Evolution and ecology of associations between Drosophila and their parasitic nematodes
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ProQuest Information and Learning 300 North Zeeb Road, Ann Artrar, Ml 48106-1346 USA 800-521-0600
EVOLUTION AND ECOLOGY OF ASSOCIATIONS BETWEEN DROSOPHILA
AND THEIR PARASITIC NEMATODES
by
Steven John Perlman
A Dissertation Submitted to the Faculty of the
DEPARTMENT OF ECOLOGY AND EVOLUTIONARY BIOLOGY
In Partial Fulfillment of the Requirements For the Degree of
DOCTOR OF PHILOSOPHY
In the Graduate College
THE UNIVERSITY OF ARIZONA
2002 UMI Number 3065509
A UMI
UMI Microfomi 3065509 Copyright 2002 by ProQuest Information and Leaming Company. Ail rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code.
ProQuest Information and Leaming Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 2
THE UNIVERSITY OF ARIZONA ® GBADDATE COLLEGE
As members of the Final Examination Committee, we certify that we have read the dissertation prepared by Stpvpn .Inhn Pprl.nan entitled Evolution and Ecology of a«;«;r>riafinnc Sotwoon
Drosophna and their paragitir npmatnrip^
and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy
Dlt? '
Wayni^Maddi son Date
Therese Markow %ate
Nanicyj^Twan Date V ( '?S T (Ij^ward Ochman Dat«V¥
Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to the Graduate College.
I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement.
f ^ / C Di^secifttion-J^ector Date John Jaenike 3
STATEMENT BY AUTHOR
This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.
Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgement of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgement the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained by the author.
SIGNED: 4
ACKNOWLEDGMENTS
I would first like to acknowledge my dissertation advisor John Jaenike for being a truly excellent mentor. He has always set an impressive example, both as a gentleman and a great biologist. I thank my committee - Wayne Maddison, Teri Markow, Nancy Moran and Howard Ochman, in particular for all of their support after John and the lab moved back to Rochester from Tucson. The following people made the lab a nice place to work - Irene Dombeck, Jake Russell, DeWayne Shoemaker, Kelly Dyer, Heather Mallory and Paula Campbell. Heather Mallory was a terrific helper and Kelly Dyer taught me a lot about molecular methods. Irene Dombeck was a great source of friendship and support. Jake Russell was my great sounding board and friend, both in Rochester and in Tucson. In Tucson, the following people were very supportive and helpful - Lacey Knowles, Allen Gibbs, Andrew Holyoake and Tamar Erez. I benefited greatly from discussions with Joe Watkins, Sergei Kosakov, Cheryl Vanier and David Maddison on phylogenetic and statistical methods. I learned a lot of nematology from Patricia Stock. 1 also thank the EEB and CIS staff for all of their help. I thank Andy Beckenbach, Jeff Birdsley, Irene Dombeck, Irene Eijs, Ray Huey, Masahito Kimura, Clyde Sorenson and Chuck Taylor for collecting insects, and Fern Forest Nature Center in Florida for permission to collect. David and Jaye Houle (Florida State U.) sheltered me and my flies in the days after Sept. II, 2001. Greg Spicer (San Francisco State U.) sequenced the Drosophila mtDNA, and DeWayne Shoemaker sequenced some of the nematode mtDNA while in the lab in Rochester. My research was generously funded by the following: at the U. of Arizona - the Center for Insect Science, the NSF Research Training Grant in the Analysis of Biological Diversification, the department of Ecology and Evolutionary Biology, and the Flinn Foundation (through the Mathematics department), as well as the American Museum of Natiural History and National Science Foundation grants to John Jaenike. I thank friends and family scattered far and wide, for making me feel very loved. Finally, I acknowledge Stephanie Weinstein, who makes everything wortliwhile. 5
TABLE OF CONTENTS
I. LIST OF ILLUSTRATIONS 6
II. LIST OF TABLES 9
III. ABSTRACT II
IV. CHAPTER I: INTRODUCTION 13
V. CHAPTER 2: PRESENT STUDY 24
VI. REFERENCES 30
APPENDIX A. .ASSOCIATIONS BETWEEN MYCOPHAGOUS DROSOPHIU
AND THEIR HOWARDUL-i NEMATODE PARASITES : A WORLDWIDE
PHYLOGENETIC SHUFFLE 34
APPENDIX B. POTENTIAL HOST RANGES OF DROSOPHIU - PARASITIC
NEMATODES: .AN EXPERIMENTAL PHYLOGENETIC .ANALYSIS 78
•APPENDIX C. EVOLUTIONARY DYNAMICS OF VIRULENCE IN DROSOPHIU
.AND THEIR PARASITIC NEMATODES 123
.APPENDIX D. COMPETITIVE INTERACTIONS AND PERSISTENCE OF
TWO NEMATODE SPECIES THAT PARASITIZE DROSOPHIU RECENS... 163
.APPENDIX E. ACTUAL VERSUS POTENTIAL HOST RANGE OF A PARASITIC
NEMATODE OF D/?050P///iL4 193 6
LIST OF ILLUSTRATIONS
Figure A.l, Neighbor-joining ITSl tree of the Howardula ciade termed 'Drosophila
parasite ITSl' 71
Figure A.2, Maximum likelihood 18S tree of Howardula 72
Figure A.3, Number of first, second and third position transitions and transversions as
a function of pairwise maximum likelihood genetic distances for mtDNA Howardula
sequences 73
Figure A.4. Maximum likelihood mtDNA tree for the 'Dro^op/z/Za-parasite ITSl'
Howardula clade 74
Figure A.5. Maximum likelihood tree using Drosophila COL II and III sequences 75
Figure A.6, Distribution of known nematode parasitism in the quinaria and testacea
species groups 76
Figure A.7, Associations of Howardula nematodes and Drosophila species 77
Figure B. 1. Infection success for European and North American H. aoronymphium,
Japanese H. cf. aoronymphium. P. nearcticus, H. neocosmis, and Howardula sp. F. 111
Figure B.2, Infection success (relative to that in the standard host species) plotted
against the genetic distance between the standard and experimental host species ... 112
Figure B.3, Host suitability to infection by a parasite as a function of the genetic
distance between the natural parasite of the host species and the test parasites 113
Figure B.4, Nematode motherworm sizes for A) North American H. aoronymphium,
B) Japanese H. cf. aoronymphium, C) H. neocosmis, and D) Howardula sp. F 114 7
Figure B.9, Fecundity of females infected with nematodes, for A) North American H.
