University of Cincinnati

UNIVERSITY OF CINCINNATI

Date: 15-Dec-2009

I, Aaron Greene , hereby submit this original work as part of the requirements for the degree of: Master of Science in Biological Sciences It is entitled: Heritable Behavioral Resistance to Natural and Novel Ectoparasites in

Drosophila melanogaster

Student Signature: Aaron Greene

This work and its defense approved by: Committee Chair: Michal Polak, PhD Michal Polak, PhD

Iain Cartwright, PhD Iain Cartwright, PhD

Stephanie Rollmann, PhD Stephanie Rollmann, PhD

5/28/2010 863 Heritable Behavioral Resistance to Natural and Novel Ectoparasites in Drosophila melanogaster

A thesis submitted to the

Graduate School

of the University of Cincinnati

In partial fulfillment of the

requirements for the degree of

MASTER OF SCIENCE

In the Department of Biological Sciences

of the College of Arts and Sciences

AARON VILAS GREENE

B.S. Biological Sciences

Mansfield University, November 2001

Committee Chair: Dr. Michal Polak

Abstract

Ectoparasites affect many different organisms, are naturally abundant, and have been

shown to decrease fitness and drive host evolution. However, few studies have

estimated heritable variation in resistance to ectoparasitism, and none have tested the

effects of those defenses on other ectoparasites. The threat of parasitism can be so

costly that potential hosts develop several lines of defense to protect themselves from

parasitism. The ability to resist novel enemies, such as ectoparasites, has been shown

to carry fitness costs. However, possessing traits that effectively resist or limit the threat

of multiple enemies potentially could benefit the host by minimizing the costs associated

with resistance. The current paper details the presence of behavioral resistance

potentially utilized by Drosophila melanogaster to defend against ectoparasitic mites .

This study reports the results of artificial selection for increased resistance in Drosophila melanogaster to the ectoparasitic mite, Macrocheles subbadius , which is a known enemy of some Drosophila species. My work also investigates the effectiveness of the selected resistance against the natural enemy Gamasodes queenslandicus . Selection was applied to the pre-attachment phase, thereby targeting behavioral defensive traits.

Realized heritability ( h2) of resistance against Macrocheles is estimated at 0.06 (SE

0.015). Results also demonstrate that flies selected for increased resistance to M. subbadius also have improved resistance to G. queenslandicus. This study demonstrates the evolutionary potential of generalized behavioral defenses against ectoparasite attack in D. melanogaster .

Acknowledgements

I would like to first thank my advisor, Dr. Michal Polak for his guidance and support. I would also like to thank my committee members Dr’s Stephanie Rollmann and Iain

Cartwright who both offered their invaluable expertise and advice.

The Department of Biological Sciences at the University of Cincinnati provided financial support for this project.

I would also like to thank my lab mates, Brooke Hamilton, Karl Grieshop and Dr.

Arash Rashed for their support and insightful talks throughout the course of my research.

Lastly, I would like to thank my family and friends who gave me support and encouragement throughout this process.

Table of Contents

Abstract……………………………………………….……………………………………….. i

Acknowledgements …………………………….….………………………………………… ii

Introduction …………………………………………………………………………………… 1

Thesis Overview………………………………………………………………………. 4

Methods ……………………………………………………………………………………… 5

Collecting Organisms and culturing………………………………………………..... 5 Resistance Behaviors…………………………………………………………………. 6 Selection Lines………………………………………………………………………… 7 Response to Selection and Heritability…………………………………………….... 9 Resistance against Gamasodes ……………………………………………………... 10 Fly Mortality within Chambers………………………………………………………... 10 Mechanisms of Defense…………………………………………………………….... 11 Statistical Analyses……………………………………………………………………. 12

Results …………………………………………..…………………………………………….. 14

Resistance Behaviors………………………………………………………………… 14 Resistance to Selection and Heritability...... 16 Fly Mortality within Chambers……………………………………………………….. 17 Mechanisms of Defense……………………………………………………………... 18

Discussion …………………………………………………………………………………….. 18

Resistance Behaviors…………………………………………………………………. 19 Response to Selection and Heritability……………………………………………… 21 Mechanisms of Defense………………………………………………………………. 22 Resistance against Gamasodes……………………………………………………... 23

Conclusions …………………………………………………………………………………... 26

References ……………………………………………………………………………………... 28

List of Tables and Figures

Figures:

Figure 1: Infestation Chamber…………………………………………………………… 32

Figure 2: Flick frequency…………………………………………………………………. 33

Figure 3: Decamp frequency…………………………………………………………….. 33

Figure 4: Prying Frequency……………………………………………………………… 34

Figure 5: Rolling Frequency……………………………………………………………... 34

Figure 6: Divergence in Resistance through 13 Generations………………………... 35

Figure 7: Contrast in Resistance to M. subbadius ……………………………………... 36

Figure 8: Contrast in Resistance to G. queenslandicus ……………………………….. 37

Figure 9: Estimated Heritability of Resistance………………………………………….. 38

Tables:

Table 1: Incidences of Resistance Behaviors…………………………………………. 39

Table 2: Mortality Rates within No Mite Chambers……………………………………. 40

Table 3: Resistance Assays to M. subbadius ………………………………………….. 41

Table 4: Resistance Assays to G. queenslandicus……………………………………. 43

INTRODUCTION :

Parasites are ubiquitous in the environment, and affect many organisms at some stage of their life (Price 1980). Parasites are defined as organisms that live on

(ectoparasites) or in (endoparasites) other organisms and that exploit their hosts as a resource for their own nutrition, longevity, and reproduction (Sheldon and Verhulst

1996). During their interaction with hosts, parasites can decrease host fitness (Price

1980; Ewald 1994) and through the selection they impose, can drive rapid host evolution and alter host population dynamics and host population genetic structure (e.g.

Burdon 1980; Dwyer et al. 1980; May and Anderson 1983).

Predicting host evolutionary response to the effects of parasite – imposed selection on the evolution of a host population requires an understanding of the heritable genetic variation in host defensive traits (Endler 1986; Polak 2003). One approach to examining the heritable nature of parasite resistance in insects is the use of artificial selection experiments. The central idea behind using artificial selection in this context is that a significant response to artificial selection on a particular trait demonstrates the presence of additive genetic variation for that trait in the original

(base) population (Falconer and Mackay 1996). Several examples of artificial selection for increased resistance against parasites exist in the literature. The mosquito,

Anopheles gambiae , has been used to show significant response to selection for resistance against the malaria parasite, Plasmodium (Collins et al. 1986). Kraaijeveld &

Godfray (1997) successfully selected for resistance against two species of parasitoid

wasps in Drosophila melanogaster . More recently, D. nigrospiracula was successfully selected for increased resistance to the ectoparasitic mite, Macrocheles subbadius

(Polak 2003). Thus, the evidence suggests that there may often be genetic variation for resistance in natural populations, but little is known about the heritable genetic basis of resistance against ectoparasitism.

