explainhg variation in immune defense against parasitic water mites.
Christopher Paul Yourth
A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Zoology University of Toronto
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Abstract of a thesis submitted in conformity with the requirements for the degree of Master of Science. Depanment of Zoology University of Toronto
Christopher Paul Yourth. 201 This thesis tests predictions of the emerging theory of ecological irnmunology using variation in immune expression of Lestes dryas Kirby. Lestes focipatus Rambur. Lestes unguiculatus Hagen and Lestes congener Hagen to a generalist parasitic water mite.
A rrenums planus Marshall. Immune responses of the four lest id species were compared as they relate to prevaîence and intensity of mite infection; these measures of parasitism did not fuliy explah ainong-species variation. Within-species variation in immunity of L. forcipatus was reiated to time of season, but not to host body size or asymmetry, measures of host condition. When L. fmipatus were aliowed to respond to Sepahdex beads at a fuced temperature across season. no seasonal pattern in irnmunity was observed and a positive correlation between condition and immune response in males was detected.
These results implicate seasonal variation in temperature as being a major factor in determining immune responsiveness of lest id damselfiies. iii
Acknowledgments
Numerous people deserve thanks for their contributions to this thesis. Primarily, 1 thank Dr. Mark Forbes who has been an exemplary role mode1 and CO-supervisor.and my other CO-supervisorDr. Robert Baker for his guidance and fuiancial support. 1 also thank my adjunct supervisor Dr. Angela Lange for her feedback and suggestions. 1 am indebted to Dr. Bruce Smith who is an inexhaustible source of information on insects, mites, lab techniques, statistics and everything else. 1 feel very fonunate for Bruce's involvement in this thesis.
1 thank Dr. Raleigh J. Robertson. Frank Phelan and theù staff at the Queen's University Biology Station, for maintainhg such a welcoming environment for education and research. Chapter three would not have been possible without the field and lab assistance of Marc Lajeunesse and his plucky sidekick Laborlux K, microscope of Dr.
James Fullard. I am gratehil for the many hiends 1 have made there over my the there, of these 1 especialiy thank Kit Muma, Bruce Smith, Heather McCracken, Tim Demmons and Alissa Moenting.
1 also thank Luc Bussiere, Clint Kelly and my labmate Tmdy Watson for their feedback on various aspects of my thesis, and the rest of my confederates here in the Biology Group for the welcome distraction they have provided.
Finally, 1 thank my parents Harold and Elaine Yourth, for their unwavering support.
Chapter 1 of this thesis fomthe basis of the manuscript: Yourth, C.P., Forbes,
M.R., and Smith, B.P., 2001. On understanding variation in immune expression of the damseiîlies Lestes spp.. Canadian Journal of Zoology 79:8 15-82 1). This research was supported by a Naturaï Sciences and Engineering Research Grant to MRF. Chapter 2 forms the basis of the manuscript: Yourth. C.P., Smith, B.P., and Forbes. M.R.. Immune expression in a damselfiy is related to time of season. not to fiuctuathg asymmetry or host sue. (Accepted Mar. 2001. Ecological Entomology). This research was supported by a Natural Sciences and Engineering Research Grant to M.R.F. I thank the anonymous reviewers of these manuscripts for their helpful comments. Table of Contents .. Abstract ...... *...... *...* ...... *..* ...... * .... * ...... 11 .. . Acknowledgements ...... iii Table of Contents...... v
List of Tables ...... vtii.m. List of Figures ...... ix General Introduction ...... 1 Chapter One: On understanding variation in immune expression of Lestes spp.
damselflies...... 7 Abstract ...... 8 Introduction...... 9
Relevant natural history of t-k host-parasite association ...... 1L
Methods ...... m...... e...... m...... 12 Censuses to examine patterns of mite infestation and emergence dates ... 12 Preparations for viewing mite stylostomes or feeding tubes ...... 14 Results ...... 15 Do males or females or species differ in mite infestation? ...... 15
Do emergence dates diner across species and does this account for
variation in mite prevalence? ...... 17 Are dead mites asswiated with melanized stylostomes? ...... 17 Does immune responsiveness differ across species? ...... 18 Discussion ...... f 8 Literature Cited ...... 23 Chapter Two: Immune expression in a damselfly is related to time of season. not to fluctuating asymmetry or host size ...... 31
Abstract ...... , ...... 32 ...... Introduction ...... 33 Relevant natural history of the host-parasite association ...... 35 Methods ...... 36 Results ...... 39
Patterns of mite infestation ...... , ...... 39
Causes of mite death ...... , ...... , .... 39 Immune responsiveness in relation to gender...... 39 Immune responsiveness of males and fernales in relatio r.
to other factors...... 40
Discussion ...... 41 Literature Cited ...... 44 Chapter Three: Pattern of immune expression in a damseifiy is not explained
b y seasonal differences in allocation to immunit y ...... 56
Abstract ...... 57 Introduction ...... 58
Methods ...... 60 Survey and collection ...... 60 Scoring of immune responses ...... 62 Analyses ...... 63
Results ...... 64 vii
Survey results ...... 64 Condition, immune response. and date of emergence ...... 65 Discussion ...... 66 Literature Cited ...... 69 Sumrnary and Conclusions ...... 74 viii
List of Tables
Chapter One
Table 1: Comparisons of prevalence of infection for males and females of eac h species ...... *....*...... 26 Table 2: Comparisons of median intensity of infection for males and females
of eac h species ...... 27 Table 3: Proportions of infected hosts responding for males and females of each species ...... 28
Chapter Two
Table 1: Comparisons of Julian dates of capture and winglengths of responders and non-responders ...... 50 Table 2: Comparisons of absolute wing celi asymmetry of responders and non-responders...... 5 1 List of Figures
Chapter One
Figure 1: Proportion of individuals responding in relation to prevalence of mite infestation ...... 29
Chapter Two
Figure 1: Light micrograph of nomand encapsulated Arrenurur planus Stylostomes...... 52
Figure 2: Relationships between residual numbers of dead mites and date of sampling ...... 54 Chapter Three
Figure 1: Relationships between condition and Julian date of capture. residual number of beads melanized and condition, and residual number of beads melanized and Julian date of capture ...... 72 General introduction Ecological immunology is concemed with how hosts contml parasite infections and reduce the associated fitness costs (Sheldon and Verhulst. 1996). This field considers the fact that immune responses require host resources and that in some situations the immune response may be more taxing to the host than the cost of the parasite. However. whether the host is losing resources to the parasite or to the response. these resources are not available for purposes such as reproduction. Thus it is expected that host immune responses will be optimized and traded off against other hinctions.
These optima should differ depending on condition of the hosts and costs of the specific immune response and costs of the other funciions of the host. As a result, individuals within a population of hosts are expected to employ different tactics in mitigating the impact of parasites.
Although many investigators of ecological immunology study birds (e.g. Ots et al.. 1998; Horak et al.. 1999; Ilrnonen et al.. 2000; Noms & Evans. 2000; Raberg et al..
2000). the insect melanization response (described later) has proved to be a fertiie system for investigating iife history trade-offs with irnmunity. Variation in immune expression of insects, as is tme for any character of an organism, is attributable in part to genetic variation; the majority of investigations into the role of genetics in immune expression of insects use Drosophila and their parasitoids as a models (Hughes & Sokolowski. 1996;
Kraaijeveld & Godfray, 1997; Feiiowes et al.. 1998). Th&thesis attempts to address some of the non-genetic factors. which may govem the immune expression of lestid damselflies. These factors include the cost/risk of parasitism, the costs and benefits of
responding as well as the costs of maintainhg an immune delense in the absence of
parasitism. For example, if the risk of parasitism is low. and the cost to maintain a
defense is high. the most adaptive tactic may be to redwe investment in immunity to a minimum (Sasaki & Godfray. 1999). Likewise the most adaptive degree of investment, for an infected host. will depend on the benefit:cost ratio of the immune investment
(Behnke et al.. 1992). with the total energy diverted to parasitism and response king minimized.
Determinhg how selective pressures rnay influence the immune function of organisms within a population requires some knowledge of the system in question. It is important to know the variation in parasitism and immunity observed in nature as weil as the costs of parasitism and response. The variation in parasitism is known for this system
(see chapter one). Previous studies in sirnilar systems indicate mites can reduce flight ability. fecundity, mating success, and survivorship (Lanciani, 1983; Smith. 1988;
Forbes, 199 1; Forbes & Baker, 199 1; Léonard et al., 1999). Costs have also been shown in the maintenance of immunity in Drosophila (indicated by reduced cornpetitive ability of parasitiod resistant kva when competing with normal lama for limited food resources;
Kraaijeveld & Godfray, 1997; Feliowes et al.. 1998) and of response in Bum ble bees
(indicated by reduced survival when responding to inert Unmune elicitors during starvation, as compared to non-responding individuals: Moret & Schrnid-Hempel, 2000).
Given this Wormation. I can attempt to explain the variation in the immune responses of lestid damselflies. Chapter One describes variation in immune responses based on risk of parasitism.
In this chapter, immune responsiveness of four species of lestid damseiflies are compared
on the basis of prevalence and mean intensity of infestation by Arrenurus planus Marshall mites.
Chapter Two investigates the importance of size, asymmetry. and date of capture
in predicting immune responsiveness of Lestes forcipatus to A. planus mites. The size of the damselfly may affect the amount of resources available for immune responses.
Asymmetry has been iinked to condition (Leung & Forbes, 1996) and may also affect responsiveness. Additionally, date of capture may affat degree of response of damselflies to mites since melanin is used for both immune responses and thennoregulation and since early emerging damselfles are more %ely to experience cooler temperatures (Corbet. 1999).
