Canadian Journal of Zoology

Parasitism, immune response and egg production of the hastulatum

Journal: Canadian Journal of Zoology

Manuscript ID cjz-2016-0146.R2

Manuscript Type: Article

Date Submitted by the Author: 10-Jan-2017

Complete List of Authors: Kaunisto, Kari; University of Turku, Department of Biology Kaunisto, Pipsa; 2) Parks & Wildlife Finland, Southern Finland Ilvonen, Jaakko; Turun Yliopisto Suhonen, Jukka;Draft Turun Yliopisto

ODONATA < Taxon, Coenagrion hastulatum, Gregarine, Host-parasite Keyword: interaction, Water mite, Damselfly egg production

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Parasitism, immune response and egg production of the damselfly Coenagrion hastulatum

Kari M. Kaunisto 1, Pipsa Kaunisto 2, Jaakko J. Ilvonen 3, and Jukka Suhonen 3

1) Zoological Museum, Biodiversity Unit, University of Turku FI-20014, Turku,

Finland.

2) Parks & Wildlife Finland, Southern Finland, Metsähallitus, Turku, Finland.

3) Department of Biology, UniversityDraft of Turku FI-20014, Turku, Finland.

Correspondence to Kari Kaunisto

Address: Biodiversity Unit, Zoological Museum, FI-20014 University of Turku, Finland.

E-mail: [email protected]

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Abstract

Theoretical models predict that parasites reduce reproductive success of their hosts, but very few empirical studies have given support to this. Using the damselfly Coenagrion hastulatum

(Charpentier, 1825), we tested how immune response, wing length, and the number of both endo- and ectoparasites affect egg production of host . The study was conducted with four different populations in Southwest Finland. We found a negative association between endoparasitic gregarines and the number of host eggs. Furthermore, immune response increased with the number of water mites, but decreased with the number of eggs.

Contrary to previous studies with other damselfly species, the number of ectoparasitic water mites did not affect the number of eggs. Moreover, wing length, used as an indicator of individual size, was not associated with egg numbers. The negative effect of gregarine parasites on egg numbers is likely to Draftaffect the composition of host populations, i.e. damselflies that show higher resistance to these endoparasites will have more of their offspring represented in subsequent generations. In future, more experimental research on the varying effects of different parasite species on the number of eggs is needed.

Key words: , parasite, gregarine, water mite, Coenagrion hastulatum , damselfly egg production, host-parasite interaction

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Introduction

Parasitism is the most common consumer strategy in nature (Lafferty et al. 2008). Thus, a

great deal of research has been devoted to understanding how the host’s life history traits

have been shaped by investments into parasitic defense mechanisms (Poulin and Forbes

2012). Parasites utilize their host’s resources, while the host tries to minimize parasite

infections with e.g. immune responses. Consequently, a tradeoff prevails between the costs of

immunity and other fitness parameters, such as lifespan and reproduction (Hamilton et al.

1990; Graham et al. 2011). However, demonstrating the costs of parasitism on a host’s fitness

has been difficult, because parasites may mainly infect hosts that are already in poor

condition, which in turn may reduce host reproductive success and lifespan (Tompkins and

Begon 1999). As a consequence, detailsDraft on how parasites affect their host’s fitness on a large

scale still remain largely unclear.

In this study we used damselflies (Zygoptera) as the model organism, since they are

frequently parasitized by ectoparasitic water mites (Hydrachnidia) and endoparasitic

gregarines (Actinocephalidae, Eugregarinorida) (Di Sabatino et al. 2002; Desportes et al.

2013; Ilvonen et al. 2016). Parasitized damselflies respond to water mites behaviorally, by

increasing grooming and lifetime movement distance (Forbes and Baker 1990; Leung et al.

1999; Allen and Thompson 2010; Suhonen et al. 2010 b), or physiologically, by trying to

encapsulate the mite's feeding tube using melanin (Forbes et al. 1999). In general, the number

of water mites decreases towards the end of the flight season, because water mites have

engorged themselves and then detach near water (e.g. Rolff and Martens 1997). Water mites

are shown to lower mating success, decrease flight ability, reduce male condition and

survival, and increase the host's energetic demands of maintaining and mounting immune

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responses (Forbes et al. 2002; Schilder and Marden 2006; Lawniczak et al. 2007).

