well throughout the sampling period, data obtained from them were not included in the calculation of the proportion of sexes within the population. Since pan traps are designed to attract gravid females upstream, data obtained from these samples could potentially skew the results toward the female sex, creating a sampling bias. Emergence traps,

however, are designed to collect all black flies emerging from the streams, regardless of sex. Due to time constraints, individual adult black flies were not identified to species level, and this analysis thus represents the proportion of sexes present in the total simuliid population of adult black flies over an entire emergence (spring/summer) season.

2.4. Monthly variation of parasite-host populations

2.4. 1 . Collection

Seasonal variation of the parasite-host interactions was analyzed by surveying larval and/or pupal simuliid populations over a 12-month period. Specimens were

collected once a month (as per section 2. 1 .2 and 2. 1 .3). Mean monthly air temperature data was obtained from Environment Canada's Climate Data Almanac (Environment

Canada) from February 2005 to January 2006.

60

2.4.2. Sample processing

Larvae and, when present, pupae were processed as per sections 2.2. 1 and 2.2.2.

Simuliid infection status was recorded as being either uninfected or mermithid-infected.

To understand the parasite-host species interactions, monthly trends in the mermithid and simuliid populations were assessed over the 12-month period.

2.5. Mermithid identification

Since mermithid nematodes collected in this study were all immature, that is, still

in the parasitic stage of their lifecycle, it was virtually impossible to provide accurate morphological identifications. Mermithids collected from the simuliid hosts were thus identified using DNA barcoding; nematode colour was also recorded as a potential identification tool. Although primer sequences for the various mermithid sf)ecies were not published at the time of this study, restricted access to these primers was made available through Dr. Brad Hyman at the University of California at Riverside. DNA was extracted from a representative of each mermithid colour morph from each simuliid species using

SigmaKit©. DNA was then lyophilized and sent to the University of California at

Riverside for PCR amplification and sequencing using methods similar to Vandergast and

Roderick (2003). Three primer sets were used in the mermithid identification process; nuclear 18S ribosomal DNA, nuclear 18S D3 ribosomal DNA and the highly conserved mitochondrial COI sequence. Resulting sequences were compared to a mermithid data set compiled by graduate students of Dr. Hyman.

61

2.6. Larval sex determination

Sex of mermithid-infected simuliid larvae was determined by examining the

sexually dimorphic gonads morphologically and cytologically. Larvae were characterized

as being early in development, lacking any trace of gill histoblast formation, or late in development, exhibiting histoblast formation. The latter are referred to throughout this thesis as "pharate pupae" while the former are "early larvae".

2.6.1. Staining and dissection

To examine the sexual developmental characteristics of mermithid-infected

Simuhidae, larvae were Feulgen stained as per Hunter (1987). Larvae were dissected in

4°C Camoy's fixative (Appendix 1) in a small watch glass. A slit was made along the ventral midline using minuten pins and fine forceps, exposing the gut and salivary glands.

After dissection the larvae were placed into individual wells containing room temperature distilled water for 20 minutes. While the dissected larvae were in the water, 5 mL of IN

HCl was preheated to 60°C in corked 15 mL glass vials. After 20 minutes in distilled water, the dissected larvae were removed from the water and blotted on paper towel to remove excess water.

The larvae were then transferred to the vials containing the 60°C 1 N HCl, and incubated for exactly nine minutes for mild acid hydrolysis. Larvae were then quickly removed from the acid and transferred into another glass vial containing 4°C Feulgen stain (Appendix I). These vials were placed in a dark drawer and left for three hours for staining. Larvae were removed from the Feulgen stain and placed into another vial containing 5 mL sulphur dioxide water (Appendix I) for nine minutes to remove any

62

precipitated stain. The sulphur dioxide water was then poured off from the vial, and larvae were rinsed three additional times with cold tap water. After rinsing, the glass vials

were filled with cold tap water, corked and refrigerated at 4°C for at least 1 2 hours

(overnight).

After 12 hours larvae were ready for gonad examination. Stained larvae were removed firom the refrigerator and placed onto a clean dissecting dish containing cold

Camoy's fixative under a Leica® WILD M3B dissecting scope. Using fine forceps, larval head capsules were removed and lateral head length and head width were measured using an ocular micrometer (Figure 15). The remainder of the decapitated larva was then dissected along the ventral midline. Any nematodes present in the larval haemocoel were removed and measured (length and width). Specimen voucher slides were made using

Fisherbrand® superfrost slides with 22x22x1 mm cover slips. A drop of Euparal was placed onto the middle of the slide, and the larval head capsule along with the mermithid(s) was placed into this drop. The voucher slides were laid flat and allowed to set for 24 hours at room temperature, after which another drop of Euparal was added and

the cover slip gently applied. The voucher slides were then laid flat in a drying oven at

40°C for approximately four weeks to allow the Euparal to set, followed by permanent storage in a slide box at room temjjerature.

2.6.2. Gonad morphology

Larval morphological sex was determined by examining gonad morphologies of infected and uninfected black fly larvae from both early and late developmental stages under a Leica Wild M3B stereo microscope. Following mermithid removal, the gut was

63

then removed from the larva, exposing the gonads on the dorsal underside of approximately the sixth abdominal segment (Crosskey 1990). The gonads were identified individually as being either a morphological ovary or testis (Figure 16) (Alencar et al.

2001). If a larva contained either two ovaries or a single ovary, it was considered to be morphologically female. Likewise, if a larva contained either two testes or a single testis,

it was considered to be morphologically male. Larvae were scored using a system wherein "O" indicates the presence of an ovary and "T' indicates the presence of a testis.