aoronymphium, B) Japanese H. cf. aoronymphium, C) H. neocosmis, D) P. nearcticus,
E) Howardula sp. F 118
Figure C.1, Natural associations between the four D. testacea group flies and their
nematode parasites 156
Figure C.2, Mean number of mature eggs carried by female flies as a function of
infection status 157
Figure C.3, Effect of motherworm number on host female fecundity 158
Figure C.4. Survivorship of infected and uninfected flies 159
Figure C.5. Effect of motherworm number on host adult survivorship, for two host
parasite combinations 160
Figure C.6. Number of offspring sired by infected and uninfected testacea group
males 161
Figure C.7, Model of the evolution of virulence in testacea/aoronymphium
associations 162
Figure D. I, Number of mature eggs in one-week old female Drosophila recens,
of varying infection status 189
Figure D.2, Mean survival of adult Drosophila recens, of var>ing infection status. 190
Figure D.3, Mean size of H. aoronymphium motherworms in one-week old Drosophila
recens, in single and mixed infections 191
Figure D.4, Number of Parasitylenchiis nearcticus F1 offspring in single and mixed
infections 192 8
Figure E.l, Survival of infected and uninfected D. cardini and D. acutilabella .... 218
Figure E.2, Mean number of D. cardini and D. acutilabella emerging from single and
mixed jar treatments 219 9
LIST OF TABLES
Table A. I, Nematodes included in phylogenetic analysis 65
Table A.2, Drosophila species included in the study and their Howardula parasites 67
Table B. I, Nematodes included in experimental infections and phylogenetic analysis
106
Table B.2, Drosophila species included in the study and their Howardula 107
Table C.l, Number of motherworms per fly in experimental infections 149
Table C.2, Female fecundity as a function of host species, infection treatment and
host species by treatment interaction 150
Table C.3. Adult mortality as a function of host species, infection status, and a host
species by infection status interaction 151
Table C.4, Fertility of D. testacea group males as a function of infection status ... 152
Table C.5. Effect of H. aoronymphium nematodes on fertility of wild D. neoiestacea
males 153
Table D.l, Percent infection by Howardula aoronymphium and Parasitylenchus
nearcticus and relative density of H. aoronymphium motherworms in Drosophila
recens 186
Table D.2, Howardula aoronymphium and Parasitylenchus nearcticus infection rates
as a function of host sex, infection density, infection with or without the other
nematode species, and replicate vial 187
Table E.l, Prevalence of H. neocosmis in wild-caught Florida flies 213
Table E.2, Relative densities and motherworm sizes of H. neocosmis in experimental 10
LIST OF TABLES - Continued
infections of strains of D. acutilabella and D cardini 214
Table E.3, Motherworm number as a function of host species, strain, sex and vial 215
Table E.4, Motherworm size as a function of host species, strain, sex and
motherworm number per fly 216 11
ABSTRACT
In this dissertation, the evolutionary and ecological determinants of host range of nematode parasites (Tylenchida: Allantonematidae: Howardula, Parasitylenchus) of mushroom-breeding Drosophila (Diptera: Drosophilidae) are examined. These nematodes are horizontally transmitted, obligate parasites, often with severe effects on host fitness.
Phylogenetic analysis of Drosophila and Howardula DNA sequences shows little congruence between host and parasite phylogenies. with frequent host colonizations and
losses. Dro5op/j/7a-parasitic Howardula are not monophyletic. with host switches occurring between Drosophila and distantly related mycophagous sphaerocerid flies.
Molecular analysis reveals eight new Howardula species.
The ability of five nematode species to infect and develop in 24 ta.\onomically
diverse Drosophila species is assessed. All nematode potential host ranges but one are
large, even for host specialists. Novel hosts that are distantly related from the native host
are less likely to be infected, but among closely related hosts there is variation in
susceptibility. Potential host ranges differ greatly between related parasite species. Most
novel infections do not cause reductions in host fecundity, with the exception of P.
nearcticus. Thus, Drosophila-nematode associations are dynamic, and appear to be
driven by a combination of repeated opportunities for host colonization due to shared
mushroom breeding sites, and large nematode potential host ranges.
Recent colonization of novel host species may explain the striking differences in
virulence observed in natural Drosophila-nematode associations. For example, Nearctic 12
species of the Drosophila testacea group are more severely affected by infection than
Palearctic species, including complete female sterility. Cross-infection experiments reveal that virulence is evolutionarily labile in testacea-Howardiila associations, and that high levels of virulence manifested in some host-parasite combinations are due to a lack of host resistance.
Finally, ecological determinants of host range are considered. First, competition between a generalist and a specialist parasite of D. recens is assessed by comparing nematode infection success and reproduction in single and double infections. Second, differences between the actual and potential host ranges of Howardula neocosmis are documented. This parasite appears restricted to D. acutilabella in nature, yet successfully
parasitizes the closely related, microsympatric D. cardini. Neither differential virulence
nor competitive interactions between hosts explain the host range differences. 13
CHAPTER 1: INTRODUCTION
It is now recognized that parasites can have profound effects on the evolution and ecology of their hosts (Little 2002), and on the structure of ecological communities.
E.xamples of the importance of parasites are widespread, including large scale outbreaks
of pathogens and their effects on host population dynamics and community structure (e.g.
Kohler and Holland 2001), the success of various biological control programs (e.g.
Bedding 1984), and the high levels of nucleotide diversity in host genes that affect
resistance to parasitism (e.g. Hedrick 1994).