Furthermore, a previous study by Rolff (1999) found that the number of eggs oviposited decreased with the abundance of water mite ectoparasites on the damselfly Coenagrion puella (L., 1758).

Other common parasites of the odonates are endoparasitic gregarines. Gregarine infection occurs when damselflies ingest gregarine oocysts, either attached to their prey (Åbro

1976) or via drinking water contaminated with gregarines (Córdoba-Aguilar and Munguia-

Steyer 2013). Consequently, the number of gregarines is likely to increase over the lifetime of damselflies as they feed (Åbro 1976; Locklin and Vodopich 2010b). Once ingested, gregarines aggregate mainly to the posterior of the host’s gut, where they develop and reproduce (Desportes et al. 2013). PreviousDraft studies have found rather inconsistent results for the effects of gregarine parasites on their hosts. For example, Siva-Jothy and Plaistow (1999) found negative effects of gregarines on their host’s energy resources. More recently, Marden and Cobb (2004) found that damselfly individuals with inferior mating strategy had more gregarines, indicating a physiological cost for the host. Interestingly, Canales-Lazcano et al.

(2005) found a negative impact of gregarines on egg numbers, while Locklin and Vodopich

(2010a) found no such association between gregarine infection and egg production. Finally, as the gregarines do not seem to be associated with an energetically costly immune response

(Córdoba-Aguilar et al. 2006; Honkavaara et al. 2009; Kaunisto and Suhonen 2013), it is occasionally reported that these endoparasites are fairly harmless to their hosts (e.g. Hecker et al. 2002).

In this study our main goal was to untangle the relationship between parasite load, immune response and egg production, all of which are energetically costly and therefore under

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resource allocation tradeoffs. Consequently, we tested for the Northern Damselfly

Coenagrion hastulatum (Charpentier, 1825), how immune response, wing length as an

indicator of individual size, and the number of both endo- and ectoparasites affect egg

production of host damselflies of four different populations in Southwest Finland.

Material and methods

Study species and fieldwork

Our study species Coenagrion hastulatum is one of the most common damselfly species in

Finland, having a long flight season in Northern Europe, from June to September, and found

throughout Finland close to varying types of freshwater bodies (Dijkstra 2006; Suhonen et al.

2010 a). Moreover, it harbors both endoparasiticDraft water mites and ectoparasitic gregarines (e.g.

Kaunisto and Suhonen 2013) making it an optimal species for studying the costs of

parasitism.

We collected 63 sexually mature Coenagrion hastulatum females from four different

populations in Southwest Finland in 2010 (Table 1). Each site was sampled on consecutive

days (10.-13. June 2010) in order to minimize the temporal variation in e.g. parasite numbers

(Table 1.). All sample sites were mainly open areas with freshwater wetland. Each female

was caught from a copulation wheel, i.e. a joined position together with a male damselfly

performed before mating, in order to maximize the likelihood of sexual maturity and

consequently a similar stage of egg production between study individuals. Only adult females

of nearly the same age were used in this study to minimize the possible effect of host age on

parasite load (Rolff and Martens 1997). Host age was determined by the stiffness of the

wings (as described in Plaistow and Siva-Jothy 1996) and by the coloration of the bodies,

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since adult damselflies become more brightly coloured than immature individuals as they mature. After being caught, damselflies were placed into individual plastic containers with a piece of wet paper towel to avoid dehydration of specimens. Each container was then placed inside a cool box and transported to the laboratory within four hours.

Laboratory work

To evaluate an individual damselfly's immune response, we challenged the damselfly’s immune system by inserting a piece of nylon filament through the third segment of the abdomen of each female. This method is commonly used to measure invertebrate immune responses (Córdoba-Aguilar et al. 2006; Rantala and Roff 2007; Rantala et al. 2010; Nagel et al. 2011). Prior to insertion, the filaments were smoothed with fine sandpaper, knotted and sterilized with 99,6 % alcohol, (lengthDraft of the inserted end was 2,0 mm, diameter 0.16 mm).