Each observed gonad was designated its corresponding letter (O or T); thus larvae with two ovaries would be scored "OO" (Figure 16a), while larvae with only one ovary present would be scored "O". Likewise, larvae with two testes present would be scored

"TT" (Figure 16b), while larvae with only one testis would be scored "T'. If no gonads

were present in the larva, it was scored as "None".

2.6.3. Genetic sex

To confirm the morphological sexes observed in the dissected simuliid larvae, the genetic sex of the larvae was examined in infected and uninfected black fly larvae from both early and late developmental stages. Genetic sex was determined by examining the

chromosomes of the gonads during meiosis I. To accomplish this, the morphologically identified gonads were transferred using fine forceps and a dissecting needle from the

dissected larva into a drop of 50% acetic acid (Appendix I) in the centre of a

Fisherbrand® superfrost slide. A 22x22x1 mm Fisherbrand® cover slip was lowered over the gonad being careful not to introduce any air bubbles. The slide was then placed under a paper towel, and pressure from a thumb was applied to the slide for approximately three to four seconds to squa.sh the nuclei, making the chromosomes more visible. A drop of

64

2% aceto-cannine (Appendix I) was applied using a hypKxiermic needle to the comers of the cover slip to seal the slide. The sUde-mounted gonads were then examined using oil immersion at lOOOx magnification with a Leitz DMRBE microscope to determine the genetic sex of the larva by characterizing the meiotic chromosomal condition of the

gonads, that is, if the gonads were chiasmate or achiasmate (Figure 17) at Meiosis I.

These sUdes were laid flat in a freezer at -20°C overnight, followed by permanent storage in a slide storage box at -80°C. Digital photographs were taken of the meiotic chromosomes for voucher purposes.

Gonad chromosomes were scored as being chiasmate (exhibiting crossover), achiasmate (not exhibiting crossover), or unscorable (no condensed chromosomes were visible) (Figure 17). The genetic and morphological identification results were then compared, with the morphological sexes being either female (OO/O) or male (TT/T).

65

a)

66

H)

b)

Figure 17. Chromosomal condition of larval simuliid gonads during Meiosis I. Chiasmate meiosis (a) is typical of female simuiiids, while achiasmate meiosis (b) is typical of male simuliids. Gonads

without visible condensed chromosomes were "unscorable'' (c).

67

2.7. Statistical analyses

All statistical analyses were performed with SPSS version 12.0 for Windows

(SPSS Inc., Chicago, IL). For all tests, P values of 0.05 or less were considered to indicate a significant effect.

2.7.1. Monthly variation

To examine if mermithid infection was equally abundant throughout the annum in susceptible black fly species, intraspecies monthly variation of mermithid infection presence was assessed. Specifically, variation from month to month was assessed by comparing the frequency of uninfected and mermithid-infected larvae and/or pupae present per collection date. Only months with non-zero frequencies of black flies were used in these calculations. Data were analyzed statistically using the Chi-square test for independence with the Monte Carlo method. Monte Carlo simulations are commonly applied to randomly sampled populations that are not normally distributed, have different variances and consist of a small sample size with respect to the biological population size

(Ludbrook 2000. Smeeton and Sprent 2001, Sokal and Rohlf 1995). Ten thousand Monte

Carlo simulations were generated using SPSS (SPSS Inc., Chicago, IL) to determine if the observed intraspecific monthly infection frequencies differed from those expected by a random model. Monte Carlo simulations generated a sample distribution of the monthly

intraspecies infection frequencies by resampling rather than enumerating all possible data orderings for exact probabilities (McCreadie et al. 1997, Sokal and Rohlf 1995). The observed Chi-square values from the data were then compared to the distribution of the test statistic values under random re-sampling.

68

2.7.2. Gonad moqjhologies

Two questions were asked with respect to the gonad morphologies of the uninfected and mermithid-infected black fly larvae. First, does infection status influence gonad morphological frequencies? Gonad morphologies in uninfected and mermithid- infected specimens were compared within each developmental stage (uninfected early larvae versus mermithid-infected early larvae; uninfected pharate pupae versus mermithid-infected pharate pupae).

Second, does developmental stage influence gonad morphological frequencies?

Gonad morphologies of early larvae and pharate pupae were compared, keeping infection status as an independent variable (uninfected early larvae versus uninfected pharate pupae; mermithid-infected early larvae versus mermithid-infected pharate pupae).

Gonad morphological data were analyzed statistically using two-tailed Fisher-

Freeman-Halton exact tests. The Fisher-Freeman-Halton (F-F-H) exact test is an extension of the Fisher exact test into an R x C table, used to examine the significance of associations between multiple variables from small sample sizes (Ludbrook 2000,

Smeeton and Sprent 2(X)1).

2.7.3. Gonad chromosomal conditions

Two statistical questions were also asked of the data resulting from examination of the chromosomal conditions of simuliid larvae. First, does infection status influence the chromosomal conditions of larval black fly gonads of the same morphological sex?

Frequencies of each gonad chromosomal condition (achiasmate, chiasmate or unscorable)

69

in uninfected and mermithid-infected specimens were compared at each developmental

stage, keeping morphological females and males separate. A morphological female had either one ovary or two ovaries present, while a morphological male had either one testis or two testes present.

Second, does developmental stage influence frequency of each chromosomal condition? The frequencies of gonad chromosomal condition in early larvae and pharate pupae were compared for uninfected and mermithid-infected specimens. Gonad chromosomal condition data were analyzed statistically using the same test as the gonad morphological data using two-tailed Fisher-Freeman-Halton exact tests (Lundbrook 2000,

Smeeton and Sprent 2001).