Whether parasites will have both short (i.e. ecological) and long-term (i.e. genetic,
and therefore evolutionary) effects on their hosts will depend on five important factors.
First, parasites must negatively affect host fimess. Second, a significant proportion of
hosts must be infected; infection prevalence of infection will strongly depend on the
mode and efficiency of u-ansmission. Third, the nature of genetic variation in the host's
ability to resist parasitism, and in the parasite's ability to counter-resist hosts must be
considered. Fourth, the age of association must be assessed. And finally, the host range
of a parasite will strongly affect the long-term outcome of an association. Of these five
factors, host range is probably the least understood, and is the main focus of this
dissertation. The goal of this dissertation is to understand what determines host range in
parasitic nematodes of Drosophila flies. We still have little understanding of why
parasite species are found on certain host species and not others. This question is
particularly timely, considering the growing importance of emerging diseases, i.e. those
that have recently colonized new hosts as a consequence of changing environmental 14
conditions (Daszak et al. 2000). Often our knowledge of host range is restricted to lists of parasite species found on hosts.
The most important ecological consequence of host range is parasite-mediated competition. A parasite that is more virulent to some host species than others can affect
the structure and composition of whole communities, by either maintaining or excluding
certain hosts (Park 1948; Price et al. 1986; Kohler & Wiley 1997; Tompkins et al. 2001).
For e.xample. a parasite can contribute to the coe.xistence of two hosts if it is more
virulent to the one that is a better competitor. In addition, a virulent parasite can e.xclude
a host if it is maintained in another species in which it is more benign. This has
important conservation implications, as parasites can increase the risk of extinction of
endangered species if they are maintained in another host which serves as a reservoir (van
Riper et al. 1986; McCallum and Dobson 1995).
Parasite host range also has a number of important evolutionary consequences, the
most important of which relates to host specificity. A parasite's population genetic
structure will be largely determined by the genetic structure of its hosts (Johnson et al.
2002). Thus, the potential for tight coevolutionary interactions will be weakest for a
host-generalist parasite, as host-specific adaptation will be restricted in parasites that
continually encounter different hosts (Jaenike and Dombeck 1998). For example,
multiple host species may explain departures from optimal virulence (Jaenike 1996a;
Davies et al. 2001). However, genetic correlations in adaptation to multiple hosts will
affect the strength of diffuse coevolution (coevolution involving more than two species)
(Iwao and Rausher 1997). 15
A parasite's actual host range is determined by two distinct components. First, a host must be intrinsically suitable for parasite infection, development and transmission
(Solter and Maddox 1998). This intrinsic suitability defines a parasite's potential host range, and is determined by physiological and biochemical properties of the host. A parasite's actual host range, however, is a subset of its potential host range and is determined by a number of ecological factors which can restrict the use of what would otherwise be a perfectly suitable host. Ecological factors that can limit a parasite's actual host range include geography, competing parasite species (Dobson 1985), and host density (Anderson and May 1978). The recent, widespread movement of organisms among and between continents is providing many opportunities for the establishment of new host - parasite associations. These emerging diseases are parasites with broad potential host ranges but which previously lacked the ecological opportunities to colonize novel hosts. Identifying the factors other than sympatry that both permit and prevent the successful establishment of novel associations presents a clear challenge to biologists.
The goal of this dissertation is to understand both the evolutionary and ecological determinants of host range of parasitic nematodes of mushroom-breeding Drosophila. In particular, this study is one of the first to examine the role of host and parasite phylogeny in determining a parasite's potential host range. My study asks the following specific questions. 1) What is the history of associations between Drosophila and their nematode parasites? Are they based on long-term lineage-specific associations, or has there been recurrent establishment of new associations and/or disappearance of existing ones? 2)
What is the relationship between a parasite's potential and actual host ranges? How 16
important are host and parasite phylogeny in determining the ability of a parasite to successfully infect a novel host? 3) What determines the expression of virulence in current and novel host-parasite associations? 4) How does competition between different nematode species affect host associations in nature?
Mushroom-feeding Drosophila and their parasitic nematodes
Mushroom-feeding Drosophila, particularly from the quinaria and testacea species groups, are among the most abundant visitors to decaying mushrooms in temperate and boreal forests, and are highly polyphagous (e.g Kimura and Toda 1989;
Wertheim et al. 2000). These flies are commonly infected by Howardida and
Parasitylenchus allantonematid nematodes. The Drosophila-ncmaiode association is an ideal model for studying host-parasite evolution, particularly the determinants of host
range, for several reasons. First, the nematodes are obligate parasites with direct life
cycles (i.e. no intermediate hosts). They often have severe effects on host fitness, causing
reductions in female and male sterility, adult survival, and male mating success (Jaenike
and Perlman 2002). Each moiherworm developing within a fly represents an independent
successful infection, such that rates of parasitism can be easily measured in the field and
lab. Both hosts and parasites can be maintained in the lab, and experimental infection
assays have been developed. Finally, there is much variation in both host range and
virulence of Drosophila-ncmatode associations in the wild.
Inseminated female Howardula nematodes infect fly larvae by piercing through
their cuticle with a stylet (Welch 1959). When the adult fly emerges, the nematode 17
begins releasing infective juveniles into the hemocoel of the fly, which are shed from the ovipositor and anus of the fly onto new mushrooms, where the nematodes mate and continue the cycle. The life cycle of Parasitylenchus is similar, except that it has an additional sexual generation inside the fly (Siddiqi 2000). Motherworms produce a few offspring that first mate inside the fly and then produce infective juveniles which are shed to mushrooms. Howardula and Parasitylenchus transmission thus requires survival of
the host. Unlike most parasitoids and many herbivorous insects, in which host
acceptance and development in that host are carried out by the mother and her offspring,
respectively (e.g. van Klinken 2000; Morehead and Feener 2000). in Howardula and
Parasitylenchus^ the same individuals (inseminated females) are responsible for both
infection of the host and successful development inside it. This greatly simplifies
considerations on the evolution of host range.