Each filament was left inside the damselfly for exactly 12 hours, incubated at 20°C and in indirect light. Afterwards the filaments were gently removed with fine forceps. The degree of encapsulation response, and hence the magnitude of immune function, was measured as grey values of photographed filaments (for details, see Kaunisto and Suhonen 2013). Thus, individual immune response values consisted of a combination of the thickness of the cell layer and the darkness of the cells formed around inserted filaments, caused by a melanization reaction of the immune response. The higher the value (the darker the cell layer), the more efficient the immune response is assumed to be. This method of comparing cell layer thickness and the melanization reaction of cells has been shown to associate strongly with an individual’s ability to react against natural pathogens (e.g. Rantala and Roff

2007).

After measuring the immune response in the laboratory, the number of ectoparasitic water mites was counted from the surface of each damselfly individual using a stereomicroscope

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(Olympus SZX 16). Scars left by detached water mites (scars described in e.g. Smith 1988)

were also carefully checked for, but none were found.

After counting ectoparasites, the intestinal tract was exposed by slicing the abdomen, and the

number of endoparasitic gregarines and the number of eggs (both developing and mature)

were counted. In addition, we measured the length of each hind wing to the nearest 0.01 mm

with a digital caliper to evaluate the body size of females.

Statistical analyses

A generalized linear model was used to test which variables were associated with the number

of eggs (Table 1.). The number of eggs was used as a dependent variable, while the number

of water mites, gregarines, the immune response and the mean wing length were included as

covariates and the population as a fixedDraft factor. Negative binomial errors and a log link

function were used because our data was aggregated. The scale parameter was set to a fixed

value of 1 and the model effects were estimated using type III sums of squares. The

generalized linear model was also repeated after omitting individuals with less than 10 eggs,

to check for the possibility of some individuals having laid eggs before being caught. Finally,

Spearman’s rank correlation was used to determine the relationship between the number of

eggs and immune response. All analyses were performed using the IBM-SPSS statistical

package, version 23.

Results

A total of 63 Coenagrion hastulatum females were sampled for this study (Table 1.). Mean

water mite prevalence was 76.2% (95% CL = 63.8 - 86.0) and intensity 31.9 (95% CL = 23.0

- 40.9; n = 48). Mean gregarine prevalence was 96.8% (95% CL = 89.0 – 99.6) and intensity

81.2 ( n = 61; 95% CL = 59.5 – 102.8). Wing length was on average 17.3 mm (S.D. = 0.62;

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range = 15.9 - 18.8 mm). The mean number of eggs was 81.7 (S.D. = 65.9; range = 2-320).

Immune response value was on average 24.9 (the higher the value, the darker the cell layer around the filament) (S.D. 8.6; range = 10.5-53.6).

The number of eggs decreased with increasing number of gregarines (Wald = 5.986; DF = 1;

P = 0.014; Fig. 1, Table 2). However, egg numbers were not affected by water mite numbers, wing length, the strength of the immune response or the population (Table 2).

The immune response value was positively associated with the number of water mites (Wald

= 14.413; DF = 1; P < 0.001; Fig. 2). Furthermore, when treated separately the number of eggs was negatively associated with immune response ( rs = -0.262, p = 0.038).

Manipulating the data to omit individuals with less than 10 eggs did not affect our results significantly (individuals with less thanDraft 10 eggs removed from the data, GLM: Wald = 5.685,

DF=1, P=0.017), indicating that our study individuals had not laid considerable numbers of their eggs before being caught.

Discussion

In this study we found a negative association between gregarine load and the number of the host’s eggs. However, we found no differences in egg numbers between populations, and neither wing length, nor the number of water mites affected the egg numbers. We also found that the immune response was positively associated with water mite load while associating negatively with the number of eggs.