70

3. Results

3.1. Collections

A total of 33 collections in 2004 and 30 collections in 2005 were obtained at

Costello Creek, and 29 collections in 2004 and 36 collections in 2005 at Mud Creek.

3.2. Specimen processing

Throughout the spring and summer of 2004 and 2005, mermithid-infected larvae

(n = 172 in 2004, n = 553 in 2005) and pupae (n = 245 in 2005) were collected. Total numbers of simuliid specimens collected throughout the spring and summer of 2005 are

presented in Appendix 11, while the percentage of mermithid-infected simuliid larvae

and/or pupae collected in 2005 are presented in Appendix III. Mermithid-infected adult black flies were collected using pan traps in 2004 (n = 45) and 2005 (n = 49), emergence traps in 2004 (n= 49), sweep netting and manual aspiration in 2005 (n = 13, combined).

Appendix IV outlines the number of each mermithid colour variety that parasitized simuliid larvae, pupae and/or adults throughout the spring and summer of

2005. Clear mermithids were only found in P.fuscum/mixtum (n = 43), C. dacotensis (n =

5) and S. truncatum/venustum/rostratum (n = 9) larvae, as well as in S.

truncatum/venustum/rostratum pupae (n = 2). White mermithids were present in P.

fuscum/mixtum (n = 277), St. mutata (n = 13), S. vittaium s.l. (n = 4), C. dacotensis (n =

40), and 5. truncatum/venustum/rostratum (n = 280). Green mermithids were found in S.

vittatum s.l. (n = 3), C. dacotensis (n = 39), and S. truncatum/venustum/rostratum (n =

71

46). Pink mermithids were present in P. fuscum/mixtum, (n = 21), St. mutata (n = 5), C. dacotensis (n = 26), and S. truncatum/venustum/rostratum (n = 4). Yellow mermithids were found in P. fuscum/mixtum (n = 19), St. mutata (n = 9), and S.

truncatumAfenustum/rostratum (n = 2).

3.3. Adult sex proportions from emergence trap collections

Throughout the spring and summer of 2004, 4589 adult black flies were collected

in emergence traps at Costello Creek. This total population was comprised of 3 1 .8% female, 67.8% male and 0.4% intersex adult black flies. From this population, 29 individuals (0.6%) were infected by mermithid nematodes. Mermithid-infected individuals were female (69.0%), male (3.5%) or intersex (27.6%) adult black flies.

3.4. Monthly variation of parasite-host populations

Seasonal infection trends are pre.sented in Figure 1 8, and mean monthly temperatures for Algonquin Provincial Park are seen in Appendix V. The larvae of/*. fuscum/mixtum are univohine and were collected from October through to May (although two lone specimens were collected in August); mermithid nematodes were present in this

species from December to May. St. mutata is also univoltine; larvae were collected from

November through to May, with mermithid infections present from January to May. S. vittatum s.l. is multivoltine with larvae having been collected in three separate cohorts from November to April, June and August; however, mermithid infections were only

72

present in November and June. C. dacotensis is univoltine; larvae were found in the

streams from May to June, hosting mermithid infections in May only. Larvae were

collected from the S. truncatumAenustum/rostratum complex from May through to

October, with infection occurring in June, July and August (Figure 18).

Significant monthly variation of mermithid infection prevalence was found in P. fuscum/mixtum (x^(8] = 20.673, P = 0.021), St. mutata (x^6j = 38.27, P < 0.001) and 5.

truncatumAenustum/rostratum (x^[7i = 68.710, P = 0.008). In contrast, no significant

monthly variation in was found C. dacotensis (x^[2] = 0.859, P = 0.614) and S. vittatum

si- (xVl = 3.310^ P = 0.695) (Figure 18).

73

P.fuscum/mixtum (x" =20.673. P=0.021)

« M JO 3>| •s %

4>|

3) i Mu _2

"S tx

1»

flD C 12S YD

15 » o SD * Z

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74

More frequent estimates of mermithid infection abundance in larval and/or pupal

Simuliidae were collected every 48 hours from April 30 to August 18, 2005. Data from these collections demonstrate similar trends to the monthly collections data; however, they cannot be combined with the monthly collection data due to different sampling timelines. The total number of mermithid-infected black fly larvae and/or pupae per

simuliid species collected throughout the spring and summer of 2005 is presented in

Figure 19. The total number of simuliid larvae and/or pupae per simuliid species

collected during the spring and summer of 2005 are presented in Appendix II, and the

percent infection present in each simuliid species is presented in Appendix III. According

to data obtained from the 48-hour collecting intervals (Figure 19), mermithid infection is present in P. fuscum/mixtum from April 30, the first sample date of the 48 hour collecting season, until May 22, with an infection peak on May 10. St. mutata harboured mermithids from April 30, the first collection date as well, until May 20 with a peak occurring on

May 10. 5. vittatum s.i, the third overwintering species, harboured mermithids on May

10 and from June 15 to June 23. Mermithid nematodes were found in C. dacotensis

collected from May 1 8 to May 30, with infection peaking on both May 22 and 24. The S. truncatum/venustum/rostratum complex harboured mermithid parasites from May 22 through to August 2, with two peaks occurring approximately three weeks apart on May

26 and June 15.