Most of our understanding of parasite resistance comes from studying endoparasites (e.g. parasitoids). However, ectoparasites make an ideal model for use in this field for several reasons. First, ectoparasites are abundant in natural communities (Marshall 1981), and they have been shown to damage a variety of host fitness traits (Forbes and Baker 1991; Polak 1996). Ectoparasites also are known vectors of many parasitic diseases of animals, including humans, and so can have significant economic, veterinary and medical consequences (Lehmann 1993; Marshall

1981). Perhaps the most advantageous practical aspect of utilizing ectoparasites in ecological and evolutionary studies is the benefit of visual confirmation of parasitism. In systems utilizing other forms of parasites such as parasitoids and entomophagic nematodes (i.e., endoparasites), infection determination often requires the need to sacrifice the host.

Two common behavioral strategies animals use to prevent infestation and

minimize the cost of ectoparasite infestation are active avoidance and removal of

ectoparasites (Hart 1994). In bats, for example, studies have shown that increased

grooming behavior occurs in species affected by higher densities of ectoparasites (ter

Hofstede and Fenton 2005). There have been very few studies to determine the

resistance behavior of an invertebrate host to a potential ectoparasite. However, the

honeybee, Apis mellifera, has been observed to express self-grooming and allogrooming behavior (Peng et al. 1987) in populations affected by the parasitic mite,

Varroa destructor . In addition, worker bees have been shown to identify drone brood

cells infected with the ectoparasites, expose the cell, and destroy the mites (Martin et al.

2001), or remove the infected larvae from the hive (Boecking and Spivak 1999).

The general aim of my project is to evaluate the evolutionary potential of resistance to ectoparasitic mites in Drosophila melanogaster Meigen. This study conducts an artificial selection experiment to test for the presence of additive genetic variation in behavioral ectoparasite resistance in D. melanogaster. The ectoparasite used in my selection experiments is a species of mite in the family Macrochelidae,

Macrocheles subbadius (Berlese). Although not a known, natural parasite to D. melanogaster, M. subbadius offers more control over infestation rate in the laboratory compared to D. melanogaster’s natural enemy, the mite Gamasodes queenslandicus

Halliday and Walter, which belongs to the family Parasitidae (Halliday et al. 2005).

Drosophila melanogaster and G. queenslandicus mites are known to be associated in northeastern Australia. In preliminary experiments I conducted with D. melanogaster to familiarize myself with the dynamics of infestation by G. queenslandicus , it was found that the rate of infestation was so rapid (mites are very agile and move to colonize flies very rapidly) that the flies became overwhelmed by mites so quickly that it was difficult to recover unparasitized flies from experimental chambers. Thus, prior to commencing artificial selection using M. subbadius as the agent of selection, I tested whether the same behavioral traits used to resist infection by M. subbadius were also used against

G. queenslandicus . Since the artificial selection experiment I wished to conduct requires a cohort of unparasitized flies to be reliably collected each generation, it was desired to use a more slow-moving species of mite exhibiting lower rates of infestation. Therefore,

Macrocheles was used as a surrogate ectoparasite for means of artificial selection.

Macrocheles mites attach to Drosophila by penetrating the integument with their mouthparts, causing the formation of a 'scar' at the site of attachment (Polak 1996;

Halliday et al. 2005). Scars are small melanized regions of encrusted fly haemolymph that seeps from the mite-inflicted wound. Gamasodes queenslandicus produces similar scarring to those inflicted by M. subbadius on the North American fruitfly D. nigrospiracula Patterson & Wheeler (Polak 1996). Normally a predator (Axtell 1961), M. subbadius causes a reduction in both the survival and fecundity of D. nigrospiracula hosts. Gamasodes queenslandicus mites probably also consume haemolymph while attached to flies (though this has not been demonstrated experimentally), and should be considered an opportunistic parasite of Drosophila and not simply phoretic (Halliday et al. 2005). Although both these species of mites depend on their hosts for transport to the patchy habitats in which they feed and reproduce (e.g. fruit, dung, fungal sporocarps), they use a variety of insect hosts (Polak 1996; Halliday et al. 2005).

Evidence shows that negative effects on host species are more likely to occur when parasites have alternative carriers (Herre 1993). Thus, M. subbadius and G. queenslandicus have similar life histories and expected effects on host ecological and evolutionary dynamics.

THESIS OVERVIEW:

My study selected two replicate lines of D. melanogaster for increased resistance

to the ectoparasite M. subbadius . The first step of the research is to test whether flies do

respond behaviorally to mites and to describe the specific behaviors flies use to defend

themselves against ectoparasitism. In a second step, I test whether flies respond similarly to M. subbadius and G. queenslandicus in order to ascertain whether M.

subbadius can serve as an adequate surrogate agent of selection in my artificial

selection experiment I conducted in the laboratory. The third and main goal of my thesis

is to use artificial selection to test whether heritable genetic variation exists in the

behavioral traits used to resist ectoparasitism. In the event of a significant response to

selection, I also sought to examine the consistency in response between males and

females, and to estimate the realized heritability of resistance against mites. I also

tested whether the traits selected provide cross-resistance against a second species of

ectoparasite, G. queenslandicus . Finally, I evaluate the possibility that greater activity levels in the resistant lines are the cause of the divergence in resistant and control lines that resulted from artificial selection. The results of my thesis work are then discussed in the broader context of host-parasite evolutionary ecology.

METHODS:

Collecting Organisms and culturing

The base population that served as the source of the selected lines was derived from a sample of 100 field-caught female flies collected in northeastern Australia (Cape

Tribulation) in 2005. The base population was mass-cultured in the laboratory in 12

200ml bottles per generation, at 25˚C day and 22˚C night temperature cycle, and on a

12L: 12D photoperiod. Adults were aspirated into a cone of sterile (autoclaved) twisted tissue inside the bottle and were removed from the bottles after 48 hours. After

emergence, flies were identified and sorted by sex, and held in vials containing standard molasses-yeast medium.