Our results concur with a previous study by Canales-Lazcano et al. (2005), reporting a negative relationship between the number of eggs and gregarine burden on the damselfly

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Enallagma praevarum (Hagen, 1861). There are several possible explanations why gregarine

numbers correlated negatively with the number of eggs. Firstly, since the exoskeleton of

is near rigid and both eggs and gregarines require space inside the host, it seems

obvious that a large number of both gregarines and eggs cannot physically fit inside the same

female. Secondly, the energy resources consumed by the gregarines could reduce

energetically costly egg production. This kind of tradeoff has been previously reported by

Córdoba-Aguilar et al. (2003), showing that damselfly Calopteryx haemorrhoidalis (Vander

Linden, 1825) females with more gregarine parasites produced less wing pigmentation, which

is energetically costly. Furthermore, this energy deficit may be amplified if a parasitized

female tries to compensate the energy deficit by increasing prey consumption, which may

subsequently increase parasitism if the prey happens to be a vector of gregarines (Åbro 1976;

Gonzalez-Tokman et al. 2011). In contrastDraft to these negative effects of gregarines on their

hosts, e.g. (Hecker et al. 2002) found no harmful effects of gregarines on the host damselfly

Enallagma boreale (Selys, 1875). These varying results on the impact of gregarines may

originate from a simple reason: that the gregarines between different studies may represent

different species. Currently, there are some 1600 described species of gregarines reported

(Desportes et al. 2013). However, there are some indications that the number of gregarine

species infecting damselfly species could be quite low (Cielocha et al. 2011).

In this study, we also found a positive association between the number of water mites and the

strength of the immune response of female C. hastulatum damselflies. In other words,

females that have more water mites also have a stronger immune response. A similar result

was previously found during an inter-population comparison of average immune responses

(Kaunisto and Suhonen 2013). This positive association between water mite burden and

immune response could originate from local adaptation to chronic stress from parasite

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burden, indicating initially higher immune responses in heavily parasitized populations.

However, a recent interspecific comparative study found no association between water mite parasitism and encapsulation response, while phylogeny and host body weight seemed to play a role in determining the strength of the immune response of an odonate species (Ilvonen and

Suhonen 2016).

On another front, immune priming, e.g. improved protection of the host after a second encounter with the same parasite or pathogen, could play a role in our results. If immune priming increased the effectiveness of the immune response while decreasing its costs, former parasite encounters could partly explain our results on the association between water mite load and immune response. Unfortunately, there are only a few studies reporting the cost of immune priming in invertebrates (reviewedDraft in Little and Kraaijeveld 2004) and there is no information indicating whether the better effectiveness (i.e. parasite killing) of immune priming actually decreases the costs.

As suggested by Mlynarek et al. (2015), odonate hosts may apply a general or a specific defense strategy depending on how many different water mite species the host comes in contact with. It is possible that our study species C. hastulatum , being a widespread species, relies on a general strategy and thus perhaps has a lower encapsulation response against water mites. This implies that, even though the encapsulation response seems to be a direct protective mechanism against water mites, the number of observed parasites may not completely explain immune response levels.

In the invertebrate immune response, phenoloxidase (PO) converts tyrosine-based precursor molecules to melanin in an energetically costly process. The melanin forms around

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pathogens, killing them (Lawniczak et al. 2007). Previous studies on the tradeoff between

hosting water mites and a costly immune response have been rather inconsistent. For

example, Joop et al. (2006) found that Coenagrion puella females with an enhanced immune

function (higher haemocyte counts and PO activity) did not have a decreased number of

water mites. On the other hand, Kaunisto and Suhonen (2013) showed that damselfly

populations with higher water mite burden have considerably higher immune responses

(encapsulation responses). Interestingly, egg production in females may increase PO levels,

which in turn may affect the females’ response towards water mite parasitism (Robb and

Forbes 2006). Moreover, research by Rolff (1999) found a negative correlation between the

number of eggs laid and water mite load for the damselfly Coenagrion puella . In contrast, we

did not find a similar negative association between water mite parasitism and egg numbers in

this study. This inconsistence could originateDraft from e.g. different life histories between the

study damselflies and/or water mite species, as well as differences between study sites.

Additionally, water mites are known to detach from their damselfly hosts during oviposition

(Rolff and Martens 1997) and therefore, even though we only included damselflies from a

tandem wheel, as well as included only individuals of a similar age in our study, the

detachment of water mites could potentially affect the observed results.