FinaiIy, Chapter Three presents immune response data for L. furcipatus assayed through the experirnental injection of Sephadex beads and assessrnent of the resulting encapsulation. Individuals were aîiowed to respond to the beads at a contmiled temprature, testing whether the patterns of immunity uncovered in chapter two are the result of differential investment in immunity across the season. Literature Cited
Behnke. J.M.,Barnard, C.J. & Wakelin, D. (1992) Understanding Chronic Nematode
Infections: Evolutionary Considerations. Current Nypoiheses and The Way
Foward. International Journal for Parositology, 22,86 1-907.
Corbet, P.S. (1999) Dragonjlies: Behuvior and Ecology of Odonatu. Comeil University
Press. Ithaca, New York. 829 pp.
Fellowes, M.D.E.. Kraaijeveld. AR.. & Godftay. H.C.J. (1998) Trade-off associated
with selection for increased ability to resist parasitoid attack in Drosophila
melanogaster. Proceedings of the Royal Socieq of London Series B, 265. 1553-
1558.
Forbes. M.R. (1991) Ectoparasites and mating success of male Enallagma ebrium
damseiflies (Odonata: Coenagrionidae). Oikos, 60,336-342.
Forbes, M.R.L. & Baker, R.L. (1991) Condition and fecundity of the damselfly.
Enallagma ebrium (Hagen): the imponance of ectoparasites. Oecologia, 86,335-
34 1.
Horak, P.. Tegelmann, L., Ots, I., & MnUer. A.P. (1999) Immune hinction and survival of
great tit nestlings in relatio to growth conditions. Oecologiu, 121, 3 16-322.
Hughes, K. & Sokolowski, M.B. (1996) Natulai Selection in the Laboratory for a change
in Resistance by Drosophila melanogaster to the parasitoid wasp Asobara tabido.
Journal of Insect Behavior, 9,477-49 1.
Ilmonen, P., Taarna. T., & Hasselquist, D. (2000) Experimentally activated immune
defence in female pied flycatc hers results in reducd breeding success.
Proceedings of the Royal Society of London B. 267,665-670. Kraaijeveld. AR. & Godfray. H.C.J. (1997) Trade-off between parasitoid resistance and
larval competative ability in Drosophila melanogaster. Nature. 389. 278-280.
Lanciani. C.A. (1983) Overview of the effects of water mite parasitism on aquaticinsects.
Research Needs for Development of Biological Control of Pests by Mites (cd. by
M. Hoy, O. Cunnigharn and L. Knutson), pp. 8690. In Agriculncral Erperimental
Station of University California Publication 3304. Berkeley. California.
Léonard, N.J.. Forbes, M.R. & Baker, R.L. (1999) Effects of Limnochares americana
(Hydrachnidia: Limnocharidae) mites on üfe history traits and grooming
behaviour of its damselfly host. Enallagrna ebrium (Odonata: Coenagrionidae).
Canadian Journal of Zoology, 77. 16 15- 1622.
Leung. B. & Forbes, M.R. (1996) Fluctuating asymmetry in relation to stress and fttness:
effects of trait types as revealed by me ta-anai ysis. Écuscience, 3, 4004 13.
Moret, Y. & Schmid-Hempel, P. (2000) Survival for Immunity: The Price of Immune
System Activation for Bumblebee Workers. Science, 290, 1166- 1 168.
No mis. K.. & Evans, M.R. (2000) Ecological immunology: Life history trade-Offs and
immune defense in birds. Behavioural Ecology. 11, 19-26.
Ots, 1.. Murumatgi, A., & Hôrak, P. (1998) Haematologicai health state indecied of
reproducing Great Tits: methadology and sources of natural variation Funcrional Ecology, 12,7W-707.
Raberg, L., Niisson. J.-A., Ilmonen, P., Stjernman, M.. Hasselquist, D. (2000) The cost of
an immune response: vaccination reduces parental effort. Ecofogy Leners. 3.382-
386. Saski, A & Godfray, H.C.J.(1999) A mode1 for the coevolution of resistance and virulence in coupled host-parasitoid interactions. Proceedings of the Royal Sociery
of London B, 266.455-463.
Sheldon, B.C. & Verhulst, S. (1996) Ecological immunity: costly parasite defenses and
trade-Offs in evo lutionary ecolog y. Trends in Ecology and Evofution, 1 1, 3 17-
321.
Smith. B.P. (1988) Host-parasite interaction and impact of larval water mites on insects.
Annual Review of Entowwlogy, 33,487-507. Chapter One:
On understanding variation in immune expression of Lestes spp. dameiflies Abstract Immune ability and expression have been viewed as Me history traits influenced by factors such as iikelihood of being parasitized, intensity and costs of parasitism. and trade-offs associated with immune expression. In this chapter. 1 show that different patterns of parasitism by a generalist ectoparasite (Arrenurus planus Marshall)
(Anenuridae: Hydrachnida) do not explain fully the variation in immune expression across four species of sy mpatk lest id damselflies (Lest idae: Zygo ptera). The immune response was melanotic encapsulation of mite feeding tubes, which was associated with dead mites. Within species. no gender bias in immune expression was evident. One oft- exploited species did not mount immune respom against attending iarval mites. while the th-e other species showed variable expression. Of the species show ing immune expression, the species with the highest prevalence and intensity of infestation had a significantly higher proportion of individuals responding immunologically to mites. Thus current infestation levels only partially predict immune investment; a consideration of timing of emergence of different species suggests that season may be an important predictor of immune investment. htroduction
One main goal of the field of ecological immunology is to understand which (or
when) hosts should commit resources, the, and energy to defense against parasites and
pathogens (Sheldon and Verhulst 1996). Studies on variation in resistance or in
susceptibility are typically done at the within-species level. 1 suggest that studies of generalist parasites attacking suites of closely rehted species present another approach to
testing theory and to targeting questions of interest for funher within-species studies. 1
illustrate these points through a detailed study of the extent to which males and females of four rehted and syntopic species of damselflies (Lestidae: Zygoptera) are differentiaiîy parasitized by the ectoparasitic water mite, Arrenurus planus Marshall (Arrcnuridae:
Hydrachnida). 1 then test whether differentiai parasitism is predictive of differential immune responses.
This central prediction is easily tested for my study system. Larval mites colonize
larval hosts before actual eclosion to adults and host responses to mites occur before host
reproduction. In damselflies, host responses are melanotic encapsulation of mite feeding
tubes (Abro 1979.1982). Thus for damselfiies. differences in relative timing of
parasitism or variation in reproductive effort should not codound responses arnong
species. Other stud y systerns have been used to address whether dzferences in age- specific reproductive effort of hosts carry a cost of increased parasitism: some observational and experimental studies provide support, whereas others do not (reviewed in Mgller 1997). Another factor deemed important to immune responses is the cost of parasitism.
The physiological costs of parasitism by individual larval mites are probably similar for the damselfly species considered herein for two reasons. Fist, only one species of parasite is king considered. In fact, A. planus is the only mite species exploithg these damselflies at my study site. Second, there is considerable size overlap between these damselîlies (Walker 1953). Notwithstanding. the costs of parasitism may differ among species depending on differences in intensities of infestations. nius. 1 consider not only the prevalence of parasitism, but also intensity of infestation once parasitized.
1 suggest that the absolute costs of resistance should be similar across species if the same immune mechanism is deployed (Le. melanotic encapsulation of feeding tubes).
Cost of resistance for invertebrate hosts has been shown or strongly inferred (e.g.. KUnig and Schmid-Hempel 1995; Kraaijeveld and Godfray 1997; Siva-Jothy et al. 1998).
Although the same resistarne mechanism is deployed in damselflies, its deployment may have varying costs across species due to differing trade-offs mediated by unknown ecologicai factors. 1contend that cornparison of species differing in their ecology may provide clues about trade-offs with immunity.
1 scored differences in parasitism by mites in two ways: proportion of hosts infested and numbers of mites on infested hosts (termed prevalence and intensity, sensu stricto Bush et al. 1997). I also considered emergence dates of hosts as temporal co- occurrence of hosts and larval mites has been implicated in host species use for other mite-insect systems (e.g.. Smith and McIver 1984a; Forbes and Baker 1991). 1 included seasonal data to see if 1 could explain variation in prevalence of parasitism. Irrespective of the initial cause(s) of differences in parasitism, my main objective was to investigate whether measures of immune expression were predicted by measures of parasitism. Relevant natural history of the host-parasite association
1 examine the suitability of Lestes congener Hagen, Lcstes dryas Kirby, Lestes
forcipatus Rambur. and Lestes unguiculatus Hagen as hosts for the water mite. A. planus.
Awenums planus is a generalist parasite, exploiting a small number of Sympetrum
dragonfly species and a similarly small number of Lestes damselfly species. While only
parasitizing approximately eight species in two disparate taxa, the common ünk is that
these hosts are the oniy odonates in the region that inhabit temporary autumnal and vernal
woodland pools (Wiggins et al. 1980). Arrenums planus is the only parasite of odonates
in temporary waters. and is an obligate temporary pool speciaüst. These four species
emerged from the same pond and were parasitized by only one mite species. namely, A.
Colonization of odonate hosts by Arrenurus spp. mites occurs in a series of steps
during which the hosts may mount defenses. Larval mites colonize odonate larvae and
larvae can groom and reduce attachment rates (Forbes and Baker 1990; Leung et
al. 1999). Larval mites are phoretic on odonate larvae; once larval hosts emerge and start
to eclose, the larval mites abandon the cast exoskeleton and crawl ont0 the newly- formed imago. Mites cling to the host with their palpal claws. and won pierce the host's cuticle using their cheiicerae (Smith 1988). Anchored to the host by the chelicerae, the mite secretes an acellular mucopolysaccharide that foms a stylostome (a blind-ended feeding tube: Smith 1988).