A bacteriophagic nematode extracted from horse dung (from a farm 5 km west of

Independence, KY) was cultured in medium within 4-qt plastic tubs. The culture medium

was comprised of autoclaved wheat bran and wood chips, corn meal, and inactive

yeast. The medium in each jug was aerated daily by inversion; tipping the tubs upside

down and shaking the mixture. In addition to aeration, fresh bran medium was regularly

cycled into the tubs by removing dried or molded portions and adding fresh moistened

medium. Gamasodes queenslandicus mites were harvested from the bodies of D.

melanogaster flies collected from the exposed flesh of jackfruit Artocarpus heterophyllus

(Moraceae) at Cape Tribulation, Australia.

Cultures of the ectoparasitic mite, Macrocheles subbadius, were collected from the bodies of D. nigrospiracula flies from necrotic cacti east of Phoenix, AZ (specific

locales provided in Table 1 of Polak 2003). Macrocheles subbadius was also cultured on

bran medium with nematodes, but separately from G. queenslandicus . Both species of

mites were reared in the laboratory on a 12L: 12D photoperiod, and a 26˚C day and

22˚C night temperature cycle.

Resistance Behaviors

In the first two phases of the research, I tested whether flies respond behaviorally

to contact by mites, and whether the same resistance behaviors are elicited in the host

by both mite species. To explore any shared behaviors, individual D. melanogaster flies

were exposed to G. queenslandicus mites within infestation chambers and were

observed utilizing avoidance and resistance behaviors in response to attack by ectoparasites.

Data on potential behavioral resistance traits were gathered by observing the

response of flies aspirated into a circular (22mm in diameter) plastic observation

vessels with mites. A Canon ® XLI camera with a 7x lens was used to record the

interactions between fly and mites within the vessel from a distance of 70mm. Twelve

mites were collected from a sample of culture medium and were placed into the vessel

using a paintbrush. Interactions were observed via video playback on Apple iMovie

software. Magnified 7X, each unique fly response to mite stimuli was identified and

tallied during the exposure until parasitism occurred or ten minutes (600 seconds)

elapsed. Exposure time is time elapsed in seconds from when flies were first exposed

to mites to either parasitism or 600 seconds. Fly responses expressed in the presence

of mite stimulus were compared to those of control flies, recorded within the same

observation chambers but in the absence of mites.

Behaviors in D. melanogaster elicited by interactions with G. queenslandicus

were compared to behaviors expressed by D. melanogaster in interactions with M.

subbadius.

Selection Experiment

From this base population, two replicate selection lines were derived, along with

paired unselected control lines. Each line was maintained in four bottles each

containing 40 males and 40 females. The two selected lines were independently

selected for behavioral resistance against M. subbadius mites. Within 6 hours of

emergence, virgin males and females were collected from bottles and placed separately into vials, containing standard molasses-yeast medium. At 3 days old, 50 flies of each sex, from both selection lines, were placed into eight separate experimental infestation chambers (Figure 1) for 24 hours. The sexes were exposed separately and no food resource was present within chambers to minimize competitive interactions (Polak

2003). The chambers consist of 300 ml Ball® Mason Jars (Alltrista Corp., Muncie, IN) lined at the bottom with plaster of Paris and containing bran medium with mites (Polak

2003). Coarse paper towel was used to cover the mouth of the jar and then sealed using a canning ring. A hole was pierced through the paper towel using a pencil. Using a small spatula, a space was excavated into the medium and flies were gently aspirated into this space. The infestation chambers were inverted at a 45˚ angle and stored in an incubator at 26˚C: 22˚C L: D cycle for 24hrs. After exposure, all surviving flies were removed from chambers by aspiration and observed for signs of parasitism: mites attached to flies and mite-induced scars. Unparasitized and unscarred flies, which together comprised the putatively resistant group of flies, were used to seed the next generation of the selection experiment.

A control line was maintained in parallel to each selected line, and derived from

the same base population as the selection lines. The two control lines were exposed in

the same infestation chambers as selected flies, but in medium only.

After a 24h exposure period, control lines were removed from the chambers and

used to seed the next generation of the control line. The number of individuals used to

seed a given control line were exactly the same as that used to seed its paired selected

lines. In this way, inbreeding effects between selected and control pairs of lines was minimized (Polak 2003).

Response to Selection and Heritability

Assays to estimate the response to selection within each line were performed by contrasting resistance between the selected lines (R1 & R2) and their corresponding controls (C1 & C2). An assay consisted of aspirating flies from both the resistant line and its paired control line into a common infestation chamber and contrasting proportions of parasitized flies between lines (Polak 2003). Using a pair of Vannas micro scissors, a small clip to the tip of one wing was used to identify the flies as per their line. The wing that received the clipping was alternated across replicate infestation chambers. Wing clips were conducted 24 hours before the start of the assay under light

CO 2. Multiple infestation chambers were used for each line. Paired groups varied from

10 to 40 flies of each treatment and paired groups within chambers were always equal in numbers. Assays were performed starting at generation three and again every other generation following, until the completion of the experiment.

Jointly exposing selected and control lines to mites within the same chamber allowed for the minimization of unwanted variation in mite density and substrate humidity, between chambers. Flies were exposed to mites for 24 hours before being removed by aspiration. Following exposure, all surviving flies were removed from chambers via an aspirator, identified by wing-clip, counted and observed for evidence of parasitism (i.e., attached mite or mite-induced scarring). Difference in prevalence of infestation was calculated for each chamber by subtracting the proportion of infested or

scarred control flies and subtracting the proportion of infested selected flies. Prevalence for each group was transformed to mean liability of infestation following Falconer and

Mackay (1996, p. 301). Assuming similar variances of liability of each group, response to selection was calculated as the difference in mean liability of resistance between lines

(Hill 1972). Thus, resistance to mites is modeled here as a threshold character with two phenotypic classes (i.e., affected or not) (Falconer & Mackay 1996).

Resistance against Gamasodes

At the completion of the selection experiment, I tested the hypothesis that traits artificially selected for resistance to Macrocheles would also produce effective cross- resistance to Gamasodes. To test this hypothesis, flies were placed under light CO 2 and assigned an identifying wing clip (L or R), which was alternated between the lines, before aspirating control and selected flies into a common chamber packed with bran medium containing either Macrocheles or Gamasodes mites. The proportion of infested selected flies was then contrasted to controls. The degree of divergence in infestation, between selected and control lines, was compared between flies exposed to

Macrocheles versus Gamasodes . In the case of cross-resistance, lines selected against

Macrocheles should exhibit resistance also against Gamasodes .