Because multiple adult Drosophila, belonging to several species (Jaenike and
James 1991), can emerge from the same individual mushrooms, there are many
opportunities for horizontal transmission of parasites both within and among host species.
This has important consequences for the host range and virulence of the nematodes.
First, the high potential for colonization of novel host species suggests that host switches
should be an important force in the evolution of Drosophila-ncmdlodc associations.
Second, because of the opportunities for horizontal transmission of the nematodes, we
would expect the nematodes to significantly reduce host fecundity (Jaenike 1996a). This
is because parasite reproduction will occur at the expense of host reproduction, with host
aggregation within mushrooms ensuring a pool of potential hosts. 18
Phylogenetic determinants of host range
Although there are extensive records of actual host-parasite associations in nature, there have been few experimental studies of the phylogenetic determinants of host range.
These have generally shown that the ability of a parasite to infect a novel host species decreases with host genetic distance (Futuyma et ai. 1995; Moore and Gotelli 1997; Reed and Hafner 1997; Nishigushi et al. 1999; Morehead and Feener 2000). This is not surprising, as distantly related hosts are likely to represent an environment very different from that to which the parasites are adapted. These studies however, have considered only a small number of potential host species, and have been usually restricted to only one or rvvo parasite species.
It is unclear, however, how host suitability for parasitism will decline as a function of genetic distance from the natural host. If infection is characterized by rapid biochemical and physiological evolution by parasites and counter-evolution by hosts, then closely related host species will show significant variation in susceptibility to specific parasites. For example, the ability of Leishmania trypanosomes to successfully
infect their phlebotomine sand fly hosts appears to be largely determined by rapidly evolving and variable cell surface phosphoglycan molecules (Sacks 2001). .Among
closely related phlebotomine sand flies that harbor Leishmania. phylogeny would
therefore not be a good predictor of host switching potential. Clearly, whether or not
phylogeny is an important determinant of infection success will strongly depend on the
mechanisms and evolutionary rates of host resistance and counter-resistance by the
parasites. 19
Another approach to understanding the potential for host switching by parasites has been to compare the phylogenies of hosts and parasites. Indeed, a major recent goal of comparative biology has been to determine whether phylogenies of associated organisms, such as hosts and parasites or symbionts (Huelsenbeck et al. 1997, 2000), and plants and pollinators or herbivores (Farrell and Mitter 1990; Weiblen 2001), are generally congruent with each other. Congruence indicates that such associations transcend speciation events and are, therefore, relatively old. and that hosts and parasites are cospeciating. Many factors can lead to incongruence of host and parasite phylogenies. Including colonization of new host species (host switching events), parasite e.xtinction or release of certain hosts or host populations from parasitism (sorting events), and parasite speciation in the absence of host speciation (duplication events) (Page 1994).
Such a comparison can be used to estimate the frequency of host switches and to identify
host lineages that are prone to colonization, as well as the degree to which such host shifts are constrained by phylogeny (Huelsenbeck et al. 2000; Ricklefs and Fallon 2002).
Thus, phylogenies that are highly congruent with each other indicate that little or no host- switching has occurred over e.xtended periods of time.
Phylogenetic analysis is also useful in identifying important factors that determine
host switching. For example, Becerra (1997) showed that plant chemistry was a better
predictor of host switching by specialist herbivorous chrysomelid beetles than plant
phylogeny. Tompkins and Clayton (1999) recently showed that barb size in the feathers
of swifilets was a better predictor of host suitability for louse parasites than host 20
phylogeny. Thus, in these cases, specific attributes of the hosts, rather than overall genetic similarity, govern a parasite's host range.
One of the few generalities to have emerged from studies of host-parasite cospeciation is that the most important determinant of congruence is parasite transmission mode (Herre et ai. 1999). Most cases of phylogenetic congruence between hosts and symbionts are those where the symbiont is vertically transmitted (i.e. from mother to offspring) within the host population (but see Nishiguchi et al. 1998). For example, some microbial endosymbionts of organisms such as aphids and deep sea clams e.xhibit congruent phylogenies with their hosts (Moran et al. 1995; Peek et al. 1998).
Congruent phylogenies can also arise when the parasite has very little opportunity for dispersal to new hosts, as has been found in chewing lice parasites of vertebrates such as pocket gophers (Hafner and Nadler 1988). However, even these highly restrictive kinds of associations can depart from cospeciation, as evidenced by the lack of phylogenetic congruence in the vertically transmitted Wolbachia, which are bacterial reproductive parasites of arthropods (Werren et al. 1995), and chewing lice of seabirds (Paterson et al.
2000).
The majority of parasites however, including Dr050/7/i/7a-parasitic nematodes, are horizontally transmitted (i.e not strictly from mother to offspring), and there are several reasons why their phylogenies are unlikely to be congruent with those of their hosts.
Many parasites infect more than one host species (Woolhouse et al. 2001).
Ta.xonomically related parasites can infect distantly related hosts (Siddiqi 2000), which can only occur via host switching events. Novel host-parasite associations in the wild. 21
such as emerging diseases, are also increasingly common (Daszak et al. 2000).
Nevertheless, all parasites, even horizontally transmitted ones, must successfully locate, infect, develop and reproduce within their hosts. This would seem to require such a degree of host-specific adaptation that one would expect parasites to be restricted to certain host lineages, with few switches to distantly related hosts. Thus, a comparison of host and parasite phylogenies can be used to estimate the frequency of host switches in horizontally transmitted parasites, as well as the degree to which such shifts are constrained by host phylogeny (Ricklefs and Fallon 2002). In conjunction with experimental infections (Nishiguchi et al. 1998), this approach may also help us to
understand what determines the origin and fate of novel infections. It is this approach - a
combination of experimental infections in a phylogenetic context, that is taken to
understand the evolution of associations between Drosophila and their horizontally
transmitted parasitic nematodes.
E.\planation of thesis format
The research in this dissertation examines the evolutionary and ecological
determinants of host range in parasitic nematodes of Drosophila flies. The dissertation
consists of five manuscripts which are included as appendices.