75

»# « ////////////// ///

— P. fuscLrrymydim — p Sf. mutata

-A S tnncatLm^enustirr/rcBtrstLm • S. \MtetLm -m— C dacatensis

Figure 19. Total number of mermithid infected Simuliidae coUected from Algonquin Provincial Park between April 30 and August 18, 2005. No coUections were made between June 29 and July 27, 2005.

76

3.5. Mermithid Identification

Dr. Brad Hyman (University of California at Riverside) identified two subfamilies

of mermithid nematodes (Mesomermithinae and Gastromermithinae) in the simuliid

specimens collected from Algonquin Provincial Park using molecular techniques. Within

the subfamily Mesomermithinae, three branches were identified: Aranimermis,

Strelkovimermis and Romanomermis. The Aranimermis branch is known to contain

Mesomermis and SimuUmermis, two known simuliid parasites. Strelkovimermis is closely

related to Aranimermis, but composes a different branch (Vandergast & Roderick 2003)

that contains a known mosquito (Diptera: Culicidae) parasite. Finally, the Romanomermis

branch is known to contain Isomermis, also a known simuliid parasite. The second

subfamily, Gastromermithinae, contains three separate branches, in this case

characterized as being three separate species: Gastromermis sp. A, Gastromermis sp. B

and Gastromermis sp. C.

Table 1 outlines which mermithid species were collected from each simuliid

species. The frequencies of mermithid colour morphs within their respective simuliid

species associations are seen in Table 2 with a detailed account of the number of

mermithids of each colour variant parasitizing each simuliid species in Appendix IV. A

preliminary distance tree (Figure 20) illustrates the divergence of the mermithid species

with respect to the highly conserved COI sequences (Brad Hyman, personal communication), illustrating species groupings within the two subfamilies. s-s

5 5

I

CVI, ^Z" C o CM CO (O

c

CO

.<0

CVJ. p in

^ (0 w o

g -~;

i I -I

CO

a

•§ CO

I I 3 I

O O u

x:

2

79

Gastromermis "C"

Gastromermis spp. B

Gastromermis spp. B

Gastromermis spp. A

Romanomermis spp.

Romanomermis spp.

10 PAM Aranimermis spp.

Figure 20. Preliminary distance tree based on COI sequences of mermithids recorded from black flies in Algonquin Provincial Park; data obtained from Dr. Brad Hyman (University of California at Riverside).

80

3.6. Larval sex determination

3.6.1. Morphological sex determination

The gonad morphology of P. fuscum/mixtum and S. rostratum/venustum/

truncatum specimens at the same developmental stages was affected by mermithid

infection at both the early larval stage (F-F-H [4] = 34.99, P < 0.001, Figure 21) and the

pharate pupal stage (F-F-H ,4] = 57.81, P < 0.001, Figure 22).

Reorganization of the data to consider the effects of developmental stage revealed

that gonad morphology did not differ between uninfected early larvae and pharate pupae

(F-F-H [4j= 8.18, P = 0.085, Table 3), nor between mermithid-infected early larvae and

pharate pupae (F-F-H [4j = 7.36, P = 0.1 13, Table 3).

81

Nbne

I Uhnfected Infected

Figure 21. Frequencies of observed gonad morphologies in mermithid-infected and uninfected early instar P. fuscum/mixtum and S. truncatum/venustum/ rostratum larvae. Morphologies were classified as having two ovaries (OO), one ovary (O), two testes (TT), one testis (T) or no gonads present (None). Morphologies of infected versus uninfected specimens differed (F-

F-H ,41 = 34.99, P < 0.001). An asterisk (*) indicates a zero count

82

Figure 22. Frequencies of gonad morphologies in mermithid-infected and uninfected P. fuscum/mixtum and S. truncatum/venustum/rostratum pharate pupae. Morphologies were classified as two ovaries (OO), one ovary (O), two testes (TT), one testis (T) or no gonads present (None). Morphologies of infected versus uninfected specimens differed (F-F-H [4j= 57.81, P < 0.001). An asterisk (*) indicates a zero count

83

Table 3: Comparison of the number of gonads of each morphology present in P. fuscum/mixtum and S. rostratum/venustum/truncatum early larvae versus pharate pupae. Separate tests were done for each infection status group. Fisher-Freeman-Hahon exact tests found no significant difference exists between the two developmental stages, in both uninfected and mermithid- infected specimens.

Infection Developmental Gonad Morphologies* (n) F-F-H Status stage 00 O TT T None df exact P

Uninfected

84

3.6.2. Genetic sex determination

Mermithid infection influenced the chromosomal conditions of P. fuscum/mixtum

and 5. rostratumA>enustum/truncatum specimens at both the early larval (female: F-F-H [2]

= 10.2, = male: = P 0.003; F-F-H [2] 39.5, P < 0.001) and pharate pupal (female: F-F-H [2]

= 7.1, P = 0.020; male: F-F-H (2) = 19.1, P < 0.001) developmental stages (Table 4), regardless of morphological sex.

The data was reorganized to consider the effects developmental stage had on chromosomal conditions in the simuliid specimens. Chromosomal condition did not differ

between uninfected early larvae and pharate pupae (female: F-F-H |2] = 0.96, P = 0.792;

male: F-F-H [2] = 2.45, P = 0.205), nor between mermithid-infected early larvae and

pharate pupae (female: = 1 1 F-F-H |2] .2 9, P = 0.539; male: F-F-H [2] = 1 .380, P = 0.5 1 8), regardless of morphological sex (Table 5).