Fly Mortality within Chambers

To determine if death rates within chambers could have been the result of factors

other than the mites, we aspirated resistant and control flies into common chambers

without mites for a 24-hour period, in groups of 30 flies per treatment. Both sexes were

assayed in separate chambers. The total number of chambers used was 18. Death rates of flies within chambers were contrasted between selected and control lines. In this way, deaths in the two groups not attributable to mites could be determined. This experiment also allowed me to verify that it was the presence of mites per se that caused the separation between resistant and control lines; in these chambers without mites I expected no separation between resistant and control lines. A t-test was used to contrast proportion dead flies between selected and control lines. Only a single test was conducted because selected and control lines were pooled across replicate selection experiments.

Mechanisms of Defense

Since active avoidance of ectoparasites is a potential form of pre-attachment behavioral resistance, I tested the hypothesis that selected lines were resistant to mites because they exhibited higher activity levels, and thus were a more difficult target for the mites. The activity rates of D. melanogaster were observed and tracked within empty

54mm diameter Petri dishes or arenas using Ethovision® XT software (Noldus). D. melanogaster were aspirated individually into observation arenas and tracked. Paired control and selected flies from each line were systematically assigned testing arenas and were recorded at the same time for twenty minutes. The activity level of selected and control flies were recorded for the following parameters: the distance each fly moved during the trial, how much time (s) was spent in motion, the frequency each fly started and stopped and velocity (mm/s) of the movement was calculated and compared to controls. Movement duration examined the time spent moving by individual flies of

each line over a 20-minute trial. Testing included four trials each performed between

1pm and 4pm.

Statistical Analyses

Resistance Behaviors

Behaviors expressed in response to mite stimuli were contrasted among experimental categories using analyses of covariance (ANCOVAs). For each analysis, the factor was “Mite type” (i.e., no mites present in chamber (control flies), Gamasodes present, and Macrocheles present), and the response was incidence of one of 4 fly behaviors, which were analyzed in turn. The covariable was exposure time. The data for all behaviors were checked for normality using the Shapiro-Wilk test and the data were reasonably close to normal ( W=0.5-0.97). Post-hoc tests were conducted using the Tukey-Kramer method. The level of replication was individual fly. Behaviors utilized by D. melanogaster in response to attack by Macrocheles were also contrasted to behaviors expressed by flies exposed to Gamasodes and to control flies using separate,

pair-wise t-tests.

Response to Selection and Heritability

A two-factor analysis of variance (ANOVA) was conducted in which the response

variable was the difference in proportions between selected and control flies. The two

factors were generation and sex. The level of replication was individual bottle.

After 13 generations of selection the data were analyzed for this generation using

a t-test. The data that were analyzed were the proportion of flies parasitized. The level

of replication was, again, individual bottle, in which both control and resistant flies were exposed simultaneously.

A regression model was also constructed in which generation (i.e., 3, 5, 7, 11, and 13) was the continuous independent variable, and the difference in proportion between selected and control lines was the response variable. Bottle, again, was the level of replication and the total sample size was 48. Difference in resistance between sexes was tested using a t-test.

Because selection was applied to both males and females, the mean realized heritability of resistance was taken as the slope of the regression line relating response to selection (as the difference in mean liability, see above) on generation number

(Falconer & MacKay 1996, Polak 2003).

Resistance against Gamasodes

At the terminus of the selection experiment, differences in resistance to G.

queenslandicus by selected and control flies were contrasted using a t-test. The data

were proportions of flies infested, and assay chamber was the level of replication. The

total sample size was 16 (total number of bottles).

Fly Mortality within Chambers

A t-test was conducted on proportion of flies found dead between control and

selected lines that had been placed into chambers without mites.

Mechanisms of Defense

Individual ANOVAs were conducted, for each line, contrasting one of four measures of activity between selected and control flies. The independent variable in these analyses was “Mite type” and the response variables tested in turn were: distance traveled, velocity, movement duration and, movement frequency. Sexes were pooled since there were no significant differences in resistance between males and females. A total of 72 flies were tested, 18 from each of the four lines (i.e.; the two selected and paired controls).

Software

JMP, Version 7, (SAS Institute Inc.) was used for all statistical analyses. An α value of 0.05 was used for each test.

RESULTS

Resistance Behaviors

D. melanogaster exposed to M. subbadius mites were found to utilize several repeated behaviors to resist or avoid attack by mites, of these behaviors four were identified for further scrutiny. These behaviors were: decamping from the substrate, rolling, tarsal flicking, and prying (Table 1). Tarsal flicking was observed to be any drumming or punching of a fly’s forelimbs onto an attacking mite. Decamping was defined as any movement that took the fly and all of its appendages from the substrate.

Prying behavior is the act of pushing or extension of a fly’s limbs between themselves

and the mite, and occurred when a mite and fly were actively engaged but the mite was not yet attached (Figure 4). Rolling occurred when the fly would tip over onto its back.

ANCOVA on the incidence of tarsal flicking, with exposure time as covariable, revealed a significant effect of the factor “Mite Type” ( F2,26 =3.54, P=0.044; Figure 2). A post-hoc Tukey-Kramer test revealed a significant difference between Gamasodes - exposed flies and controls ( P<0.05). However, a Tukey-Kramer post-hoc test revealed

no difference between Gamasodes-exposed and Macrocheles -exposed flies in mean

incidence of flicking (P>0.05). Subsequent pair-wise tests confirmed a significant

difference between Gamasodes -exposed flies and controls ( t17 =2.30, P=0.03), and lack of a significant difference between Gamasodes -exposed and Macrocheles -exposed flies

(t17 =0.70 P=0.49).

ANCOVA on the incidence of decamping, in turn, with exposure time as covariable, revealed a significant effect of “Mite Type” ( F2,26 =6.63, P=0.0047; Figure 3).

Post-hoc Tukey-Kramer tests revealed a significant difference between Gamasodes - exposed flies and controls ( P<0.05), but again, no difference between Gamasodes and

Macrocheles -exposed flies in mean incidence of decamping (P>0.05). Subsequent

pair-wise tests confirmed a significant difference between Gamasodes -exposed flies and controls ( t17 =3.46, P=0.0030), and again, a lack of a significant difference between

Gamasodes -exposed and Macrocheles -exposed flies ( t17 =0.10 P=0.92).

ANCOVA on the incidence of prying, with exposure time as covariable, revealed a significant effect of the factor “Mite Type” ( F2,26 =10.70, P=0.0004; Figure 4).