In Appendix One, Howardula parasite diversity is assessed by molecular
characterization of nematodes dissected from wild-caught flies. I reconstruct the
phylogenetic relationships of the Drosophila hosts and their Howardula nematodes, using
mitochondrial and nuclear ribosomal DNA sequences. I ask whether host and parasite 22
phylogenies are congruent, and if Dro5opA/7a-parasitic Howardula are monophyletic.
This work was done in collaboration with Greg Spicer at San Francisco State U.. who sequenced the Drosophila mtDNA, and DeWayne Shoemaker who provided eight of the
Howardula mtDNA sequences, while at the University of Rochester. I obtained all of the
remaining DNA sequences and did all of the phylogenetic analysis.
In Appendi.x Two. 1 explore the role of host and parasite phylogeny in
determining a parasite's potential host range, by infecting 24 taxonomically diverse
Drosophila species with five nematode species. 1 compare parasite infection success,
motherworm size (as an indirect measure of parasite fitness), and host female egg number
(as a measure of parasite virulence) in native and novel hosts.
In Appendix Three, I use experimental cross-infections to examine what
determines virulence in the associations between flies in the Drosophila lesiacea group
and their parasites in the Howardula aoronymphium species complex. 1 measure the
effects of both native and novel parasites on multiple components of fitness - female
fecundity, male fertility, and adult survival.
In Appendix Four. I ask what is the role of competition between parasite species
in shaping host range patterns. I compare parasite infection rate, reproduction, and
virulence of two parasites of D. recens - H aoronymphium, a host generalist, and P.
nearciicus, a specialist known only from this host species in the wild, in single and in
mixed species infections.
In Appendix Five, I use a combination of field collection and experimental
infections to document differences between the actual and potential host ranges of a 23
recently described parasite, Howardula neocosmis. This nematode has been collected in
D. aciitilabella^ but not in the closely related and microsympatric D. cardini. However, I show that D. cardini is a perfectly suitable host for this nematode in the lab. 1 then ask whether these host range differences are due to differential virulence or to competitive interactions between the two host species. 24
CHAPTER TWO: PRESENT STUDY
The methods, results and conclusions of this study are presented in the papers appended to this thesis. The following is a summary of the most important findings in these papers.
Appendix One. Associations between mycophagous Drosophila and their Howardula nematode parasites: a worldwide phylogenetic shuffle
One goal of macroevolutionary studies of hosts and parasites has been to determine the degree to which the evolutionarj' histor>' of parasites depends on that of their hosts (Huelsenbeck et al. 1997). These studies have been mostly restricted to situations where parasites have little opportunity for dispersal or horizontal transmission
(e.g. Hafner and Nadler 1988); congruence between host and parasite phylogenies is
therefore very high, indicating that these associations transcend speciation events and are
thus evolutionarily old. In Appendi.x One, I use phylogenetic analysis of host and
parasite DNA sequences to characterize the associations between mushroom-breeding
Drosophila and their Howardula nematodes, and to assess nematode diversity.
Drosophila mitochondrial DNA (subunits I, II and III of cytochrome c o.xidase) and
Howardula mitochondrial (cytochrome c oxidase subunit I) and nuclear ribosomal (18S
and ITSl) DNA sequence was analyzed. This work was done in collaboration with Greg
Spicer at San Francisco State University, who sequenced the Drosophila mtDNA.
DeWayne Shoemaker, a researcher in the Jaenike lab at the University of Rochester,
contributed eight of the Howardula mtDNA sequences to the study. 25
Molecular analysis reveals eight new species of Howardula. Host and parasite phylogenies are highly incongruent, despite the fact that many of the nematodes are ecological host specialists, known from only one host in the wild. Associations between
Drosophila and Howardula are characterized by many, sometimes rapid, host switches.
Dr050/7/i/7a-parasitic Howardula are not monophyletic, with host switches occurring between Drosophila and distantly related mycophagous sphaerocerid flies. There is also evidence for some phylogenetic association between parasites and hosts, with some nematode clades associated with certain host lineages. Thus. Drosophila-Howardula associations are dynamic, and appear to be driven by a combination of repeated opportunities for host colonization due to shared mushroom breeding sites, and large nematode potential host ranges.
.Appendix Two. Potential host ranges of Dro5op/://a-parasitic nematodes: an experimental phylogenetic analysis
While there is much data on the actual host ranges of parasites in nature, very little is known about their potential host ranges, in particular the conuibution of host phylogeny to a parasite's capacity to colonize a new host species. Given the importance of host colonization evident in Drosophila and nematode associations. I assessed the current host-switching potential of these nematodes. In Appendix Two. I report the results of controlled experimental infections of 24 mushroom-breeding Drosophila that are widely distributed taxonomically, with five different nematode species. I quantified nematode infection success, motherworm size (as a measure of parasite fitness inside 26
hosts), and the effect of nematodes on female host fecundity (as a measure of parasite
virulence) in novel hosts. This is one of the only studies to assess the importance of both
host and parasite phylogeny in determining a parasite's potential host range.
The potential host ranges of all of the nematodes are much larger than their actual
host ranges in nature, even for parasites with only one known host species in the wild.
Novel hosts that are distantly related to the native host are much less likely to be infected.
However, among closely related hosts, there is variation in susceptibility to specific
parasites. In addition, potential host ranges differ greatly between related parasite
species. All nematode species that successfully infected novel hosts produced infective
juveniles in these hosts. Most novel infections did not result in significant reductions in
the fecundity of female hosts, except for P. nearcticus. This host specialist significantly
reduced the fecundity of all novel host species it successfully infected, sterilizing all
quinaria group hosts, only one of which is a host in nature.