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87

4. Discussion

4.1. Collections

To survey peak mermithid infection periods throughout the spring and summer

seasons, specimen collection every 48 hours from early spring through to late summer

was adequate since mermithid presence did not appear to fluctuate substantially in a 48

hour period. Sampling commenced earlier in 2005 than in 2004, and it was evident that

the frequency of mermithid infections was quite high in simuliid populations early in the

spring immediately following snow melt. Winter temperatures and environmental

conditions influence infection prevalence throughout the season (Colbo 1982). However,

while mermithids do exhibit annual variation in infection prevalence, infection rates

remain relatively stable over several years (Colbo 1990).

4.2. Specimen processing

To survey black flies of all life .stages exhibiting mermithid-infection, continuous

monitoring procedures were necessary throughout the spring and summer seasons. Also,

it was necessary to process the samples as soon as possible upon return to the field

laboratory at the Wildlife Research Station. This reduced the number of mermithids that exited the simuliid hosts in the collection containers prior to processing, thus preventing

skewed infection frequencies from being observed. Refrigeration of samples prior to

processing al.so helped to prevent this.

88

Camoy's solution is commonly used as a preservation medium among simuliid

researchers, since it effectively fixes polytene chromosomes for cytogenetic analyses

(Hunter 1990). Specimen storage in 95% ethanol, however, proved to be most efficient

for preserving simuliid and mermithid specimens from all life stages for both molecular and morphological examinations in this study. Refrigeration along with changing the

95% ethanol that the larvae are preserved in at least once ensures the preservation of

DNA for molecular work.

4.3. Adult sex proportions from emergence trap collections

In this study the observed proportions of each sex of uninfected adult black flies was male-biased. While simuliid larvae were not sexed by species in this study, in

uninfected larval black fly populations the proportion of sexes is typically approximately

50:50 (Adler et al. 2004). Generally, male simuliids have a shorter larval development time, pupating and emerging into adults prior to female counterparts, commonly referred to as protandry (Torre-Bueno 1985). Early collections of adults are thus likely to be male- biased, mid season collections to be 50:50 and late season collection of adults might be female-biased. From a theoretical standpoint, a 50:50 proportion of males and females

would .still be anticipated by combining early-, mid- and late-season data. Results from this study, however, suggest that despite continuous sampling (where sex bias should even out) more males than females were present. Lower than expected female frequencies may be, in part, an indirect consequence of longer developmental times (Rubstov 1989).

Increased larval development times in black fly females could make them more

89

susceptible to larval mortality caused by predation, parasites, deficiency of available

nutrients in the water and environmental pressures (Adler et al. 2004, Chmielewski and

Hall 1993, Colbo and Porter 1980).

Data obtained from mermithid-infected adult simuliids indicate a trend deviating from the uninfected adult data towards female bias, thus indicating that mermithids

altered the expected proportion of sexes of infected black flies. These data support previous findings that mermithid infections can alter the proportion of sexes within a

population (Mondet and Bernard 1981, Bailey et al. 1977, 1974, Ebsary and Bennett

1973, Gordon et al. 1973). If the proportion of sexes within a population is skewed sufficiently towards one sex, the reproductive potential of a simuliid population may decline, ultimately reducing the number of black flies present.

4.4. Monthly variation of parasite-host populations

Simuliid species in Algonquin Provincial Park vary in life history. P.fuscum/ mixtum and St. mutata are univoltine, coexisting and overwintering in streams as larvae,

and emerging as adults in early spring. While not as prevalent, 5. vittatum s.l. also

overwinter as larvae within the same habitats. 5. vittatum s.l., however, is a multivoltine species, with the early spring generation followed by at least two other generations, one

in late spring and one in late summer (Wood et al. 1963). C. dacotensis is a univoltine

species, present in the streams from mid to late spring. S. truncatum/venustum/rostratum is dominant in the waterways from late spring through to late summer. While composed

of three separate cytologically distinct species in Costello and Mud Creeks, S.

90

truncatumAenustum/rostratum is considered as one entity for the purpose of this study.

This complex is composed of two broadly overlapping (in space and time) univoltine

species, S. truncatum and 5. venustum, followed by the multivoltine S. rostratum (Hunter

1990).

Since both P. fuscum/mixtum and St. mutata have similar life histories, they are

expected to be susceptible to parasitism from the same mermithid species. The

overwintering 5. vittatum s.l. larvae are also expected to harbour the same mermithid

species as P. fuscum/mixtum and St. mutata. The mid-spring cohort of 5. vittatum s.l., in contrast, coexist with C. dacotensis; these two species are thus likely to be infected by the

same mermithid species. Thus, if the species associations of mermithids and simuliids are

based solely on the life histories of the host, then 5. vittatum s.l. will harbour multiple mermithid species over the duration of the year. Similarly, multiple mermithid species are expected to infect S. truncatum/venustum/rostratum. Note that the species of this complex

were grouped, thus the observed double peak in mermithid infection within S. truncatum/venustum/rostratum could be attributed to parasitism occurring in separate black fly species. Identification of the simuliid species using cytogenetic analysis and/or molecular barcoding of these simuliid specimens would be necessary to confirm this hypothesis.

4.5. Mermithid identification

Data indicate that colour is not, for the most part, suitable to use as a mermithid

identification tool (Table 1). 5. vittatum s.l. is infected by green Romanomermis, while 5.