In addition to playing an important role in immune response, phenoloxidase (PO) and

tyrosine are also key compounds in the reproductive process, involved in the tanning and

hardening of the egg chorion in insects (Walker and Menzer 1969; Li and Christensen 1993;

Bai et al. 1996). This may create a tradeoff in reproducing females when resources are

limited: PO and tyrosine that are allocated to egg production cannot be used for encapsulation

in an immune response. In turn, this may explain why the number of eggs was negatively

associated with immune response in our study. To further untangle this, future research

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should be directed to experimentally examining immune response components and egg production under varying levels of parasite infection.

In conclusion, even though gregarine parasitism is occasionally considered to be fairly benign to a host, gregarines appear to strongly associate with the number of eggs produced by their host damselfly. This reduction in egg numbers may have an effect on the fecundity of parasitized host individuals, thus potentially affecting the host population dynamics, as previously shown in birds (Hudson et al. 1998). In future, since heterozygosity is linked with lower parasite burden (Kaunisto et al. 2013), the next step could be the genetic analysis of females to assess the impact of overall genetic diversity on parasite load and egg production ability.

Draft

Acknowledgements

This study was supported by Ella and Georg Ehrnrooth Foundation’s grant to KMK. The authors would like to thank two anonymous reviewers, who provided constructive notes to a previous version of this work. We would also like to thank Tapani Hopkins for proofreading the manuscript. The authors have no conflict of interest to declare. Neither the data nor the text of our manuscript have been used in other articles or books, whether published, in press, submitted, or soon to be submitted elsewhere.

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Figure captions

Fig. 1 The relationship between number of eggs and number of endoparasitic gregarines of

Coenagrion hastulatum (Charpentier, 1825) (n = 63) females. Black dots represent observed

values, open dots values predicted by the model. The equation of the line is: log 10 (number of

2 eggs) = 2.82 (±0.15) – 0.31 (±0.08) * log 10 (number of gregarines), r = 0.19. The figure

shows values that have been back-transformed from logarithmic values.

Fig. 2 Relationship between the number of water mites and encapsulation ability of damselfly

Coenagrion hastulatum (Charpentier, 1825) (n = 63) females. The higher the encapsulation

value, the darker and thicker a cell layer the individual is able to form around a nylon filament during 12 hours of incubation.Draft

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Figure 1.

350

300

250

200

150 Number of eggs of Number 100

50 Draft

0 0 100 200 300 400 500 Number of gregarines

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Figure 2.

120

100

80

60

Number of mites of Number 40 Draft 20

0 0 10 20 30 40 50 60 Encapsulation

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Table 1. Parasitism of Coenagrion hastulatum (Charpentier, 1825) sampled from four populations in Finland. " n" represents the number of individuals of each location sampled. Confidence intervals (CI 95%) for prevalence ( P) and intensity ( I), as well as for the minimum and maximum number of parasites are shown after their respective values.

Population Water mites Gregarines Name Coordinates n P (95% CI) I (95% CI) Min/Max P (95%CI) I (95% CI) Min/Max Paimio 60°27'N, 22°47'E 16 100 (79-100) 47 (26-68) 2/114 94 (70-100) 82 (13-152) 0/500 Masku 60°32'N, 22°9'E 15 87 (60-98) 14 (4-24) 0/59 100 (78-100) 46 (31-61) 11/110 Turku 60°30'N, 22°16'E 16 19 (4-46) 3 (0-7) 0/34 94 (70-100) 79 (52-105) 26/175 Säkylä 60°55'N, 22°47'E 16 100 (79-100) 33 (20-46) 3/84 100 (79-100) 105 (59-152) 0/330 Total 63 76 (64-86) 24 (17-32) 0/114 97 (89-100) 79 (57-100) 0/500

Draft

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Table 2. Generalized linear model of the variables tested against egg numbers of the host damselfly Coenagrion hastulatum (Charpentier, 1825). Statistically significant result shown in bold type.

Wald df P (Intercept) 2.089 1 0.148 Population 4.977 3 0.173 Number of water mites 1.452 1 0.228 Number of gregarines 5.986 1 0.014 Wing length 0.026 1 0.872 Immune response value 0.219 1 0.640

Dependent Variable: Number of eggs Model: (Intercept), Population, number of water mites, Number of gregarines, wing length, immune response value

Draft

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