Gamasodes -exposed flies revealed significant difference compared to controls in post- hoc Tukey-Kramer tests ( P<0.05). Post-hoc Tukey-Kramer analysis showed no

difference between Gamasodes and Macrocheles exposed flies in mean incidence of prying (P>0.05). Pair-wise tests again confirmed a significant difference between

Gamasodes -exposed flies and controls ( t17 =4.18, P=0.0006), and lack of a significant difference between Gamasodes -exposed and Macrocheles -exposed flies ( t17 =0.30

P=0.77).

Differences in mean incidence of rolling, with exposure time as covariable, however, was not significant among levels of the factor “Mite Type” ( F2,26 =2.01, P=0.15).

Subsequent Tukey-Kramer post-hoc tests also revealed no differences between the pairs of means ( P>0.05). Pair-wise tests again confirmed non-significant differences

between Gamasodes -exposed flies and controls ( t17 =1.88, P=0.078), and lack of a significant difference between Gamasodes -exposed and Macrocheles -exposed flies

(t17 =0.75 P=0.47). Although significant differences were not found in mean rolling frequency between levels of “Mite types”, the pattern of expression across these levels

(Figure 5) was similar to that of the other response variables (see above).

In summary, the above behavioral data indicate that flies exhibit significantly greater rates of specific behaviors when exposed to Gamasodes mites. It is therefore concluded that D. melanogaster express defensive behaviors when exposed to mites.

Moreover, flies exposed to the two types of mites, did not differ in the rates of behaviors they expressed. Therefore Macrocheles represents an appropriate surrogate ectoparasite for Gamasodes for selection purposes.

Resistance to Selection and Heritability

Infestation rates, expressed as proportion of flies infested, among selected and control flies, are presented in Table 3. ANOVA on the difference in the proportion of infestation between selected and control lines revealed a significant effect of generation

(F5,41 =4.39, P=0.003), and a non-significant effect of sex ( F1,41 =0.00, P=0.98).

Regression analysis, with difference in proportion as the dependent variable,

revealed a significant effect of generation number ( F1,46 =17.56, P=0.0001); the difference increased with generation number (Figure 6), indicating resistance increased in the selected line over the course of the experiment.

After 13 generations of selection (i.e., at the terminus of artificial selection), selected flies were more resistant to ectoparasitism than controls ( t15 = 3.35, P=0.005;

Figure 7).

Table 4 presents the rates of infestation by G. queenslandicus , as proportion flies infested, between selected and control flies from both lines. Data from all 16 assay chambers and both sexes are shown. At the completion of artificial selection, flies selected for resistance against M. subbadius were significantly less susceptible to infestation by G. queenslandicus than control flies (t-test contrasting proportion flies infested between selected and control lines: t-30 = 2.45, P=0.02, Figure 8). These data strongly suggest that selection applied to flies for increased resistance against

Macrocheles mites conferred cross-resistance against Gamasodes queenslandicus , the

natural enemy of D. melanogaster .

The mean realized heritability of ectoparasite resistance against Macrocheles was taken as the slope of the regression line relating response to generation number, and is estimated at 0.06 ± 0.015 SE (Figure 9).

Fly Mortality within Chambers

A t-test indicated no significant difference in the mortality rates of selected and control flies within chambers with no-mites ( t14 =1.12, P=0.85; Table 2).

Mechanisms of Defense

ANOVA on differences in total distance traveled between selected and control flies showed no significance in the total distance traveled (how far they moved over a twenty minute interval) than control flies, for both lines (Line 1: F1,34 =2.48, P=0.12; Line

2: F1,34 =1.63, P=0.21). Selected and control flies did not move at significantly different

velocities (mm/s) (Line 1: F1,34 =2.57, P=0.12; Line 2: F1,34 =2.65, P=0.11). ANOVA also

revealed no significant differences in movement duration between selected and control

flies (Line 1: F1,34 =3.002, P=0.099; Line 2: F1,34 =1.86, P=0.18). The data indicate that there is also no differences in the frequency of movement between selected flies and paired controls (Line 1: F1,34 =0.05, P=0.82; Line 2: F1,34=0.228, P=0.64). Means are not provided as none of the above contrasts were significant.

DISCUSSION:

The main objectives of the present study were to employ artificial selection to test

for the presence of heritable genetic variation underlying behavioral resistance to

ectoparasitic mites in D. melanogaster , and to test whether the selected resistance traits

provide increased resistance against a different species of ectoparasitic mite. The

results reveal the presence of heritable resistance to ectoparasitism in D. melanogaster .

My findings show also that flies selected for increased resistance to the surrogate

ectoparasite M. subbadius do indeed have increased resistance to G. queenslandicus.

Thus, D. melanogaster possesses behavioral defenses that are effective against both G.

queenslandicus and M. subbadius mites. The selected resistance does not appear to

be strongly linked to an increase in general activity rate (i.e., hyperactivity) by selected

flies over control flies, suggesting that D. melanogaster possesses specific behavioral

traits that it can use to protect itself from attack by ectoparasitic mites.

Resistance Behaviors

In general, a first line of defense against mites involves non-specific behavioral mechanisms that reduce the probability a mite contacts its host (Malcolm 1992) and moves to its site of attachment on the fly’s body (Polak 2003). Downstream forms of defense relate to a potential suite of physiological and biochemical mechanisms (e.g., induction of haemolymph effectors) that act on the mite to reduce the duration of time it remains attached and feeding from its host (e.g., Wikel 1996). Once a mite attaches to its host there is little the fly can do behaviorally to dislodge it, although there exists variation among hosts in the amount of time a mite remains attached to its host (or in apparent host “palatability” Wikel 1996), suggesting the existence of genetic heterogeneity underlying post-attachment forms of defense against ectoparasitism.

Moreover, the fitness costs from ectoparasitism are not only dose-dependent, but are

also related to the duration of mite attachment (Polak 1997, 1998; Polak & Starmer

1998), implying that there is selection for defense at both these stages of the interaction. For example, melanization occurs where mites attach and feed, suggesting a systemic host response (Polak & Markow 1995). Secretion of antiparasitic peptides into the hemolymph may also occur (Hoffmann & Reichhart 1997). Thus, it should be emphasized that there are several forms of resistance traits of flies likely to be expressed and be under selection that were not addressed by my research, and that may be the focus of future fruitful research. For example, it would be interesting to test the degree to which the effectiveness of the behavioral forms of defense I detected are traded for resistance expressed at later stages of the fly-mite interaction. Such a trade- off, if genetically based, could act to maintain genetic variation for defensive traits generally (e.g., Mitchell-Olds and Bradley 1996).