91

truncatunUvenustum/rostratum is infected by green Strelkovimermis. Though both mermithid species are from the same subfamily (Mesomermithinae), they are not from the same branch or species. Mature green mermithids harboured by Simuliidae have also been previously identified as Gastromermis sp. (Welch 1962). Identification of the simuliid specimens ftx)m this complex to the species level using cytotaxonomy or

molecular barcoding is necessary to determine if it is just one sibling species from this complex that harbours Strelkovimermis and Gastromermis sp. C, or if the mermithid species parasitize separate sibling species from within this black fly complex. Also, clear mermithids appear in virtually all simuliid host species (Appendix IV), and thus being

"clear" can not be used as a species indicator. Molecular identification of the mermithid species appears to be the most feasible and trustworthy method of parasitic mermithid species classification.

Previous studies have noted that simuliid species with similar life histories, occupying the same streams at the same time, tended not to be parasitized by mermithids at the same time (Colbo and Porter 1980, Ebsary and Bennett 1973, 1975). Data from this study, however, indicate that black flies with different life histories are infected by the

same mermithid species. P. fuscum/mixtum & S. vittatum s.l. both harbour Gastromermis

sp. A, S. vittatum s.l. & C. dacotensis both harbour Romanomermis. Black flies with similar life hi.stories harbour mermithid species from different subfamilies. The univoltine

black fly species P. fuscum/mixtum and St. mutata exhibit similar life histories, overwintering together in the same habitats; however, P. fuscum/mixtum harbours

Gastromermithinae, while St. mutata harbours Mesomermithinae. Three species

harboured two mermithid species from different subfamilies. S. truncatum/venustum/

92

rostratum harboured Strelkovimermis and Gastromermis sp. C, C. dacotensis harboured

Romanomermis and Gastromermis sp. B, and S. vittatum s.l. harboured Romanomermis and Gastromermis sp. A.

The mermithid species Neomesomermis flumenalis has previously been found to parasitize both P. fuscum/mixtum and 5. venustum in Newfoundland (Ebsary and Bennett

1973, 1975). Neomesomermis flumenalis, however, was not found to be present in any of the species parasitized by mermithid nematodes in Algonquin Provincial Park. Perhaps the mermithids in these early studies were not identified accurately; Welch acknowledged

that some mermithid identification practices were inadequate (Welch 1962). It is also possible that geographic distribution plays a key role in parasite-host interactions.

Separate geographic locations could result in different mermithid and/or simuliid species presence, potentially altering host specificity. Data obtained from this study in Ontario differed from findings of a study in Newfoundland (Colbo and Porter 1980). In both cases, P. fuscum/mixtum and St. mutata had similar life histories, cohabiting the same streams throughout the winter and early spring. In Ontario, these simuliid species groups harboured two separate mermithid species, but in Newfoundland P. fuscum/mixtum harboured mermithid parasites while occupying the same stream as St. mutata that were free of mermithid infection. Molecular identification found that Ontario P.

fiiscum/mixtum harbour Strelkovimermis .sp., while St. mutata \\dibo\\T Aranimermis sp.

While mermithids from the Newfoundland study were not identified, it is possible that, in this case, Strelkovimermis sp. occur in both Ontario and Newfoundland, while

Aranimermis sp. potentially occur in Ontario but not Newfoundland.

93

While no significant difference was noted in the monthly variation of mermithid frequencies in C. dacotensis and 5. vittatum s.L, these species do play a role in mermithid transmission within the streams. C. dacotensis is an early season univoltine simuliid species that, unlike the early season univoltine P. fuscum/mixtum and St. mutata species, do not over-winter as larvae. Ova from this species eclose in the early spring, with larval development complete by late May or early June. C dacotensis is the only simuliid species to harbour Gastromermis sp. B; furthermore, C. dacotensis and S. vittatum s.L are

the only species to harbour Romanomermis. Also, S. vittatum s.L does not exclusively harbour one mermithid species, co-harbouring Romanomermis with C. dacotensis, and

Gastromermis sp. A with P. fuscum/mixtum. Since monthly mermithid presence in S.

vittatum s.L does not vary significantly, it is possible that some mermithid species have undergone an evolutionary adaptation to parasitize multiple host species. Such an adaptation could result from factors limiting mermithid development, such high

competition for a suitable host. Perhaps Romanomermis only parasitize S. vittatum s.L if there are not sufficient numbers of C. dacotensis hosts in the stream. Likewise,

Gastromermis sp. A. might infect S. vittatum s.L when P. fuscum/mixtum host presence is limited. Previous studies have demonstrated the benefits of long-term monitoring (Colbo

1990, Colbo and Porter 1980), and the influence of winter temperatures and environmental conditions on infection prevalence throughout the season (Colbo 1982).

Thus, to gain a more accurate interpretation of the infection rates and parasite-host species interactions within a stream, a more long-term .study must be implemented.

Pamsite-host species interactions throughout the annum can be represented using a seasonal timeline. Mermithid species infection patterns within the multi- and univoltine

.

94

simuliid species are presented in Figure 23, incorporating mermithid parasitism of simuliid larvae, pupae and adults over the course of a 12-month period.

4.6. Larval sex determination

4.6. 1 Morphological sex determination

The developmental stage at which feminization is initiated was determined by examining gonad morphology and meiotic chromosomal condition in mermithid-infected black fly larvae. Morphological feminization is initiated in mermithid-infected black

flies early in larval development, prior to gill histoblast formation, since gonad morphologies of uninfected and mermithid-infected black flies differ significantly at both early and late developmental stages. Note that although the same data used to test effects of developmental stage and infection in two separate tests, even the most conservative

Bonferroni correction does not alter the results (Sokal and Rohlf 1995).

4.6.2. Genetic sex determination

The chromosomal condition at meiosis of mermithid-infected simuliid larvae

differs significantly from their uninfected counterparts during both early and late

developmental .stages. Mermithid-infected morphological males exhibit either male

(achia.smate meiosis) or female (chiasmate meiosis) chromosomal conditions, while

uninfected morphological males only exhibit the characteristic male achiasmate meiosis.