Some dragontly species attempt to neutralize one or more stylostomes through melanotic encapsulation (Abro 1979, 1982). In other species such as Synpetrum intemum Montgomery. the host appears to aggregate haemocytes at the site of stylostome
formation (Forbes et 01.1999) similar to other insects parasitized by mites (cf. Davids
1973). If resistance is not mounted and the mite engorga successfblly, it drops off the host when the host retums to the water for reproduction simikir to other mite-damselfly associations (RoH and Martens 1997). nie mite then continues through its nymphal and adult stages as a fixe-living aquatic predator on microcrustaceans. On some hosts. scars
are clearly visible following mite detachment (Forbes 199 1). In this study, damselflies
which had just gained their mites (newly-emerged) are included; damselflies which had
matured and had mites but not mite scars, are also included (but see below for an exception).
Methods
Censuses to examine patterns of mite infestation and emergence dates
1 caught teneral or pre-reproductive damselflies to elucidate pattems of infestation and reproductively mature damselflies to elucidate pattems in engorgement success.
Collections were done at Yeze~ac'sPond (44'32' 12"N, 76O22'55"W), located on the
Hiida and John Pangman Conservation Reserve of the Queen's University Biological
Station or QUBS faciiities. 1 made 5 1 trips to Yezerinac's Pond between 28 May and 5
August 1998. On each trip, 1 netted damselfiies and used a 20X loupe to identify them to species (foiiowing Walker 1953) and count any mites. 1 recorded gender and approximate age of each individual (cf. Walker 1953). For all species. teneerals had soft and shiny wings a soft abdomen and üttle dark pigmentation in the exoskeleton. Young to old adults al1 had rigid and duil winp young adults had melanin deposits in their appendages only, older adults had pigmentation and the oldest adult males were pruinose.
In total, 754 damselflies were used in analyses. 1 marked damselflies on wings, after processing them as described below, to preclude their re-sampling (foilowing Forbes
1991). Seventeen damselflies were old adults (5 L. unguicufutus, 2 L. congener, 10 L.
forcipatus) and had only scars and no mites. These were excluded from analyses because
1 could not score mite numbers reliably. Another 23 adults (ca. 3%) had a few scars and
mites, which were identitled as A. planus (1 L. unguiculatus, 3 L congener, 19 L
forcipatus). Thus, a few mites appeared to have detached. 1 included these adults in my
analyses because they still had many A. plantrs mites, which appear to detach more or
less simultaneously (Le. only 3% of adults were found with both scars and mites).
For L congener femaies, 14 tenerals and 11 older adults were caught; for male L.
congener, nine tenerals and 18 older adults were netted. For L. dryas females and males,
24 and 23 tenerals and 33 and 75 older adults were caught, respectively. For L. forcipatus
females and males, four and one tenerals and 37 and 49 older adults were caught,
respectively. Finally, for L unguiculatus females and males, 53 and 54 tenerals and 150
and 182 older adults were caught, respectively. These differences in sample sizes may
reflect differences in availability at times of sarnpling, more so than differences in actual densit y.
For analyses of emergence dates, only tenerals were included; these emerged within one or two days of collection. For ail 0thanalyses, all age categories were considered. 1 recorded the numbers, location of ali A. planus mites found on teneral damselfîies and engorgement success on older damseMies. Mites were alrnost invariably found on the venter of the thorax on a tubercle, or near or on the coxal plates of the legs.
Dead mites were flattened and silver in colour. Mites staning to engorge were red to
orange with their legs still visible. Mites were scored as fully engorged if theû legs were entirely obscured by theu swoilen body, and scored as partially engorged if the legs were only partially obscured (cf. Forbes et al. 1999).
Preparutions for viewing mite stylostomes orfeeding tubes
1 brought 39 damselflies with 2 1 dead mite(s) to the laboratory. 1 prepared samples for viewing stylostomes of mites (seven and six L. congener females and males, respectively; 14 and 1 1 L. forciputus females and males. respectively and one L. unguiculatus fernale). No L. dryas were found with dead mites. I stored these damselflies in glassine envelopes and reftigerated them until they were processed (within 48 h).
Preparation involved removing the head. legs, wings and abdomen. and placing each thorax in a 4.5 cm x 1.5 cm glass via1 and covering them with Andre's solution (1: 1: t chloral hydrate. acetic acid, and water by weight). 1 then stored the vials for a minimum of 48 h at ca 2VC before conducting dissections.
1 dissected damselflies by removing the dorsal half of the thorax (including the wing muscles) using no. 5 forceps and a 2.5 cm. 22-gauge syringe needle. 1then placed the ventral half of the thorax on a microscope slide with the exocuticular surface touching the slide. Mer applying glycerol to the sample and covering it with a cover slip. I examined each slide at lOOX (phase-contrast) and scanned for mites and stylostomes. 1 captured digital images of a subset of stylostomes that appeared whole. using a video camera mounted on a microscope and Snapp* software (Play inc. 1996. Snappy Video
Snapshot. Rancho Cordova U.S.A.: Play Inc.). To examine variation in successful engorgement. 1counted numbers of dead mites
found on mature males and females among dürerent host species. To see if dead mites
were the result of immune responses by the hosts. 1examined whether dead mites were
associated with partially- or whoily- encapsulated stylostomes. for the subsamples of the three host species.
Results
Do species difer in mite infestation ?
Generally, there were no gender bisses in prevalence for each species considered separately. although L. dryas females were nearly more Wrely to becorne infected than L. dryus males (x2=3.52. 1 df; p=0.06; Table 1). In cornparison, species clearly differed in the probability of becoming infested with mites. when males and females were combined
(x2=237.4;3df. p < 0.001). AU species differed from one ano ther in this respect, as the
95% confidence limits (cdculated foilowing Snedecor and Cohran. 1980) did not overlap for an y painvise comparison between species (Table 1). Lestes unguiculatus had the lowest prevaience overall(9.896. sexes combined). foiiowed by L. dryus (23.2%), then L. congener (46.2%) and fuially L. forcipatus (83.5%). Thus, 1 predicted that degree of immune expression might foilow this same pattern. if prevalence of infestation alone could select for greater or lesser maintenance or expression of immunity. Numbers (and log-transformed numbers) of mites on hosts were non-normaliy distributed (using Kolmogorov-Sminiov tests), prechiding parameuic tests for differences in mean numbers of mites between categories of hosts. Median intensities of mite
infestations did not differ between fernales and males of L. congener, L forcipatus and L.
unguiculatus. In cornparison, L. dryas females camed significantly more mites than L.
dryas males (x2=5.72.p < 0.025; Table 2). 1 excluded L. dryas from subsequent
statistical cornparisons of intensity between species, since the variation in intensity of
infestation within this species was considerable. Male L. dryas had a median intensity of
1 mite similar to L. unguiculatus males and females; femaie L. dryas had a rnedian
intensity higher than males and females of both L. unguiculatus and L. congener (Table
2).
Similar to prevalence data. the remaining thtee species showed considerable
variation in median intensity of mite infestations (Kniskal-Wallis x2=35.3,p c 0.001). 1 found that median intensities of infestation differed signifcantly only between L. forcipatus and L. unguiculatus (P < 0.05. using Dunn's multiple range test for unequal N and correcthg for ties, Zar 1996). Thus. while the prevalence data suggested a clear prediction of variation in immune expression, the intensity data would suggest that L forcipatus would differ from L. unguiculatus. 1 had a less clear predic tion with respect to other species, such as L. congener and L. dryas. Taken together, species clearly differed in prevalence of infestation, but only some species were sig nificantly different with respect to intensity of infestation. Do emergence dates differ across species and does this account for variation in mite prevalence ?
1 found significant variation in emergence dates (based on dates of capture of
tenerals) across species. using 2-Way ANOVA with gender and species as the main factors (species: F3,173= 239.5, p < 0.001. overail +=O-8 1). Gender and the interaction between gender and species did not account for significant variation in emergence dates
(gender: = a.26, p =0.68; interaction: F3,1734.51. p =0.68). For L. dryus, the mean emergence date & S.E.) was 153.7 & 0.8 days (Day l=lanuary 1. 1998). whereas for L. forcipatus and L. unguiculatus. average emergence dates were 164.0 + 2.7 and 170.3 + 0.5 days, respectively. For Lestes congener the average ernergence date was 190.4 + 1.1 days. Tukey's post-hoc cornparison indicated Lestes forciputus and L. unguiculatus showed no significant difference in mean emergence dates, but L dryas emerged sig nifîcantly earlier than these two species and L. congener emerged significant ly kter ihan al1 three species.
Emergence date did no t appear to account for sig nificant variation in prevalence of parasitism across species. L. forcipatus and Lmwiguiculatus, which overlapped in emergence dates. showed the greatest difference in prevalence.
Are dead mites associated with melmized stylostomes?
1 was able to view stylostomes associated with 86 dead mites. Al1 86 dead mites were associated with partially- (> 50%) or wholiy-melanized stylostomes suggesting that both males and females of L. congener and L forciptw are able to mount effective immune responses to mites, as are L. unguiculatus femdes (no male L. unguiculatus were studied). Lestes dryos males and femaies showed no evidence of melanotic immunity to mites (Table 3). Damselflies of the other three species had both live and dead mites. Some mites had perfatly formed stylostomes with no evidence of melanization on the stylostome itself; these mites were alive be fore preparations were made.
Does immune responsiveness differ between species ?