Flies in the presence of G. queenslandicus and M. subbadius mites expressed several shared resistance behaviors in response to attack. Observations of fly-mite interactions within infestation chambers and observation vessels show that flies avoid mite attacks with two different forms of defensive behavior; a pre-contact avoidance behavior, often displayed by decamping from the substrate, and post-contact attempts to remove an actively attacking mite through by prying or flicking the mite away with the front legs. Four of the most commonly displayed behaviors (tarsal flicking, prying, substrate decamping and rolling) were expressed in similar frequencies by flies in response to attack by both species of mite. These defensive behavioral traits may act as generalized defensive traits against multiple forms of physical attack, evidenced by their expression by flies in the presence of both ectoparasites.

Across a variety of host species, wherever ectoparasite-host interactions are studied, grooming behavior is consistently linked with resistance to ectoparasitism

(Moller 1991; Mooring et al.1996; Eckstein & Hart 2000). Host grooming, a pre- attachment defensive behavior, has also been attributed to be a major cause for ectoparasite mortality in rodents (Hawlena et al. 2007). Thus, this component of my research showed that Drosophila may be added to the growing list of host species exhibiting active behavioral defenses elicited in response to ectoparasitic arthropods.

Response to Selection and Heritability

The current study found that additive genetic variation to resistance against the

ectoparasitic mite M. subbadius exists in the population of D. melanogaster derived from

Cape Tribulation, Australia. After 13 generations of artificial selection for increased pre- attachment defensive traits, both replicate lines of D. melanogaster expressed similar

rates of divergence in resistance, compared to controls. The resistance response did

not vary between the sexes, suggesting that resistance in this population is not sex-

linked (sex linkage is the expression of a trait that is related to the chromosomal sex of

the individual, Falconer and Mackay 1996). Although flies from my two selected lines

were relatively better at preventing contact and subsequent colonization of their bodies,

resistance is clearly not 100% effective, undoubtedly in part due to the low repeatability

of behavioral traits (Hoffmann 1999). Since there were no differences in mortality rates

between selected and control flies within control chambers (No-Mites), any differences

in mite loads and in-chamber mortality can be attributed ectoparasite resistance.

In addition to this study, heritable variation in resistance to natural enemies has been found consistently in D. melanogaster (Contamine et.al 1989, Lazzaro et. al 2006,

Vijendravarma et al. 2009). The degree of this heritable resistance varies across enemies and host species. Kraaijeveld & Godray demonstrated increased resistance, through encapsulation, in D. melanogaster artificially selected for resistance to the endoparasitoid, Asobara tabida . Encapsulation is a cellular immune defense mechanism utilized by a host insect in response to invasion by an endoparasitoid. Host haemocytes form an envelope around the invading organism, which can kill and prevent parasitism by the parasitoid (Strand and Pech 1995). Selected flies had a higher probability of encapsulating invading endoparasitoids, increasing from a rate of ~5% to

~60% after five generations (Kraaijeveld and Godfray 1997).

In my study, resistance is modeled as a threshold trait with an estimated heritability of 6%, which is below the range of values reported by Polak (2003) of 11 to

18%. However, direct comparison of heritability can be misleading since selection was not made upon a single trait or set of specific behavioral responses, so that the traits selected for between the two species may have been different, contributing to the difference in heritability estimates.

Mechanisms of Defense

In the present study, pre-attachment forms of defense against ectoparasites were artificially selected for; however, it is unknown precisely which specific mechanisms of defense were selected. Although these defenses are presumed to have behavioral components to resistance, the data suggests that resistance cannot be attributed solely

to any generalized increase in activity or presence of hyperactivity. However, pre- attachment forms of ectoparasite resistance, such as tarsal flicking or increased grooming behavior, are methods potential host organisms can use to prevent infestation before an attacking ectoparasite can begin feeding. Evidence for heritable variation to pathogens in Drosophila comes mainly from studies on endoparasites and bacterial infections, where defense mechanisms of potential hosts are generally biochemical and physiological in nature (Fellowes & Godfray 2000).

There are several behavioral elements that could be the source of the significant response to selection I detected. It has been suggested that potential host organisms can regulate ectoparasite infestation by using two specific models, the ‘Programmed

Grooming Model’ (Hart et al. 1992, Mooring 1995), in which an organism will periodically groom to remove ticks and other ectoparasites before they can begin feeding. Selection upon this model should increase general grooming behavior even in the absence of mite stimuli.

The stimulus-driven model suggests that grooming is initiated by ectoparasite contact with the host organism, causing an increase in grooming behavior (Riek 1962,

Wikel 1984). Increased resistance to ectoparasitism could be a result of a more acute visual identification and response to attack, or, as suggested by Polak (2003), resistance could be caused by a heightened response to tactile stimulus, resulting in decreased response time when contacted by attacking mites.

Resistance against Gamasodes

The resistance behaviors utilized by D. melanogaster to avoid infestation against

M. subbadius , also impart increased resistance to the mite G. queenslandicus . It has been argued that in pathogen-rich environments, where hosts are likely to encounter novel infections, selection should increase immune response and therefore decrease the likelihood of novel infection (Corby-Harris and Promislow 2008). The data presented in this work support this argument.

Several ecologically important questions arise once the presence of variable heritability in any defensive trait is observed. Is the increased resistance selected for in this experiment costly? Life-history theory (Roff 1997) suggests that if there are

physiological costs associated with resistance traits, then resources invested into

defense may be traded for expression into other fitness-related traits, such as fecundity

and longevity (Gwynn et al. 2005; Fellowes et al. 1999). Consequently, in the absence

of parasitism, resistant genotypes should be negatively selected because of their

reduced relative fitness (Boots and Begon 1993). Kraaijeveld & Godfray (1997) showed

that lines of D. melanogaster selected for resistance against parasitoid wasps suffer a

reduction in competitive ability in the larval stage, as well as decreased survivability

under conditions of intermediate and severe intraspecific competition (Fellowes et al.

1998).

D. nigrospiracula selected for behavioral resistance to M. subbadius have also been shown to have decreased fecundity even in the absence of parasitism (Leung and

Polak 2007). Inbred populations have consistently been shown to have an increased susceptibility to parasitism (Arkush et al. 2002; Leung and Polak 2007). Leung and

Polak (2007) demonstrated that inbred flies exhibited lower stamina than outbred flies, resulting from increased homozygosity which compromises the ability of flies to mount

sufficient defenses to resist mite attack. Interactions between fly and mites bear an energetic cost, findings that lend support to a direct link between fly endurance and energetics and the ability to defend itself against ectoparasites.