Mermithid-infected morphological females only exhibit female chromosomal conditions

(chiasmate meiosis). mirroring their uninfected morphologically female counterparts.

95

P. fuscum/mixtum Winter Spring Summer Fall

L LP LPA

St. mutata

96

Also, based on the morphology of the gonads, both uninfected and mermithid- infected simuliid larvae do not differ in genetic status between early and late

development. The prevalence of mermithid-infected larvae with no gonads present at all, or those with gonads exhibiting no visible condensed chromosomes, suggests that mermithid infection also inhibits simuliid gonad development. Although two statistical procedures were carried out (uninfected versus infected, early larvae versus pharate pupae) that were not completely independent of one another, even the most conservative

Bonferroni corrections do not alter the results (Sokal and Rohlf 1995).

97

5. Conclusions

This study was designed to characterize parasite-host seasonal variation with

respect to species associations of the mermithid and simuliid populations. Population

monitoring and modern taxonomic approaches were used, as well larval gonad

examination to establish the developmental life stage at which feminization is initiated.

Mermithids critically interfere with survival capabilities of simuliid larvae. However,

since the development and distribution of the mermithids themselves is dependent upon

the host living at least long enough for the parasite to complete the parasitic stage of its

lifecycle, it would be advantageous for the mermithid parasites not to kill the simuliid

hosts too soon. Such an effort would ensure mermithid redistribution upstream via adult

fly dispersal. Mermithids do induce supernumerary moults and suppress histoblast

formation in the larval host, providing longer development time in the host body (Phillips

1998). Oftentimes hosts that undergo such alterations fail to pupate, resulting in host

killing. Perhaps two evolutionary stable strategies are being observed within the

mermithid populations; .some mermithids kill or alter development of the host larvae, others remain in the host through pupation, resulting in a feminized adult host.

The Evolutionary Stable Strategy (ESS) can be used to interpret the complex

interactions of the mermithid and simuliid populations. Natural selection influences

species development, with optimal behaviour resulting from a dynamic stability between

the costs and benefits of a particular outcome, with each successive successful outcome

improving on itself. An ESS cannot be invaded by a competing alternate strategy within a

population that would result in lower reproductive success (Smith 1982). Assuming that

natural selection prevents individuals that utilize strategies which result in lower payoffs

98

fiDm succeeding, a population would evolve that is resistant to any changes that would result in new traits leading to lower reproductive output. For mermithid nematodes to maintain reproductive outputs and colonize new habitats, a significant proportion of parasites must carry over into and feminize the adult host simuliids, guaranteeing transportation back upstream or to a new headwater (Colbo 1982). Such alterations of the host could be an ESS that ensures upstream dispersal, returning the mermithid nematodes to an aquatic environment to successfully complete the life cycle (Vance 1996, Colbo

1978, Colbo 1982).

Alternatively, it is possible that those mermithids which kill the host prior to adult development are able to successfully relocate and reproduce in some other way. Only in particular habitats would these seemingly premature emergence patterns be tolerated, such as a longer waterway containing numerous riffles, producing multiple simuliid populations further downstream. Upon emergence from the larval host, the mermithid may continue to drift downstream where a chance encounter with another mermithid from the same species would result in a successful mating. Once the Fl generation of mermithid preparasites hatch, they could re-enter the water and continue to drift downstream to the next simuliid population, subsequently infecting this host population, passing on their genetic information into a subsequent generation and, at the same time, dispersing into new down.stream locations.

The presence of mermithids in feminized black flies, and the lack of intersexes in uninfected black flies, indicates that mermithid parasites play a key role in simuliid feminization. The precise interactions that trigger the onset of feminization, however,

remain unknown. Additional research is required to determine whether all mermithid

99

species parasitizing Simuliidae result in host feminization, whether certain mermithid

species result in host killing rather than sex alteration, or if some mermithid species result

only in adult host feminization. By utilizing modem molecular taxonomic approaches

such questions can be answered. Furthermore, the proximate cause of feminization needs to be discovered to assist in a more complete understanding of the mermithid-simuliid

interactions. Such understanding could lead to methods with which to control black fly populations using the feminizing agent as a new and innovative instrument for biological control.

Simuliid populations can be regulated via natural control agents by inoculating the population with mass-produced organisms. Such introduced organisms can either be

foreign or naturally occurring within the population; if the situation is the latter, it would be necessary to inundate the simuliid population with the biocontrol agent. By synchronizing the control agent with the simuliid population, treatment may interrupt

normal host development, ultimately reducing the presence of black flies, disease transmission and improving the quality of life in areas plagued by Simuliidae. In the observed mermithid infections of black flies, the parasite may kill the host or lead to host feminization. If infection numbers are high enough, parasite populations may attain levels of infection sufficient to virtually eliminate host populations.

Predators as potential biological control agent have not yet been heavily investigated. Other Dipteran Families (mainly Chironomidae, Empididae, Muscidae,

A.silidae, Dolichopodidae, Phoridae, Drosophilidae and Scathophagidae) and various

Orders (Coleoptera, Odonata, Plecoptera, Trichoptera, Cru.stacea, Turbellaria, and some browsing fish) are predators of simuliids (Werner and Pont 2(X)3). Insect pathogens.

100

however, have advantages over predators as biocontrol agents. Pathogens, such as Bti, are selective, many can be mass produced on artificial media, and some are stable over long

periods of time (Lacey and Undeen 1986), all qualities of an effective and financially feasible biocontrol agent.