There were no differences between males and females in the proportion of hosts with one or more dead mites (x*values ranged from 0-0.5; P values from 0.48-1.0; Table
3). When males and females were combined, 1 found signficant variation in the proportion of hosts with one or more dead mites (x* = 55.3. df=3, P < 0.001). If 1 excluded L. dryas from tests, 1 still found significant variation across species in the proportion of host responding (x* -1 8.2, df=2. P < 0.001). Lestes forcipatus responded to mites more than L. unguiculutus, although L forci'tus and & congener did not dBer in this respect (compare 95% confidence limits for combined values from Table 3. Figure
1)-
Mscussion Several factors such as costs of parasites and costs of resistance should dictate whether or not a species evolves the abiiity to respond immunologically to a given parasite. In this study, 1 assessed whether current patterns of infestation by a generalist ectopanisite might help explain immune expression across four species of lest id damselfîies. 1chose this study system because only one mite species exploited ail host species. In addition, resistance to Arrenurus spp. mites, if deployed, is simüar between damselfly hosts (but see Forbes et al. 1999 for a different mec hanism of resistance in a dragonfly).
To my knowledge, no one has examined theoretically or empirically the relative importance of prevalence and intensity of infestation, on evolution of immunity or degree
of immune expression*These two metrics of parasitism are related generally across
species (Bush et al. 1997) and specifically for water mite-insect associations (Smith 1988). However. they need not relate such that the relative positionhg of host species is the same for both measures (this study).
Based solely on probability of being infested (Le. ptevalence), 1 predicted that
immune expression (proportion of hosts showing some degree of response) would be greatest for L forciputus, followed by L. congener, followed by L. dryus, and least likely for L unguiculatus. Based on these prevalence results combined with results on intensity of infestation, I predicted that L. forcipatus would show the greatest immune expression.
Immune expression of L. forcipotus should exceed L. unguiculatus (because of its higher prevalence and higher intewity of idestation compared with L. unguiculutus) and perhaps L congener (because L. forcipatus had higher prevalence, although not a higher intensity of infestation).
My results on immune expression indicated that aU dead mites resulted from host immune responses, ie. alî 86 dead mites examined had partially- or wholiy-encapsulated stylostomes, and none had fuliy Cormed stylostomes and appeared to have died Boom other causes. Also. at least some mites successhilly engorged on alî four species; ali mites found on L dryos encountered no resistance. That L. dryas showed no immune expression did not support my general prediction because less oft-used hosts showed resistance. The general prediction was also not supponed because responsiveness of L.
congener overiapped the high responsiveness of L. forcipatus as well as the low
responsiveness ofL unguiculatus. It is important to note, however, that L congener
differed from these two species in prevalence only and not in intensity of parasitism.
In summary, my intent was to fust test for differences in prevalence and intensity
of infestation, and then test whether these are predictive of differences in immune
investment among four syntopic damselfiies. Al1 four species aligned themselves in terms
of prevalence, but not intensity, of infection. If this same ordering for prevalence were
observed for immune investment, then this would represent only 1 out of 24 possible
orderings of species (0.01 < p < 0.05). Thus, my results do not suggest prevalence, by
itself. is strongly predictive of immune investment. Yet 1 found clear differences in
prevalence and intensity of infestation only for the L. forciputus-Lunguiculatus
comparison and this single comparison was in support of the general predic tion.
Furthermore, these species overlapped in timing of emergence, w hic h 1 now hypothesize may be an important confound of immune investment.
1 support the contention that measures of parasitism can predict immune
responsiveness and my general approach for several reasons. First, 1 know of no other example where such investigations have been undenaken. Rather, the effects and
consequences of parasitism are often studied with single specks of hosts. Tests with generalist parasites affécting several host species from the same locale are desperately
needed. With many such tests, we may be able to get a clearer picture of factors influencing parasitism and immune investment. The species 1 studied are closely reiated and similar. but &O show some differences in th& ecology. It is important to ask whether these differences might be predictive of parasitism and/or immune investment.
1 found, for example. emergence date was not a stmng predictor of patterns of infestation, contrary to other work (see references in Introduction). In other insect-mite systems, early host species may actually avoid parasites (Smith and McIver 1984a).
When temporal barriers are removed in the laboratory. early-emerging mosquito species are more susceptible to mite colonization than later-emerging species, that normally overlap temporally with the mite species (Smith and McIver 1984b). In this study, the early-emerging species was not resistant to mites. In chapter two 1 examine whether expression of resistance is seasonally dependent in species for which 1 have sufficient data. For L forciputus. later-emerging individuals are significantly more Iürely to respond immunologically (Chapter 2). This study came as a direct consapence of obsewing species differences in immune investment in relation to date.
1 still need to explain any suc h seasonal variation and here 1 present a testable hypothesis. That is. while the actual costs of melanin production and rnobilization may be the same between species. species might otherwise differ in traâe-Offs associated with this immune response. For example. melanin is used in thermoregulation of damselflies
(Corbet 1999). Early-emerging species may be more subjected to bouts of cool wet weather (Corbet 1999) and for this reason may be les willing to surrender melanin resources to immune responses. This hypothesis of a trade-Off with thermoregu htion and its effect on maturation rate has not been explored for any insect host where melanin is used to combat parasites. One fuial problem is worth coasideting. As mentione6A. phus aîso exploits
Sympemim dragonflies (Forbes et al. 1999); as such host phylogeny is not a stmng impediment to host exploitation. 1 expect that host representation may Vary across ephemeral ponds that are habitats for A. planus. Data are needed on spatial variation of host-parasite overlap. Such studies wiU help us understand whether variation in use of particular host species is predictive of host abiîity to respond to A. plonur or strength of any responses. Geographical patterns of infestation and irnmunity could prove as illuminating as seasonal comparisons. particularly if local adaptation occurs such that the age of the host-parasite association becomes an important factor (Le. the predicted outcornes may differ if the host population has not had tirne to adapt to the parasite). Li terature Cited
Abro, A. (1979) Attachment and feeding devices of water mite lame (Arrenurus spp.)
parasitic on damselflies (Odonata, Zygoptera). Zoologica Scripra 8: 22 1-234.
Abro A. (1982) The effects of parasitic water mite larvae (Amenurus spp.) on zygopteran
imagoes. Journal of Invenebrate Pathology 39: 373-38 1.
Bush, A O., Laffeny, K.D.. Lotz, J. M.. and Shostak. kW. (1997) Parasitology meets
ecology on its own ternis: Margoiis et al. revisited. Joumal of Parasitology 83:
575-583.
Corbet, P.S. (1999) Dragonflies: Behavior and Ecology of Odonata. Cornell University
Press, New York.
Davids C. (1973) The water mite Hydrachna conjecta Koenike (Acari: Hydrachnellae),
bionomics and rehtion to species of Corixidae (Hemiptera). Netherianth Journal
of Zoology 23: 363-429.
Forbes, M.R. (199 1) Ectoparasites and mating success of male Enallagmu ebrium
damsemies (Odonata: Coenagrionidae). Oikos 60: 336-342.
Forbes, M.R.and Baker, R.L. (1990) Susceptibility to parasitism: experiments with the
damseifîy Enallagmu ebrium (Odonata: Coenagrionidae) and larval water mites,
Arrenurus spp. (Acari: Arrenuridae). Oibs 58: 6 1-66.
Forbes. M.R.L. and Baker, R.L. (1991) Condition and fecundity of the damselfly,
Enallagrna ebrium (Hagen): the importance of ectoparasites. Oecologia 86: 335-
341. Forbeq M.R., Muma, K.E. and Smith. B. P. (1999) Parasitism of Synipetnun dcagonflies
by Arrenurus planus mites: maintenance of resistance panicular to one species
International Journal of Parasitology 29: 99 1-999.
KUnig, C. and Schmid-Hempel. P. (1 995) Foraging ac tivity and immuno-cornpetence in
workers of the bumble bee. Bonrbus terrestris L. Proceedings of the Royal Society
of London B 26û: 225-227.
Kraaijeveld, A.R. and God fray, C.I. (1997). Trade-Off between parasitoid resistance and larval cornpetitive ability in Drosophila melanogaster. Nature, 389,278-280. Leung, B., Forbes M.R., and Baker, R.L. (1999). Groorning decisions by damselflies,
age-specific colonization by water mites, and the probability of successful
parasitism. International Journal of Pamsitology 29: 397-402.
MgUer, A.P. (1997). Parasitism and the evolution of host life history. In host-parasite
evolution: General PRnciples und Avian Models (Chyton, D.H. and Moore, J. eds.) pp. 105-127. Oxford University Press, London.
Rouf J and Martens A (1997): Completing the life cycle: detachment of an aquatic
parasite (Arrenunis cuspidator, Hydrachnellae) frorn an aerial host (Coenagrion
puella, ûdonata). Canadian Journal of Zoolog y 75: 655659.
Sheldon, B. C.. and Verhulst, S. (19%) Ecological iinmunly: costly parasite defenses
and trade-offs in evolutionary ecology. Trends in Ecology and Evolution 11: 3 17-
Siva40t hy, M.T.,Tsu baki, Y. and Hooper, R.E. (1998). Decreased immune response as a proximate cost of copulation and oviposition in a darnselfly. Physiological Entomology 23,274-277. Smith, B.P. (1988) Host-parasite interaction and impact of larval water mites on insects.
Annual Review of Entodogy 33: 487-507. Smith. BAand McIvever SB. (1984a) nie pauenis of mosquito emergence (Dipteras
Culicidae: Aedes spp.): thek influence on host selection by parasitic mites (Ac&
Arrenuridae; Arrenrurus spp.). Canadian Journal of Zwlogy 62: 1 106-1 1 13.
Smith, B.P. and McIver S.B. (1984b) Factors influencing host sektion and successhil
parasitism of Aedes spp. mosquitoes by A rrenrurus spp. mites. Canadian Journal
of Zoology 62: 1 114- 1 120.
Snedecor, G.W.and Cochran. W.G. (1980) Statistical methods. Iowa State University
Press, Ames.
Walker, E.M. (1953) The Odonata of Al& and Canodo, vol. 1.. University of Toronto
Press, Toronto.