Appendix Three. Evolution of virulence in associations between Drosophila and their
parasitic nematodes
Parasite virulence plays a crucial role in shaping patterns in host range. Together
with rates of transmission, virulence determines the prevalence of infection within
populations, thus potentially driving host population dynamics and individual selection
pressures (Anderson and May 1978; Sasaki and Godfray 1999; Kohler and Holland
2001). High levels of pathogenicity can prevent a parasite from becoming established in
a novel host, or can severely depress populations of one host, while another less sensitive 27
host species serves as a reservoir. Such differential virulence can permit the coexistence of different host species (i.e. via parasite-mediated competition) (Price et ai. 1986).
Given the frequent and potentially rapid colonization and turnover of host
associations in Drosophila-^ssd&xiic. nematodes, what determines patterns of virulence in
both current and novel infections? While most of the novel infections in this study result
in either only modest, or no reductions in host fecundity, some novel infections result in
complete sterility (Appendix Two). In Appendix Three. I show through a detailed
analysis of associations between the Drosophila testacea group and nematodes in the H.
aoronymphium group that virulence can be highly labile. Host species show striking
differences in their responses to infection by their native nematodes, with males and
females of some species rendered sterile. E.xperimental cross-infections reveal that
female sterility is due completely to the host, with species that are sterilized by their local
nematodes sterilized by the other nematodes in the H. aoronymphium group as well.
However, reductions in host survival depend on both the host and the parasite, with some
novel infections resulting in drastically reduced adult lifespan. High levels of parasite
virulence manifested in some host-parasite combinations appear to be due to a lack of
resistance in the hosts, perhaps as a result of recent host colonization by Howardula.
Appendix Four. Competitive interactions and persistence of two nematode species that
parasitize Drosophila recens
While intrinsic host suitability (i.e. a parasite's potential host range) acts as the
primary filter through which successful infections can occur, a number of ecological 28
factors will restrict what would otherwise be a perfectly suitable association in nature.
For example, the presence of other competing parasite species can limit the distribution of parasites in nature. Parasites that share the same host species can reduce each other's densities through interspecific competition, either directly, via antagonistic interactions within the same individual host (i.e. interference competition), or indirectly, via reductions in host density (i.e. exploitation competition) (Dobson 1985). Because different species of mushroom-feeding Drosophila often share the same individual mushrooms, and because host switches are common, there is great potential for different parasite species to share hosts. Indeed, Drosophila species, such as D. recens and D. falleni, are known to harbor more than one nematode species (Jaenike 1996b; Poinar et al. 1997).
In Appendix Four. I assess the role of parasite competition on host range by performing controlled single and double infections of D. recens with its two natural parasites, the generalist H. aoronymphium, and the specialist P. nearciicus, which is only known from this host in nature. P. nearciicus has both higher rates of infection and reproduction than H. aoronymphium, completely sterilizing females in single and double
infections. In double infections, P. nearciicus is competitively superior, as H.
aoronymphium motherworms are significantly smaller than in single infections. Thus P.
nearciicus might competitively exclude H. aoronymphium if D. recens were the only host
available. The generalist H. aoronymphium is probably only maintained in D. recens due
to transmission from other, more suitable host species. 29
Appendix Five. AcUial verus potential host range of a parasitic nematode of Drosophila
Finally, in Appendix Five, I document differences between the actual and potential host ranges of H. neocosmis, a recently described nematode from Florida
(Poinar et al. 1998). This parasite is only known in nature from D. acuiilabella, yet it can successfully infect and develop in the closely related and microsympatric D. cardini. 1 experimentally show that the host range differences are probably due neither to differential parasite virulence nor to competitive interactions between hosts. It is more likely that the difference in prevalence is due to non-overlapping D. acuiilabella and D. cardini host breeding sites. 30
REFERENCES
Anderson, R. M. and R. M. May. 1978. Regulation and stability of host-parasite population interactions. I. Regulatory processes. J. Animal. Ecol. 47:219-247.
Becerra, J. X. 1997. Insects on plants: Macroevolutionary chemical trends in host use. Science 276:253-256.
Bedding, R. A. 1984. Nematode parasites of Hymenoptera. in Plant and Insect Nematodes, edited by W. R. Nickle. Marcel Dekker, Inc. New York.
Daszak. P., A. A. Cunningham and A. D. Hyatt. 2000. Wildlife ecology - emerging infectious diseases of wildlife - threats to diversity and human health. Science 287:443- 449.
Davies, C. M.. J. P. Webster and M. E. J. Woolhouse. 2001. Trade-offs in the evolution of virulence in an indirectlv transmitted macroparasite. Proc. R. Soc. London. B 268:251- 257.
Dobson. A. P. 1985. The population dynamics of competition between parasites. Parasitology 91:317-47.
Farrell. B. and C. Mitter. 1990. Phylogenesis of insect/plant interactions: have Phyllobrotica and the Lamiales diversified in parallel? Evolution 44:1389-1403.
Futuyma, D. J.. M. C. Keese and D. J. Funk. 1995. Genetic constraints on macroevolution: the evolution of host affiliation in the leaf beetle genus Ophraella. Evolution 49:797-809.
Hafner, M. S. and S. A. Nadler. 1988. Phylogenetic trees support the coevolution of parasites and their hosts. Nature 322:258-60.
Hedrick, P. W. 1994. Evolutionary genetics of the major histocompatibility complex. Am. Nat. 143:945-964.
Herre, E. A., N. Knowlton, U. G. Mueller and S. A. Rehner. 1999. The evolution of mutualisms: exploring the paths between conflict and cooperation. Trends. Ecol. Evol. 14:49-53.
Huelsenbeck, J. P., B. Rannala and Z. Yang. 1997. Statistical tests of host-parasite cospeciation. Evolution 51:410-9.
Huelsenbeck, J. P., B. Rannala and B. Larget. 2000. A Bayesian fi-amework for the analysis of cospeciation. Evolution 54:352-364. 31
Iwao, K. and M. D. Rausher. 1997. Evolution of plant resistance to multiple herbivores: quantifying diffuse coevolution. Am. Nat. 149:316-35.
Jaenike, J. 1996a. Sub-optimal virulence of an insect-parasitic nematode. Evolution 50:2241-7.