Laboratory culture of mermithid nematodes for biocontrol against Simuliidae has proven to be extremely difficult. Mermithids require precise development conditions, and

once reared, it is difficult to maintain the nematodes long enough to transfer them to an

^plication site for biological control (Bailey et al. 1977, Bailey et al. 1974). If the actual trigger that initiates feminization in the mermithid-infected black flies can be determined,

it might be possible to abandon the mermithid rearing procedures and directly administer

the feminizing agent to simuliid populations on a large scale. It is possible that sexual alteration could be initiated via a feminizing bacterium, such as Wolbachia (Werren

1997) or Candidatus Cardinium (Zchori-Fein and Perlman 2004), carried by the mermithids to the simuliid hosts.

When treating species of economic importance, such as black flies, it is essential to determine the exact stage in the lifecycle at which treatment with a control agent will

have an optimal outcome (Alencar et al. 2001). For instance, if a feminizing bacterium is

responsible for the observed feminization in Simuliidae, it is critical to fully understand the life histories of the mermithid and simuliid species within the population, as well as

knowing the developmental stage at which feminization is initiated. If treatments are applied post-initiation stage, they may have reduced or no effect. Treatment application times could be accurately predicted by regularly monitoring air and water temperatures in the host simuliid habitats and combining these data with the known developmental degree

101

days required by each black fly species (Ross and Merritt 1978). Since this study

determined that sexual alteration occurs early in development prior to histoblast

formation, simuliids must likely be exposed to the feminizing agent during the first or

second larval instar to effectively induce feminization.

Additionally, it remains unknown as to whether or not mermithid-infected adult black flies are capable of becoming gravid and producing viable offspring. Mermithid- infected adult black flies have been noted to have developed ovaries, but were killed by the mermithid parasite upon exiting the host (Colbo and Porter 1980). If successful reproduction of mermithid-infected black flies does occur, then the use of mermithid nematodes as biological control agents against Simuliidae might not be a feasible concept. Further examination of gravid female black flies to determine if any harbour mermithid infection, and whether these individuals are capable of producing viable offspring can solve this issue. The fact that feminization occurs at both phenotypic

(morphological) and genetic levels affords the possibility that intricate physiological feminization could also be occurring, not with respect to gonad structure, but to viability

of feminized individuals. Further examination of feminized adult black flies is necessary to fully understand the dynamics of such sex alterations and potential impacts to simuliid populations.

102

6. Summary

Mermithid parasites and their simuliid hosts from Algonquin Provincial Park were examined, characterizing parasite-host seasonal variation and establishing the

developmental Ufe stage at which feminization is initiated. Mermithid infection influences the phenotypic proportion of sexes within a population of adult black flies, increasing the prevalence of female and feminized adult flies while decreasing the relative frequency of phenotypic male flies in a population. A detailed analysis of seasonal variation between mermithid and simuliid populations revealed that black flies

with different life histories can harbour the same mermithid species and, conversely, black flies with similar life histories can harbour mermithids from different subfamilies.

In observed cases of a simuliid species hosting two mermithid species, these parasites were firom different subfamilies. Molecular identification revealed two mermithid subfamilies, Mesomermithinae and Gastromermithinae, present in the simuliid hosts.

Also, mermithid colour variation was not found to be a reliable species indicator.

Feminization is initiated in mermithid-infected black flies early in larval development, prior to gill histoblast formation. Mermithid-infected simuliid larvae were observed to be morphologically male (testes present) or female (ovaries present), with morphological males exhibiting either male (achiasmate) or female (chiasmate) meiotic chromosomes; morphological females were only observed as genetic females

(chiasmate). Additionally, it was found that mermithid-infection inhibits simuliid gonad development.

103

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Appendices

Appendix I Recipes

Camoy's fixative

3 parts absolute alcohol to 1 part glacial acetic acid

Feulgen stain

1. Ig basic fuchsin added to 200 mL pre-heated distilled water at 80°C

2. Cool to 60°C, add 2 g potassium metabisulphite

3. Cool to 50°C, add 10 mL IN HCl

4. Store in a dark fridge overnight

5. The next day, add 1 spoonful of Norit (decolourizing carbon), shake and filter

6. Feulgen stain is now clear, and should be kept refrigerated in the dark.

Sulphur dioxide water

1 200 mL distilled water

2. 1 g potassium metabisulphite

3. 10 mL IN HCl

4. Mix thoroughly and put in tightly stoppered bottle.

50% Acetic acid

1 part glacial acetic acid: Ipart distilled water

2% Aceto-carmine

1. 55mLdH20

2. 45 mL glacial acetic acid

3. 5 g carmine

4. Boil solution for 1 5 minutes

5. Cool

6. Filter

7. Add 1 2 mL of solution to 1 8 mL of dH20 for 2% solution.

113

Appendix 11 Total number of simuliid larvae and/or pupae collected from Costello and Mud Creeks from April 30 to August 19, 2005. No collections were made between June 29 and July 27, 2005. While S. truncatum/venustum/rostratum numbers exceeded 250 during the summer months, the y-axis reaches a maximum of n=250 to highlight rarer species numbers.

250

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114

Appendix in Percent mermithid infection according to simuliid species collected from Algonquin Provincial Park from April 30 to August 18, 2005. No collections were made between June 29 and July 27, 2005.

100

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116

Appendix V Mean monthly air temperatures for Algonquin Provincial Park, obtained from Environment Canada Climate Data Almanac.