Wiggins G.B..MacKay, R.J., and Smith LM. (1980) Evolutionary and ecological
strategies of animals in annual temporary ponds. Archive für Hydrobiologia
Supplement 76: 369-392.
Zar, J. H (1996) Biostatisticaf Analysis, 3rd ed. Prentice-Hail Inc. Upper Saddle River, New Jersey. Table 1. Number of damselflies (by species and gender) that were uninfesied (Nu) versus
infested by one or more mites (Ni). prevalence of infestation and the confidence interval
(CI) calculated following Snedecor and Cohran (1980) and Bush et al. (1997). Chi-square test statistics and associated pvalues (xZ(p))are presented for cornparisons between' males and females separately for each species. Combined values for males and females of
each species are also presented. See text for further statistical comparison among species.
sQ!xb Sexo Lestes congener females males com bined Lestes dryas females males combined
Lestes forciputus females
males
corn bined Lestes ufiguiculatus females males combined
Table 3. Results on immune responses to mites. for males and females of each species,
Nm refers to total number of damselflies with one or more mites, Nr refers to the number of damselflies with one or more dead mites. which resulted from encapsulation of mite feeding tubes. Pr refers to the proportion of damselllies responding imrnunolog ically to mites. CI is the confidence interval calculated foilowing Snedecor and Cochran (1980). Combined values for males and females of each species are also presented.
Lestes congener & & Cl
female 13 5 38.5 26.4
males 11 7 63.6 28.4
combined 24 12 50.0 20.0
Lestes dryas
female 18 O O O
mde 18 O O O
combined 36 O O O
Lestes forcipatus
female 34 25 73.5 14.8
male 42 30 71.4 13.7
combined 76 55 72-4 10.1
Lestes unguiculatus
female 21 7 33.3 20.2
male 22 7 31.8 19.5
combined 43 14 32.6 14.0 Figure 1. Roportion of individuals responding (with confidence intervals) in relation prevalence of mite infestation (with confidence intervals) for the damseiflies Lestes unguiculutus, Lestes dryas, Lestes congener, and Lestes fkpatus (from left to right).
Confidence intervals were calculated following Snedecor and Cochran (1980). Note that the proportion of individuals responding for L. dryus is zero. I a- 1 m 9 w w 4 20 40 60 80 100 Prevalence (%) Cbapter Two:
Immune expression in a damielfly is relatd to time of season, not to fluctuating
asymmtry or host size A bstract Variation in immune responsiveness within and among species is the su bject of the emerging field of ecological immunology. This study shows that individuals of Lestes forciputus Rambur ditrer in their Iikelihood of mounting immune responses, and in the magnitude of those responses. against a generalist ectoparasite. the water mite Arrenitrus planus Marshall.
Immune responses took the form of melanotic encapsulation of mite feeding tubes, occurred in the few days after host emergence, and resulted in mites dying without engorging. Such immune responses were more probable and stmnger for hosts sarnpled later rather than earlier in the season. Such responses may act as selection affecting seasonal patterns of egg hatching and larval abundance of mites. Contrary to expectation. host sue (wing length) and wing ce11 fluctuating asymmetry were not related to likelihood of immune responses The importance of season on immune expression of insects has not ken explored in detail. These results suggest possible trade-offs in docation of melanin (or its precurson) to maturation versus irnmunity. Funhermore. these results indicate the need for studies on the synergistic effets of weather and parasitism on host species that use melanotic encapsulation to combat parasites and pathogens. Introduction Emerging theory on ecological immunology treats immune expression as king
subject io trade-offs (Sheldon & Verhulst, 1996; Schalk & Forbes, 1997). One question
of interest is why individuah, populations, or species should show variation in immune responses. Researc hers have shown gene tic variability for resistance within and among
animal populations (Gregory et al., 1990; Fellowes et al., 1998a). but genetics pro babl y
do not capture the full variation in immune expression seen in nature. The work reported
here examined the extent to which individuah of Lestes forcipotus (Rambur) damselflies (Lestidae: Zygo ptera) show differential immune responses to a generaîist parasite, the
ectoparasitic water mite Arrenuw plmus Marshall (Arrenuridae: Hydrachnida). The study examined whether time of season, wing ce11 asymmetry. or a metric of host size was related to adult males or females responding immunologically to naturaliy-varying numbers of mites. Trade-offs are expected between immune system function and üfe-history traits
linked to growth and reproduction (Folstad et al., 1989; Sheldon & Verhulst, 1996).
Empirical work has demonstrated costs of resistance (Fellowes et al., 1998b, FeJlowes et al., 1999a; Fellowes & Godfray, 20). The costs and benefits of responding to parasites
(or maintainhg systems that can respond) will probably depend on other factors. These factors include the probabiîity of king parasitised. intensity of parasitism, damage that parasites might cause, and costs of mistance in the absence of parasitism (e.g. Forbes,
1993; Sheldon & Verhulst, 1996). Such factors may differ considerably within and among species because individuah can differ in exposure to parasites or in the costs of mounting immune responses. One main goal of within-species studies is to understand which hosu (or when hosts) should commit resources, tirne, and energy to defence.
The principal form of immune response in damselflies is melanotic encapsulation of mite feeding tubes (Abro. 1982; but se-Forbes et al.. 1999). Melanin has other uses. including hardening of the cuticle during the pre-reproductive period and thennoregulation (Corbet. 1999). both of which could be dependent on weather or the of season. which can influence metrics of damseifly fitness (Thompson, 1990). For temperate zone odonates, individuals that emerge eariier in the season tend to be subjected to more bouts of cool rainy weather than individuals that emerge later (Corkt, 1999). Whether emergence tirne influences cornmitment of melanin resources to immunity rather than other hinctions has never been investigated.
One might also predict that individuals in better condition are better able to resist infection or infestation. This reasoning requires that immunity is costly and there are demonstrations of a cost of immunity for insects. These include studies observing insects varying in a task and then recording responses to sirnulated parasitism such as Konig 8
Schmid-Hempel's (1995) work on bumblebees. and one study on reproduction of damseiflies limiting immune responses (Siva-Jothy et aL, 1998). ûther studies on
Drosophila (Fellowes et al.. 1998b; Feliowes et al.. 1999a; Fellowes & Godfray. 2000) have demonstrated direct costs of immunity. These include reduced female size and fecundity, potential decreased tolerance to desiccation and increased attack from O ther parasitoids.
This study examined whether a met& of body size (wing length) related to the probability of mounting an immune response. Wing cell fluctuating asymrnetry also was scored. Fluctuating asymmetry of both continuous and meristic characters has been linked to size. condition. parasitism, and fitness in many animals including damselfiies
(Bonn et al. 1996; Leung & Forbes. 1996). Furthemore. wing ceil asymmetry has been related to timing of ernergence in at least one damselfiy (Hardersen et al.. 1999).
However. strengths of fluctuating asymmetry relationships across species are highly heterogeneous and weak overall (Leung & Forbes. 1996).
Importantly, melano tic encapsulation of feeding tubes of A menurus spp. mites by damseiflies occurs early in adult life before host reproduction (Abro. 1979.1982; see below). Thus. any variable responses should not be confounded by differences in timing of parasitism or variation in reproductive effort. Some observational and experimental studies support a rektionship between reproductive effort and either increased parasitism or reduced immunity (reviewed in Mglier, 1997; Siva-Jothy et al., 1998).
Relevant natural history of the host-parasite association
Lawae of L. forcipatus damselflies are found in the same habitats (epherneral ponds) as A. plunus (Walker. 1953; Wiggins et al.. 1980). Colonisation of odonate hosts by Arrenurus spp. mites occurs in a series of steps du~gwhich the hosts may mount defences. Larvai mites fwst colonise odonate larvae; host larvae groom. presumably to reduce attachment rates (Smith, 1988; Forbes & Baker, 1990; Baker & Smith, 1997;
Leung et al.. 1999). Larval mites are phoretic on odonate larvae; once larval hosts emerge and start to eclose, the larval mites abandon the cast exoskeleton and crawl ont0 the newly formed imago. The mites cling to the host with th& palpal claws and soon pierce the host's cuticle using their chelicerae (Smith. 1988). Anchoreci u> the host by the chelicerae, the mite secretes an aceiîular mucopolysaccharide that forms a stylostome (a blind-ended feeding tube: Smith. 1988).
As mentioned. some odonate hosts attempt to neutralise one or more stylostomes through melanotic encapsulation (Abro. 1979, 1982). In other insect species. such as the dragonfly Syrnpetmm intemum, the host appears to aggregate haemoc ytes at the site of stylostome formation (Forbes et al., 1999) similar to other aquatic insects parasitised by water mites (cf. Davids. 1973). This response appears to result in collapse of the feeding tube without melanotic encapsulation (Forbes et al., 1999). Therefore, this study first assessed whether L. forciputus showed melanotic encapsulation typical of other odonates and the degree to which the death of mites resulted from immune responses.
The L. forciputus-A. planus association is similar to other odonate-Arrenunrs mite associations. If the mite engorges successfullp it drops off the host when the host retums to the water for reproduction. It continues through its nymphal and adult stages. with its active stadia king free-living aquatic predators on microcrustaceans (Smith. 1988). Host detachment is simultaneous. thus parasitised hosts usuaily have mites or have lost mites.
Evidence of past parasitism or scarring is clearly visible on some species (Forbes, 199 1).
Methods
Damsefies were surveyed to elucidate pattern of infestation by mites and to score engorgement success. Collections were made at Yezerinac's Pond (44'32' 12"N.