The data presented here indicate that possessing traits used to defend against

multiple ectoparasites can potentially help decrease the costs associated with

parasitism, especially within populations found in areas with higher densities of

parasites or increased pathogen richness, where there is an increased likelihood of

infection. However, it has been shown in D. nigrospiracula that resistance bears fitness

costs (Luong & Polak 2007, Rashed et. al 2008), possessing the genes responsible for

resistance could also be costly.

In addition to being one of the few studies to demonstrate heritable genetic

variation of resistance to parasites within natural populations, this study also seeks to

understand the general mechanisms used to resist infestation, as well as understand

the full range of resistance these defenses provide. By eliminating an increase in

general activity rates of resistant flies, future studies can focus on directly on individual

behavioral responses, which could be the source of the increased resistance in selected

flies. This study attempts to not only search for the presence of heritable resistance to

ectoparasitism in D. melanogaster , but through careful experimental design has also

discounted the potentially confounding effects of differential mortality rates and

inbreeding between selected and control flies as probable sources of the variation in

resistance. The work presented here is also one of the only studies to present heritable

resistance to multiple parasites.

Future studies can utilize the information presented here to explore any fitness

costs associated with possessing increased immunity to multiple potential ectoparasites.

It also opens the door to investigate the potential genes responsible for any defensive behaviors resistant flies possess that control flies do not. Identification of the mechanisms of defense, and the genes associated with resistance, may further our knowledge of why these traits are maintained in a variable state in natural populations.

CONCLUSIONS:

Overall, my work can be summarized as follows:

1. Drosophila melanogaster defends itself against attack by Macrocheles mites by

decamping from the substrate, using its limbs to pry off an attacking mite, tarsal

flicking to dislodge mites, and rolling onto its back to remove advancing mites.

2. There is heritable genetic variation for ectoparasite resistance in D. melanogaster

sampled from nature at Cape Tribulation, northeastern Australia.

3. Defensive behaviors elicited by Macrocheles subbadius are similar to those

elicited by Gamasodes queenslandicus, the natural ectoparasite of these flies.

Thus, the pre-attachment traits artificially selected in the present study may be a

generalized response to other natural enemies, at least to other ectoparasitic

mites.

4. The defenses used by flies and selected for using artificial selection in laboratory

chambers cannot be attributed to a generalized increase in basal activity levels,

although my results must be interpreted cautiously because of the low statistical

power owing to low sample sizes.

5. This study acts as an important first step toward fully understanding invertebrate

resistance traits, and toward explaining how genetic variation in resistance may

be maintained within populations of D. melanogaster .

6. The evidence presented increases our knowledge of behavioral responses

insects may utilize to defend themselves against ectoparasites, and possibly

pathogens that may be vectored by these mites.

7. The information contained herein may be used to better understand the

evolutionary potential of parasitism in nature.

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Figure 1: Side (A) and Cross-section (B) of infestation chamber in which flies are exposed to mites. (Polak 2003)

Figure 2. Mean Frequency of flicks (mean + SE) made by flies exposed to ectoparasitic mites compared to control flies. Numerals within bars are samples sizes, and P-values are from pair-wise t-tests.

Figure 3. Mean Frequency with which flies decamped from the substrate (mean + SE) when exposed to ectoparasitic mites compared to control flies. Numerals within bars are samples sizes, and P-values are from pair-wise t-tests.

Figure 4. Mean Frequency of prying behavior (mean + SE) made by flies exposed to ectoparasitic mites compared to control flies . Numerals within bars are samples sizes, and P-values are from pair-wise t-tests.

Figure 5. Mean Frequency of rolling (mean + SE) made by flies exposed to ectoparasitic mites compared to control flies . Numerals within bars are samples sizes, and P- values are from pair-wise t-tests.

Figure 6. Divergenc e in resistance to ectoparasitism by D. melanogaster after 13 generations of artificial selection. Divergence is calculated as the proportion of Infested Control flies minus Selected flies within infestation chambers.

Figure 7. Contrast in resistance to the ectoparasitic mite M. subbadius by D. melanogaster after 13 generations of artificial selection. Divergence is calculated as the proportion of Infested Control flies minus Selected flies within infestation chambers.

Figure 8. Difference in the proportion of Drosophila infested with G. queenslandicus mites between flies selected for 13 generations for increased resistance to the ectoparasitic mite Macrocheles subbadius and paired control flies.

Figure 9. Increase i n divergence with generations of selection for increased resistance to M. subbadius in S.D. units. Realized heritability of resistance in these lines of D. melanogaster was taken from the slope.

Table 1. Incidences of observed behaviors expressed by male D. melanogaster (taken from the base population) when exposed to mites. Flies were exposed for ten minutes or until infected. Control flies were observed in the absence of mites.

Behavioral Responses

Mite Type Trial Flicking Decamping Prying Rolling Exposure Time No Mites 1 0 2 0 0 518 No Mites 2 0 0 0 0 600 No Mites 3 0 5 0 0 345 No Mites 4 0 6 0 0 600 No Mites 5 0 1 0 0 590 No Mites 6 0 4 0 0 318 No Mites 7 0 3 0 0 600 No Mites 8 0 4 0 0 600 No Mites 9 0 1 0 0 233 No Mites 10 0 3 0 3 257 Gamasodes 1 0 3 28 1 518 Gamasodes 2 6 11 0 0 600 Gamasodes 3 11 7 33 2 345 Gamasodes 4 0 6 11 2 600 Gamasodes 5 0 2 0 0 590 Gamasodes 6 0 7 0 0 600 Gamasodes 7 9 8 29 1 318 Gamasodes 8 0 4 0 0 600 Gamasodes 9 21 10 31 4 233 Gamasodes 10 0 9 37 1 257 Macrocheles 1 6 6 28 0 511 Macrocheles 2 0 6 22 1 444 Macrocheles 3 1 7 11 0 557 Macrocheles 4 0 12 0 0 600 Macrocheles 5 0 8 0 1 600 Macrocheles 6 0 2 0 0 600 Macrocheles 7 0 5 0 0 600 Macrocheles 8 4 6 6 0 600 Macrocheles 9 0 3 17 0 413 Macrocheles 10 0 9 16 2 588

Table 2. Number found dead of D. melanogaster exposed within infestation chambers packed with nematode medium, and in the absence of mites. Exposure was for 24 hours.