Jaenike, J. 1996b. Rapid evolution of parasitic nematodes: not. Evol. Ecol. 10:565.
Jaenike, J. and I. Dombeck. 1998. General-purpose genotypes for host utilization in a Dro^o/j/jz/a-parasitic nematode. Evolution 52:832-840.
Jaenike. J. and A. C. James. 1991. Aggregation and the coexistence of mycophagous Drosophila. J. Anim. Ecol. 60:913-928.
Jaenike. J. and S. J. Perlman. 2002. Ecology and evolution of host-parasite associations: mycophagous Drosophila and their nematode parasites. Ant. Nat. in press.
Johnson. IC. P.. B. L. Williams, D. M. Drown. R. J. Adams and D. H. Clayton. 2002. The population genetics of host specificity: genetic differentiation in dove lice (Insecta : Phthiraptera). :V/o/. Ecol. 11:25-38.
Kimura. M. T. and M. J. Toda. 1989. Food preferences and nematode parasitism in mycophagous Drosophila. Ecol. Res. 4:209-18.
van Klinken, R. D. 2000. Host-specificity constrains evolutionary host change in the psyllid Prosopidopsylla Jlava. Ecol. Ent. 25:413-422.
Kohler, S. L. and W. K. Holland. 2001. Population regulation in an aquatic insect: The role of disease. Ecology 82:2294-2305.
Kohler. S. L. and M. J. Wiley. 1997. Pathogen outbreaks reveal large-scale effects of competition in stream communities. Ecology 78:2164-70.
Little, T. J. 2002. The evolutionary significance of parasitism: do parasite-driven genetic dynamics occur ex silico? J. Evol. Biol. 15:1-9.
McCallum, H and A. Dobson. 1995. Detecting disease and parasite threats to endangered species and ecosystems. Trends Ecol. Evol. 10:190-194.
Moore, J. and N. J. Gotelli. 1996. Evolutionary pattens of altered behavior and susceptibility in parasitized hosts. Evolution 50:807-19. 32
Moran N. A., C. D. vonDohlen and P. Baumann. 1995. Faster evolutionary rates in endosymbiotic bacteria than in cospeciating insect hosts. J. Mol. Evol. 41:727-731.
Morehead, S. A. and Feener, D. H. 2000. An experimental test of potential host range in the ant parasitoid Apocephalus paraponerae. Ecoi Entomol. 25:332-340.
Nishiguchi, M. K., E. G. Ruby and M. J. McFall-Ngai. 1998. Competitive dominance among strains of luminous bacteria provides an unusual form of evidence for parallel evolution in sepiolid squid-Vibrio symbioses. Appl. Env. Microbiol. 64:3209-3213.
Page, R. D. M. 1994. Maps between trees and cladistic analysis of historical associations among genes, organisms and areas. Syst. Biol. 43:58-77.
Park. T. 1948. E.xperimental studies of competition. I. Competition between populations of the flour beetles Tribolium confusum Duval and Tribolium castaneum Herbst. Ecoi Monogr. 18:265-308.
Paterson, A. M., G. P. Wallis, L. J. Wallis and R. D. Gray. 2000. Seabird and louse coevolution: Complex histories revealed by 12S rDNA sequences and reconciliation analyses. Syst. Biol. 49:383-399.
Peek. A. S.. R. A. Feldman. R. A. Lutzand R. C. Vrijenhoek. 1998. Cospeciation of chemoautotrophic bacteria and deep sea clams. Proc. Nat. Acad. Sci, U.SA 95:9962- 9966.
Poinar. G. O. Jr.. I. Dombeck and J. Jaenike. 1997. Parasitylenchus nearcticus sp. n. (Allantonematidae: Tylenchida) parasitizing Drosophila (Drosophilidae: Diptera) in North America. Fund. Appi Memat. 20:187-90.
Price, P. W.. M. Westoby, B. Rice, P. T. Atsatt, R. S. Fritz, J. N. Thompson and K. Mobley. 1986. Parasite mediation in ecological interactions. Ann. Rev. Ecoi Syst. 17:487-505.
Reed, D. L. and M. S. Hafner. 1997. Host specificity of chewing lice on pocket gophers: a potential mechanism for cospeciation. J. Mammal. 78:655-660.
Sacks, D. L. 2001. Leishmania-ssnd fly interactions controlling species-specific vector competence. Cell. Microbiol. 3:189-196.
Sasaki, A. and Godfray, H. C. J. 1999. A model for the coevolution of resistance and virulence in coupled host-parasitoid interactions. Proc. R. See. London, Series B. 266:455-463. 33
Siddiqi, M. R. 2000. Tyienchida: Parasites of Plants and Insects. Second Edition. CABI Publishing, CAB International, Wallingford UK.
Solter, L. F. and J. V. Maddox. 1998. Physiological host specificity of microsporidia as an indicator of ecological host specificity. J. Invert. Path. 71;207-16.
Tompkins, D. M., J. V. Greenman and P. J. Hudson. 2001. Differential impact of a shared nematode parasite on two gamebird hosts: implications for apparent competition. Parasitology 122:187-193.
Tompkins, D. M. and D. H. Clayton. 1999. Host resources govern the specificity of swiftlet lice: size matters. J. Anim. Ecol. 68:489-500.
Van Riper, C.. S. G. van Riper. M. L. Goff and M. Laird. 1986. The epizootiology and ecological significance of malaria in Hawaiian land birds. Ecol. .VIonogr. 56:327-44.
Weiblen, G. D. 2001. Phylogenetic relationships of fig wasps pollinating functionally dioecious Ficus based on mitochondrial DNA sequences and morpholoey. Syst. Biol. 50:243-267.
Welch. H. E. 1959. Ta.\onomy. life cycle, development, and habits of two new species of •Allantonematidae (Nematoda) parasitic in drosophilid flies. Parasitology 49:S2-\0j.
Werren. J. H.. W. Zhang and L. R. Guo. 1995. Evolution and phylogeny of Wolbachia: reproductive parasites of arthropods. Proc. R. Soc. London, B 251:55-71.