76O22'55"W). located on the Hilda and John Pangman Conservation Reserve of the
Queen's University Biologicai Station (Chaffeys Lock. Ontario. Canada). Fiy-one trips were made to the pond between 28 May and 5 August 1998. On each trip. damseiflies were nette& identified as Lfdipotus (following Walker. 1953) and their mites were
counted. using a 20X loupe. Gender and approxirnate age were recorded for each damseifîy (cf. Walker,
1953). Tenerals had soft, shiny wings, a soft abdomen and Little dark pigmentation in the exoskeleton. Young to old adults ail had rigid, dull wings. Furthermore, young adults had melanin deposits in their appendages oniy whereas older adults showed body pigmentation, and the oldest adults (for males) were pruinose. It was difficult to score accurately whether mites on tenerals were aiive or dead so tenerais were excluded from comparisons. Hosts that were not parasitised at the time of capture also were excluded from comparisons. This was necessary because althoug h presence of mite scars indicated whether older adults had knparasitised previously, it was difficult to enumerate scars and obtain an accurate count of mites previously parasitising those hosts.
Similar to other work on A. planus on other host species, dead mites on mature adults were flat and silver (Forbes et al., 1999). Mites starhg to engorge were red to orange with their legs stiîl visible. Mites were scored as hilly engorged if their legs were entirely obscured by their swollen body, and as live and partially engorged if the legs were only partially obscured by their bodies, which were not shriveled (cf. Forbes et al.,
1999). Mites were recorded as dead and faiüng to engorge or as engorging or engorged.
None of 400 mites was recorded as having engorged and later dying, indicating that mite death occurred early in the host's adult me.
Seventy-two L forciputus adults (41 males and 31 fernales) filfilied the criteria of current parasitism where engorged or engorging mites versus dead mites could be scored accurately. Of these. 47 were marked using non-toxic permanent Sanford Sharpiea pens (Sanford Corporation, Bellwood, U.S. A) on th& wings, to preclude their re-sampling
after the2 release (following Forbes 199 1). Ail 72 damselflies were processed by
enumerating live and dead mites, and by counting the number of wing cells between the
nodus and ptemstigma for both the left and right forewings (to obtain a measure of wing ceii asymmetry). Length of the right forewing also was rneasured from nodus to wing tip using digital calipers (accurate to 0.01 mm).
Twenty-five damselflies (14 females and 11 males) with one or more dead mites were brought back to the laboratory and prepared for viewing mite feeding tubes. These damseiflies were f~ststored in glassine envelopes and refngerated at Ca. 5 OC for up to
48 h. Reparation involved removing the head, legs, wings, and abdomen. What remained of the thoraces were placed in a 4.5 x 1.5 cm glas via1 and covered with
Andre's solution ( 1: 1 : 1 chloral hydrate, acetic acid, and water by weight) for a minimum of 48 h at ca 20PC before further dissection. To fbrther dissect, the dorsal half of the thorax (including the wing muscles) was removed using 00 insect pins and a 2.5-cm, 22- gauge syringe needle. The ventral haif of the thorax was placed exocuticular surface down on a microscope süde. After applying glycerol and a cover slip, each süde was examined at l0X (phase-contrasi) and scanned for mites and feeding tubes.
Statistical analyses were carried out using Systat 2.0 @ (Wilkinson, 1991) and
Jmp 3.0 @ (SAS Institute Inc., 1993). Results
Patterns of mite infestation Mites were invariably found on the venter of the thorax on a tubercle. or near or on the coxal plates of the legs. Numben (and log-transformed numbers) of mites on hosts were not distributed normally (Kolmogorov-Srnhov tests), precluding parametric tests.
A Kruskall-Wallis test showed that mite intensities did not differ between females and males (x*= 0.50, p = NS). Fernales had a median intensity of four mites (interquartile range was two to seven mites; range was one to 27 mites). Males had a median intensity of five mites (interquanile range was two to nine mites; range was one to 17 mites).
Causes of mite death
To determine whether mites died due to immune responses, the feeding tubes of
86 dead mites from 39 hosts (of which 25 were L forciputus) were examined (see
Chapter 1). AU of these mites had completely or almost completely melanised stylostomes (Fig. 1, left panel), i.e. none had fuiiy- formed stylostomes and appeared to have died from other causes. Thus, both male and female damsemies mounted immune responses to mites, but many carried both live and dead mites. Mites, alive before king placed in the Andre's solution. had perfectly formed stylostomes with no evidence of melanisation on the stylostome itself (Fig. 1, right panel). Imnuuie respomiveness in relotion tu gender
Parasitised darnselfiies were scored as either responders (if they had one or more
dead mites) or as non-responders. The proportion of responders did not differ between
males (30 of 41; 73.2%) and females (25 of 31; 80.6%; x'= 0.55, d.f. = 1, p = NS).
Immune respmsiveness of fernales und males in relation to otherfactors
A significant effect of date of sampling was found on likelihood that males responded immunologically (logistic regression, x2= 9.0; d. f. = 1; p < 0.0 1). The 11 non- responders were sampled significantly earlier in the season. compared to the 30 responders (Table 1). For females, the six non-responders were sampled earlier than the
25 responders. but this result was not sigiiificant (Table 1). Responders did not differ from non-responders with respect to wing Length or wing cell FA, for either males or females (Tables 1 & 2).
The effect of season on residual numben of dead mites, controiiing for total number of mites on hosts, also was examined. The number of dead mites related linearly to the total mites on both males and females. For males, dead mites = 0.33+0.62Xtotal mites (F = 43.7, p < 0.001, n = 41). whereas for females, dead mites = -0.95+0.78Xtotal mites (F = 64.4, p < 0.001, n = 31). if non-responders were excluded, there were still significant relationships between numbers of dead mites and total mites. For males, dead mites = 0.2 14.70Xtotal mites (F = 95.5, p < 0.001, n = 30). whereas for fernales, dead mites = -0.54 +0.77Xtotaî mites (F = 56.6, p < 0.ûû1, n = 25).
Residuals from these relationships were reîated positively to date of sampling for both males and fernales inespective of whether non-responders were included in. or excluded Erom. initial and subsequent analyses (Fig. 2). Thus. individuals with higher residual numben of dead mites were more likely to be sampled later in the season (Fig.
2).
Discussion Several studies have indicated that mites are costly to their insect hosts. For example, Polak ( 1996) showed that parasitism by Mucrocheles subbadius Berlese mites was associated with reduced fecundity of female Drosophila nigrospiracula Patterson and Wheeler. For insects, including damselflies, water mites and other parasites have been associated with reductions in flight abüity, fat accumulation, fecundity. mating success. and/or survivorship, in both observational and experimental studies (e.g.
Lanciani, 1983; Smith. 1988; Forôes, 1991; Forbes & Baker, 1991; Reinhardt. 1996;
Leonard et al.. 1999; Siva-Jothy & Plaistow, 1999). In other studies, the link between parasitism and lowered darnselfly fttness was not present (Andrés & Cordero, 1998; Rolff et al., 2000) or was ameliorated by matemal effects (Rolff.1999).
A. planus mites engorge to 140 tirnes t kir original volume compared to only five to 35 rimes for other mite species in my study region (J. M. Moran and B. P. Smith, pers. obs.). As such, one might expect fitness costs of parasitism, and immune responses toward A. planus. However, fitness costs are only one of several factors expected to be important in maintaining or mounting immunity. In the present study, date of sampiing was the only predictor of immunity and degree of that response. Why should this be the case?
The pre-reproductive period of damselflies is prolonged by cool weather (Corbet,
1999; cf. Thompson, 1990). Damselflies emerging later in the season, when conditions are warmer. may divert more melanin or precursors of melanin (see Siva-Jothy et al.. 1998) away from maturation and towards immunity. It is interesting that the earliest- emerging Lestes species at my study site (Lestes dryaî Kirby) was wholly susceptible to
A. planus. In fact, ail 93 mites found on 36 males and females engorged successfully (C.
P. Younh. pers. obs.). At present. there are no data on whether early emerging species are les likely to surrender melanin resources to immune responses, or are sim pl y incapable of elevated immune responses.
In the present study, theof season was more important than metrics of fluctuating asymmetry and host size in deteminhg probability and degree of immune responses. This is interesting, given that lestid damselflies emerging later in the season are tirne-constrained and may actually sacrifice body size for earlier emergence tirne
(Johansson & Rowe, 1999). In fact. timeîonstrained or later-emerging individuals are smaller in several other species of temperate damselflies (e.g. Forbes & Baker, 1991;
Corbet. 1999; Plaistow & Siva-Jothy, 1999). If later-emerging L. forcipotus were actuaLiy in poorer absolute condition. then these results seem counter-intuitive as these individuals invested more in immunity.
This apparent paradox could be resolved if melanin resources were not in as much demand for maturation (or temperature-dependent activities) later in the season. In a previous study contmiiing for time of season, Léonard et ai. (1999) showed that maturation the for females of the damelfiy Enallogmu ebriwn (Hagen) was slowed with experimental additions of parasitic mites. However, it is not known whether this reflected melanin allocation away from thermoregulation ancilor hardening of the cuticle and whether this response would change seasonally. Altematively, higher mean temperatures later in the season could result in higher encapsulation rates. This argument
is supported with experimental work on Drosophila subjected to two of three parasitoid
species (Fellowes et al., 1999). Ho wever, encapsulation responses to the third parasitoid showed the opposite trend (Fellowes et al., 1999).
Our results cannot be explained by age differences between responders and non- responders because dl damseülies were old adults that had matured successfully.
Additionally, the immune responses are known to occur in the fust few days of life. I am unaware of any investigations of whether melanotic responses of insect hosts are affected by natural variation in weather in relation to tirne of season. Such results will have bearing on the synergistic effects of weather and parasitism on host fitness. a topic that has not received much attention. Such seasonal effects also may place selection on female mites to lay clutches relatively early. In A. planus, srnail increases in larval abundance at the start of the season (as mites start hatching) are followed by drastic seasonal declines
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Table 2. Cornparisons of absolute wing ce11 asymmetry for male responders and non- responders and for fernale responders and non- responders. Individuals w it hin a category (e.g.. male responders) with absolute asymmetry scores > O were combined for contingency analyses.