Fly Mortality within No-Mite Chambers

Wing # found % found Chamber Sex Line Treatment Clip # of Exposed dead dead Difference 1 m 1 Selected Left 30 1 0.03 0.03 1 m 1 Control Right 30 2 0.07 2 f 1 Selected Right 30 0 0.00 0.00 2 f 1 Control Left 30 0 0.00 3 m 1 Selected Right 30 0 0.00 0.00 3 m 1 Control Left 30 0 0.00 4 f 1 Selected Left 30 1 0.03 0.00 4 f 1 Control Right 30 1 0.03 5 m 2 Selected Left 30 0 0.00 0.00 5 m 2 Control Right 30 0 0.00 6 f 2 Selected Right 30 0 0.00 0.07 6 f 2 Control Left 30 2 0.07 7 m 2 Selected Right 30 0 0.00 0.00 7 m 2 Control Left 30 0 0.00 8 f 2 Selected Left 30 0 0.00 0.00 8 f 2 Control Right 30 0 0.00

Table 3. Results of assays used to track divergence in resistance between control and selected flies against attack by ectoparasitic mites. Gen is the number of generations of selection from the base population before an assay. A difference in proportion is calculated as % infested Control - % Infested Selected.

Resistance Assays

# # infested % Infested # infested % Infested Mean of the Generation Line Rep Sex exposed Control Control Selected Selected Difference differences 3 1 1 m 10 2 0.2 3 0.3 -0.1 3 1 2 m 10 2 0.2 2 0.2 0 0.00 3 1 1 f 10 2 0.2 2 0.2 0 3 1 2 f 10 2 0.2 2 0.2 0 3 2 1 m 10 2 0.2 2 0.2 0 3 2 2 m 10 1 0.1 1 0.1 0 3 2 1 f 10 1 0.1 1 0.1 0 3 2 2 f 10 2 0.2 1 0.1 0.1 5 1 1 m 10 3 0.3 2 0.2 0.1 5 1 2 m 10 1 0.1 1 0.1 0 0.04 5 1 1 f 10 2 0.2 1 0.1 0.1 5 1 2 f 10 2 0.2 1 0.1 0.1 5 2 1 m 10 2 0.2 1 0.1 0.1 5 2 2 m 10 1 0.1 3 0.3 -0.2 5 2 1 f 10 1 0.1 0 0 0.1 5 2 2 f 10 2 0.2 2 0.2 0 7 1 1 m 10 4 0.4 1 0.1 0.3 7 1 2 m 10 6 0.6 4 0.4 0.2 0.16 7 1 1 f 10 3 0.3 3 0.3 0 7 1 2 f 10 8 0.8 4 0.4 0.4 7 2 1 m 10 2 0.2 2 0.2 0 7 2 2 m 10 7 0.7 6 0.6 0.1 7 2 1 f 10 10 1 7 0.7 0.3 7 2 2 f 10 7 0.7 7 0.7 0

Table 3. (continued)

Resistance Assays

# # infested % Infested # infested % Infested Mean of the Generation Line Rep Sex exposed Control Control Selected Selected Difference differences 9 1 1 m 20 12 0.6 5 0.25 0.35 9 1 2 m 20 13 0.65 7 0.35 0.3 0.24 9 1 1 f 20 7 0.35 8 0.4 -0.05 9 1 2 f 20 10 0.5 4 0.2 0.3 9 2 1 m 20 11 0.55 2 0.1 0.45 9 2 2 m 20 9 0.45 1 0.05 0.4 9 2 1 f 20 9 0.45 6 0.3 0.15 9 2 2 f 20 8 0.4 8 0.4 0 11 1 1 m 20 8 0.4 5 0.25 0.15 11 1 2 m 20 9 0.45 7 0.35 0.1 0.20 11 1 1 f 20 14 0.7 8 0.4 0.3 11 1 2 f 20 9 0.45 5 0.25 0.2 11 2 1 m 20 2 0.1 1 0.05 0.05 11 2 2 m 20 13 0.65 3 0.15 0.5 11 2 1 f 20 7 0.35 4 0.2 0.15 11 2 2 f 20 6 0.3 3 0.15 0.15 13 1 1 m 40 29 0.725 21 0.525 0.2 13 1 2 m 40 19 0.475 11 0.275 0.2 13 1 1 f 40 31 0.775 22 0.55 0.225 13 1 2 f 40 27 0.675 14 0.35 0.325 13 2 1 m 40 17 0.425 14 0.35 0.075 0.22 13 2 2 m 40 19 0.475 12 0.3 0.175 13 2 1 f 40 22 0.55 17 0.425 0.125 13 2 2 f 40 34 0.85 16 0.4 0.45

Table 4. Resistance to the mite Gamasodes queenslandicus by D. melanogaster selected for resistance to Macrocheles subbadius. Flies were exposed for 24 hours within infestation chambers packed with medium containing mites.

Chamber Line Treatment Sex # infested # exposed # Dead # Scarred Proportion Difference Mean 1 1 S m 0 20 8 0 0.4 0.45 1 1 C m 1 20 16 0 0.85 2 1 S f 0 20 11 4 0.75 0.15 2 1 C f 2 20 14 2 0.9 3 1 S m 1 20 10 0 0.55 -0.1 3 1 C m 0 20 8 1 0.45 4 1 S f 4 20 5 1 0.5 0.15 4 1 C f 5 20 8 0.65 0.16 5 1 S m 1 20 3 1 0.25 0.25 5 1 C m 0 20 6 4 0.5 6 1 S f 1 20 2 1 0.2 0.1 6 1 C f 0 20 5 1 0.3 7 1 S m 0 20 4 0 0.2 0.25 7 1 C m 4 20 4 1 0.45 8 1 S f 2 20 7 1 0.5 -0.1 8 1 C f 1 20 6 1 0.4 0.13 9 2 S m 0 20 11 3 0.7 0.15 9 2 C m 2 20 14 1 0.85 10 2 S f 2 20 7 1 0.5 -0.2 10 2 C f 3 20 1 2 0.3 11 2 S m 1 20 4 1 0.3 0.35 11 2 C m 4 20 7 2 0.65 12 2 S f 1 20 3 1 0.25 0.6 12 2 C f 0 20 14 3 0.85 0.23 13 2 S m 2 20 3 1 0.3 0.25 13 2 C m 0 20 9 2 0.55 14 2 S f 2 20 11 0 0.65 0.35 14 2 C f 2 20 13 5 1 15 2 S m 1 20 5 1 0.35 0 15 2 C m 2 20 1 4 0.35 16 2 S f 1 20 4 1 0.3 0.2 16 2 C f 5 20 1 4 0.5 0.20