Absolute WiDg x2 d.f. p Ceil Asymmetry O 1 2
Responders
Responden 3 3 O
Non- responders 10 12 3 Figure 1. Light rnicrograph (sale bars indicated) showing the variation in melanotic encapsulation of Arrenurus planus stylostomes by Lestes forcipotus. In the left panel. the body of a dead mite (b) and its jointed legs (mwand above b) are visible. S refers to the encapsulated stylostome, which mirs fiom the leA margin of the panel to the gnathosoma of the mite (not visible). Melanisation is apparent in the body of the mite (two patches below b). In the right panel, only the stylostome is shown: the live engorged mite had detached from the host cuticle during preparation. leaving behind a scar (mw).The stylostome (a bubble and long tube, labelled S) is fuily formed and the bulb is surrounded by a clear space indicating that the stylostome was functional.
Fi yre 2. Relationships between residual numbers of dead mites (from regressions of dead mites versus total mites, se. text) and date of sampling. Two types of analyses were performed. First. both non-responder~wd responders were included in analyses to determine residual dead mites and relationships between residual dead mites and date of sampling, for each sex separately. Second, non-responders were excluded from initial and subsequent analyses. Results are shown only for responders. Males : -6.24.13Xdate of sample (F = 17.6. p < 0.001. n = 41); fernales: -6.44.13Xdate of sample (F = 1 1.9. p <
0.025, n = 3 1). When non-responders were excluded, males: -3.34.06Xdate of sample (F
= 4.7. p < 0.05, n = 30); females: -7.2+0.î4Xdate of sample (F = 7.1, p < 0.025, n = 25).
For both males and females, individuals with higher than expected numbers of dead mites were sampled later in the season. Males
Date (1 = 28 May 1998) Chapter Three:
Pattern of immune expression in a dsnsolfïy is not expldned by seasonai àifferences
in all~tionto immuni ty A bstract
In a previous study. female and male damselflies (Lestes forcipotus Rambur) showed increased melanotic encapsulation of the feeding tubes of their parasitic mites.
(Arrenurus planus Marshall) as the season advanced. This pattern could have resulted from differences in investment in immunity by early- versus hter-emerging damseifiies. or differences in responses due to temperature variation across dates. or to other factors. Sephadex beads were injected into newly emerged damseMies collected at different times in the season to test whether immune responses varied seasonally. while temperature was held constant in a laboratory setting. Number of beads melanized was used as a metric of immune response. alter contmlling statistically for variation in the total number of beads recovered.
Condition declined seasonally for both females and males. When temperature was contmlled. the immune response of males, but not females. was positively reiated to condition, but in neither sex was there a relation between immune response and date of capture.
DüTerences in investment in immunit y by early- versus later-emerging damseiflies do not explain observed seasonal patterns in immune responses observed in nature. Funhermore, condition of individuals. which is predicted to affect immune expression, affected immune expression only for males. In nature, variables such as temperature may be more important in explaining immune expression, such that hter- emerging damselfiies in lower condition are actuaiiy more responsive. introduction
Much variation in immune responses of insects is due to genetics (Hughes &
Sokolowski 1996; Gorman & Paskewitz. 1997; Kraaijeveld & Godray, 1997; Fellowes et
al.. 1998; Fellowes & Godfray. 2000). However. environmental factors relating to good
versus poor nutrition (Leung et al. 2001). or to temperature duMg antigenic challenges
(Fellowes et al.. 1999). are likely important.
The immune response of damselflies, and many other insects. is melanotic
encapsulation. in which a foreign object within the host (such as a mite feeding tube or a
parasitoid egg) is fust sunounded by haemocytes and then melanized to isolate the ob&t. preventing any hmful effects (lackie. 1980). Whether melanization results in
death of the parasite or parasitoid. or follows its death by other means. is debatable. In at
least Lestes damselflies (4 species listed in Chapter 1). this melanization of the feeding
tubes of parasitic mites is correlated with the death of the mite (Chapter 1).
Gorrnan & Paskewitz (1997) have shown that insects respond to Sephadex beads using the same encapsulatory response used to counter parasites and parasitoids;
Sephadex beads or short lengths of nylon mono-filament. are ofken used to assay the
encapsulatory responses of insects. Responses to beads and monofdarnents have been
used to study the effects of genetic, physiological, and behavioural factors (e.g. Kan@ &
Schmid-Hempel. 1995; Gorman & Paskewitz. 1997; Siva-Jothy et al.. 1998;
Suwanchaichinda & Paskewitz. 1998; Rantala et al.. 2000). Some studies have shown
that host condition and temperature can be positively or negatively related to encapsulatory responses (Suwnachaichinda & Paskewitz 1998; Feliowes et al., 1999), and that increased foraging or reproductive activity can result in reduced immune expression (KOnig & Schmid-Hempel, 1995; Siva-Jothy et al., 1998). Such variation in host immune expression may exert variable selection on parasites.
This study explores immune expression in the damselfly Lestes forciputus (Rambur), using Sephadex beads to sirnulate parasitism. Immune responses were studied in relation to date of capture with temperature held constant. Later emerging lestid damseülies are smaller (Johansson & Rowe, 1999), and are expected to be in poorer condition (perhaps affecting allocation to immunity), as welî as being exposed to higher ambient temperatures during challenges from parasitic mites. Thus, host condition, after controlling for temperature, was ahexamined as a potential factor affecting immune expression. Given that later emerging damselflies are expected to be in poorer condition. it is counter-intuitive that they show increased immune expression in nature (Chapter 2).
This apparent paradox could be explained if later emerging individuals were subjected to higher îikelihood of parasitism and thus were expected to devote more energy to cellular immune function than earlier emerging individu&. For this reason, seasonal patterns in mite parasitism also were investigated in this study. Alternatively. seasonai increases in likelihood or intensity of parasitism may only partially relate to immune expression, which is largely controlled by other factors, such as temperature.
Our main objective was to detennine whether seasonal increases in immuno- cornpetence previously reported for L. forciparus fernales and males (Chapter 2), were still present after removing the confound of temperature following emergence, while at the same tirne considering condition of early- versus later-emerging damselflies. Methods
Survey and collection DamseMies were surveyed andlor collected at Yezerinac Pond (44O32'13"N. 76O2255"W)on the Hilda and John Pangman Conservation Reserve at the Queen's University Biological Station. Füleen trips were made to the pond between 19 June - 5 July, 2000. Sixty-nine newly-emerged females and 59 newly-emerged males were surveyed (see below). An additional 78 and 61 newly-emerged females and males
(respectively) were collected for bead injection. When needed, these two categories of individuals are distinguished as surveyed or bead-injected damseMies. AU individuals were hand netted and identified to species using a 10X loupe to examine anal appendages
(of males) and ovipositor (of females) (foilowing Walker & Corbet. 1975). Individuals were only included in samples or experirnents if they had had soft. shiny wings. a soft abdomen and little pigmentation, indicating that they had emerged within the last 48 hours (personal observations).
For surveyed damselflies. gender, length of the right forewing (a metric of size). and number of attendant mites were recorded. Wing length was measured by fust gently pressing the right forewing on a piece of paper and marking the position of the nodus and the tip of the wing and measuring the distance between these markings (to 0.01 mm) using digital calipers (Mitutoyo Inc). Surveyed individuals were marked with pigment from permanent non- toxic markers (Zig Posterman, Kuretake Co., Japan) for later identification and to preclude their re-sampling, following their released. For bead-injected damselflies, groups of fîve to 10 individuals were placed in cages (wooden frame with nylon mesh, 3ûcm X 30cm X 40cm) and returned to the lab where they were stored in a datk room at 318°C for appmximately one hout. This
procedure was meant to reduce stress caused by capture and transport. Gender, wing
length, and the number of mites were then recorded for damselflies without injuries such as crumpled wings. Wet mas of each individual was measured using a Mettler analytical
balance accurate to 0.1 mg (cf. Leung and Forbes, 1997) after which each damseifly was
placed individually into a clear plastic cup (=300ml) with = 3ml of water to prevent
desiccation. A disc of nylon mesh was skewered on a 5-cm applicator stick and forced
into the bottom of the cup but above the water so damseiflies could thus perch on the
mesh or stick and not be trapped on the surface fdm of the water. Mer initiai processing
damseiflies were left in cups for another 3-4 hours before bead injection.
Before injecting beads. 1again checked damseifïies tu ensure initial processing
did no harm. Each damselfiy was then pierced at a c 45' angle through the venter of its
thorax. using a 'ri cc 26-gauge syringe and Sephadex beads (CM C-25@ ion exchange
resin 40-120~)~previously hydrated in a generic insect saline (150m.MNaCl. lOmM KCl.
4mM CaC12, 2mM MgCb. 4mM NaHCa, SrnM HEPES pH 7.2). were then injected in
solution. The stock of beads was maintained in solution and stored in a refngerator at al1
times. Damselflies were then returned to th& cups. which were placed in an incubator ai
28°C 1 O C for 24h. Pre-reproductive damselflies will melartize stylostomes (or feding
tubes) of parasitic mites, during th& pre-reproductive period (Chapter 1). As such, at least some damselflies were expected to melanize injected beads. Thus, several steps were taken to ensure that the injection of the Sephadex beads simulated the parasitism of L. forciputus by A. planus mites as accurately as possible.
First, since A. planus mites colonize L. forciputus as larvae but do not beg in feeding until afier the damselfly emerges as an adult, only newly emerged damselflies were coilected
and injected. These damselflies have not begun foraging and are sexually immature (Corbet, 1999). thus eliminating these factors as potentiai confounds. Second, since the
mites attach almost exclusively to the venter of the thorax, aü bead injections were made
in the same region and at a sharp angle so that the beads were placed in the same tissues
that the mite feding tubes are found. Third. since any immune responses to mites occur
at the tirne of attachment when the mite stylostome fvst begins to form (see Chapters 1 & 2). only 24h were allowed for the host to respond to the beads. which were then only
counted as "melanized" if it appeared that such a response would have successfuily
disrupted the formation of a stylostome (which was estimated to be haemocytes covering
20% of a given bead). Since there is no way to place the Sephadex beads in the tissue of the damselfly without piercing the cuticle. a sham treatment assessing the effects of
trauma caused by the injection process on bead melanisation was not possible. However. this trauma is somewhat relevant as the mites themselves must pierce the cuticle to form thek feeding tubes.
Scoring of immune responres
After the incubation period. the damselflies were decapitated and legs. wings and abdomen were removed. The thorax was placed in a via) of Andre's solution (1:l: 1 mixture of water, chloral hydrate and glaciai acetic acid by weight) for 48h to clear the tissues. Mer clearing, the dorsal haif of each thorax was removed ushg a pair number 5 forceps and a 22-gauge syringe needle. The remaining haî€of the thorax was placed exocuticular side down on a ghss microscope slide. covered with a drop of glycerol and then a glas cover slip. The slides were viewed under phase contrast at lOOX magnifîcation. Slides were visually scanned and number of beads counted. The degree
of response varied but a bead was classifed as melanired if at least 20% of it was
covered w it h aggregating haemoc ytes and melanin. Numbers of stylostomes present and
numbers of collapsed. melanized stylostomes were also recorded (see Chapter 2).
Analyses Surveyed males and females were compared with respect to Julian dates of capture (an index of emergence date) using a t-Test, and the proportions carrying one or more mites versus king unparasitized. using a x2 test of association. Individuals parasitized by one or more mites were used to examine whether gender influenced the logIo-transfomedintensity of infestation (sensu Bush et al. 1997). and whether this covaried with Julian date. using ANCOVA. Transformation of intensity data was done to meet assumptions of normality.
For bead-injected individuals, 12 females were excluded from analyses because eight died during the experirnent and four had fewer than five beads recovered. leaving
66 for analysis. For males. six died and eight had fewer than 5 beds recovered, leaving 47 for analysis. Five or fewer beads recovered was chosen a prion as a cut-off point because number of beads melanized was expected to depend on numbers present. if fewer than five beads were recovered, the total possible variation in the number of beads melanized would also be lower. an artefact that would violate the assumpiion of homogeneity of residuals in rny analyses. Excluding individu* with fewer than five beads. combined with log transformation (see below), eliminated this problem. To investigate the degree of respnse to beads in relation to emergeafe date, gender. and host condition. the number of beads recovered (a îùnction of the number
present in the thorax sample and number injected) was controlled for statistically. Melanization of feeding tubes of mites might also have influenced the response to beads
and this was accounted for statistically as welL Immune response was thus scored by
frst regressing the log-transfonned number of beads melanized against the log- transformed total number of beads rmvered and the log-transformed number of
melanized stylostomes present in a General Linear Model. The residuals of this model.
representing the variation in immune response not explained by these two factors, were
used for subsequent analyses and will be referred to hereafter as response.
For both males and females, three regressions were performed relating condition to Julian date of emergence, response to Julian date of ernergence. and response to condition. Condition was simply size-corrected mass of damselflies at emergence. as detaiied below for females and males separately.
Results
Survey results
Females emerged significantly earlier (mean Julian date of emergence 176.6 f 0.5
S.E.) than males (mean Julian date of emergence 179.9 f 0.5 S.E.) (t = -4.47, d.f. = 126. p
< 0.0001). However, females and males did not differ in theu probability of becoming parasitized (x2 = 0.05 8, d. f. = 1, P = N.S.). A signifiant ANCOVA model (2 = 0.10,
F3,97 = 3.72, P = 0.014). indicated that intensities of infestation increased with Julian date
(F= 9.20, d.f. = 1, P = 0.003). but there was no difference in intensity of infestation between males and females (F = 0.003, CM= 1, P = N.S.) nor was there any interaction
between gender and Julian date (F = 0.06. d.f. = 1. P = N.S.).
Condition, immune response, îutd date of emergence.
Mass increased with wing length for both females (log (mass) = -7.96 + 1.69 X log (wing length), 2 = 0.36, Fl,ds= 35.8, P < 0.000 1) and males (log (mass) = -8.47 +
1.86 X log (wing length). ? = 0.41. Fi,&= 3 1.O. P < 0.0001). Residuals of these
regressions represent ind ividual condit ion of females and males. Condition was negatively correlated with Julian date for both femaies (condition = 0.879 -0.005 X Julian date. i = 0.06. F1.u = 4.24. P = 0.04) and males (condition = 1.739 - 0.010 X Julian date, / = 0.22. FI,&= 12.4, P = 0.001) (Fig. 1. panels a B b).
Mer 24h at conmlled temperatures in the incubator, femaies had melanized a median of 64% (range=7- 100%)of the beads recovered and males a median of 46%
(range= 0- 100%). For females. the General Linear Mode1 of log-transformed number of beads melanized in relation to log-transformed number of beads recovered and log- transformed number of stylostomes melanized was significant ( 2 = 0.55, F2.63= 39.1. P
< 0.0001), indicating that numbers of beads melanized increased with the numbers of beads recovered (F = 78.3, d.f. = 1. P < 0.01). The covariate, log transformed numbers of stylostomes melanized. was not significant (F = 1.1, d.f. = 1. P = N.S.). The median number of stylostomes melanized for females was zero. with a range of zero to th. Males showed similar results for the overall mode1 ( ? = 0.40, F2,44= 15.0. P c
0.0001). indicating that the number of beads melanized increased with the number of beads recovered (F = 23.3, d.E = 1, P < 0.0001). but not with the numbers of stylostomes melanized (F = 2.8. d.f. = 1, P = N.S.). The median number of stylostomes melanized for males was zero. with a range of zero to two. The residuals of these models represent individual response for females and males.
Response was not correlated with Julian date for females (F1~6l.a~= 1.25. P = N.S.) or males (FI.&= 0.79. P = N.S.) (Fig. 1. pane$ c & d). Response also was not correlated with condition of females (FIeac= 0.12, P = N.S.) but it was positively correlated with condition of males (response = -3.9~'~+ 2.87 X condition, ? = 0.15, F,,w = 8.18, P =
0.0064) (Fig. 1, panels e & f). Taken together, these results show later-emerging individuals are smaller and lighter at emergence in nature. At controiJed temperatures. immune response does not
Vary with date of emergence (contrary to observations in nature), but is related to condition for males.
Discussion
It is only adaptive for a host to respond to a parasite when the combined cost of response and any residual cost of the parasite is lower than the cost of the parasite in the absence of a response. However. the cos& incurred by both parasite and response wiii depend on many factors. The purpose of this study was to explain why L. forciputus damselfiies colîected later in the season had responded more aggressivel y to A. planus mites than did those collected earlier (Chapter 2). It was suspected that later emerging individuais were ailocating more energy toward immune responses. If this were true, then individuals collected later in the season should aiso be more likely to encapsulate Sephadex beads. I found thaî Lforcipatus &mueMiesshowed im covariation between date of emergence (or coiiection) and degree of immune response, after controlling for temperature during bead challenges. Thus, the seasonal pattern of immunity noted in L. forcipatus previously cannot be explained by increased investment in immunity by later- emerging individuals. It is noteworthy that increased immune response with advancing season, seen in nature, is not masked by decreases in damselfly size or condition. Such decreases in size or condition have been reported for Lestid damselfies (Johannson and
Rowe 1999; this stud y, respec tively). In other words, later-emerging damself lies are smaller, in poorer condition, and yet show higher immune responses to parasitism by mites in nature, but not in a laboratory setting. In fact, when temperature was controUed for, a positive correlation between condition and responsiveness became evident for males.
The relationship between condition and response found in males is consistent with previous studies (Suwanc haichinda and Paskewitz, 1998). Such a relationship was not found in females, possibly because females attempt to maximise their condition by accumulating resources during their prereproductive period (Anholt et al., 1991; Anholt.
1992; Anholt, 1997). If female condition is directly Linked to th& fitness, resource allocations that affect condition will be les flexible (see Collins, 1980). On the other hand, males do not gain as much weight as females during the pre-reproductive period in non-territorial damselflies (Anholt et al., 199 1; Anho It, 1992) and therefore may be more flexible in re-directing emergy to immune responses. This difference between males and femaies also may explain why Iulian date accounted for less variation in female condition than it did in maies If condition were the dnving factor in the obûerved increase in immune
expression in later-emerging damselfiies, condition would have to be higher for later-
emerging individuals, or response would have to be greater for individuals in lower condition. Since neither case is true, the ecological factors controiled for in this experiment (i.e. temperature) must be generating the seasonal increase in immunity such
that it masks other factors such as condition. The effects of temperature on insect immunity demonstrated thus far have ken variable (Suwanchaichinda and Paskewitz,
1998, FeUowes et al., 1999). However the patterns it seems to generate in this system can exert signiflcant selective pressure on the mites parasitizing the damselflies
(Chapter2; see also Forbes et al., 1999) e.g., mites emerging late in the season are Lücely exposed to low condition animals that are more responsive and this may select directionally for meronset of egg-laying in this mite species. It would be valuable to ascertain the extent to which A. planw exhibits heritable variation in breeding onset. If timing of parasitism is critical, we may expect little variation in egg-laying times within this species. Wteratum Cited
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