Wertheim. B., J. G. Sevenster, I. E. M. Eijs and J. J. M. van Alphen. 2000. Species diversity in a mycophagous insect community: the case of spatial aggregation vs. resource partitioning. J. Anim. Ecol. 69:335-351.
Woolhouse, M. E. J.. L. H. Taylor and D. T. Haydon. 2001. Population biology of multihost pathogens. Science 292:1109-1112. 34
APPENDIX ONE:
ASSOCIATIONS BETWEEN MYCOPHAGOUS DROSOPHILA AND THEIR
HOIVARDULA NEMATODE PARASITES : A WORLDWIDE PHYLOGENETIC
SHUFFLE 35
ASSOCIATIONS BETWEEN MYCOPHAGOUS DROSOPHILA AND THEIR
HOWARDULA NEMATODE PARASITES : A WORLDWIDE PHYLOGENETIC
SHUFFLE
Steve J. Perlman'*. Greg S. Spicer", D. DeWayne Shoemaker^ and John Jaenike'^
'Department of Ecology and Evolutionary Biology
Biological Sciences West. University of Arizona. Tucson. AZ, 85721
"Department of Biology
1600 Holloway Ave.. San Francisco State University. San Francisco. CA. 94132-1722
^Department of Biological Sciences
Wood Hall, Western Michigan University, Kalamazoo, MI, 49008
^Department of Biology
Hutchison Hall. University of Rochester, Rochester. NY. 14627
^corresponding author email: [email protected]
Tel: 520-626-8455
Fax:520-621-9190 36
Abstract. - Little is known about what determines patterns of host association of horizontally transmitted parasites over evolutionary timescales. We examine the evolution of associations between mushroom-feeding Drosophila flies (Diptera:
Drosophilidae), particularly in the quinaria and testacea species groups, and their horizontally transmitted Howardula nematode parasites (Tylenchida: Ailantonematidae).
Howardula species were identified by molecular characterization of nematodes collected from wild-caught flies. In addition, DNA sequence data is used to infer the phylogenetic relationships of both host Drosophila (mtDNA: COl, 11. Ill) and their Howardula parasites (rDNA: 18S. ITSl; mtDNA: COI). Host and parasite phylogenies are not congruent, with patterns of host association resulting from frequent and sometimes rapid host colonizations and losses. Drojrop/2//a-parasitic Howardula are not monophyletic. and host switches have occurred between Drosophila and distantly related mycophagous sphaerocerid flies. There is evidence for some phylogenetic association between
parasites and hosts, with some nematode clades associated with certain host lineages.
Overall, these host associations are highly dynamic, and appear to be driven by a
combination of repeated opportunities for host colonization due to shared breeding sites
and large potential host ranges of the nematodes.
Key words. - Cospeciation. coevolution, molecular systematics, mycophagy, parasitism,
phylogeny. 37
The extent to which associated organisms coevolve depends, among other things, on the duration of the association. A major recent goal of comparative biology has been to determine whether phylogenies of associated organisms, such as hosts and parasites or symbionts (Huelsenbeck et al. 1997, 2000), and plants and pollinators or herbivores
(Farrell and Mitter 1990: Weiblen 2001), are generally congruent with each other.
Congruence indicates that such associations transcend speciation events and are, therefore, relatively old. Many factors can lead to incongruence of host and parasite phylogenies, including colonization of new host species (host switching events), parasite extinction or release of certain hosts or host populations from parasitism (sorting events), and parasite speciation in the absence of host speciation (duplication events) (Page 1994).
An most important determinant of host and parasite congruence is parasite transmission mode (Herre et al. 1999). Most cases of phylogenetic congruence between hosts and symbionts are those where the symbiont is vertically transmitted (i.e. from mother to offspring) within the host population (but see Nishiguchi et al. 1998). For example, microbial endosymbionts of aphids and deep sea clams exhibit congruent phylogenies with their hosts (Moran et al. 1995; Peek et al. 1998). Congruent phylogenies can also arise when the parasite has very little opportunity for dispersal to new hosts, as has been found in pocket gopher chewing lice (Hafner and Nadler 1988).
However, even these highly restrictive kinds of associations can depart from perfect congruence, as has been shown in the vertically transmitted Wolbachia, which are
bacterial reproductive parasites of arthropods (Werren et al. 1995), and seabird chewing
lice (Paterson et al. 2000). 38
The majority of parasites however, are horizontally transmitted (i.e. not strictly from mother to offspring), and there are several reasons why their phylogenies are unlikely to be congruent with those of their hosts. Many parasites infect more than one host species (Woolhouse et al. 2001). Taxonomically related parasites can infect distantly related hosts (Siddiqi 2000), which can occur only via host switching events.
Novel host-parasite associations in the wild, such as emerging diseases, are also increasingly common (Daszak et al. 2000). Nevertheless, all parasites, including horizontally transmitted ones, must successfully locate, infect, develop and reproduce within their hosts. This could require such a degree of host-specific adaptation that one would e.xpect parasites to be restricted to certain host lineages, with few switches to distantly related hosts. A comparison of host and parasite phylogenies can be used to estimate the frequency of host switches in horizontally transmitted parasites, as well as the degree to which such shifts are constrained by host phylogeny (Ricklefs and Fallon
2002). In conjunction with experimental infections (Nishiguchi et al. 1998), this approach may also help us to understand what determines the origin and fate of novel
infections.
In this study, we examine the evolution of associations between Howardula
(Tylenchida: Allantonematidae) parasitic nematodes and their host Drosophila (Diptera:
Drosophilidae) flies. These parasites are transmitted horizontally between hosts, with
much opportunity for colonization of novel host species over evolutionary time scales.
We first assess the diversity of these Howardula by molecular characterization of
nematodes obtained fi*om wild-caught flies. We then determine and compare the 39
phylogeny of Drosophila-^ax^sxlxc Howardula with that of their hosts, and ask the following questions: 1) Are host and parasite phytogenies congruent, indicating long- term lineage-specific associations? 2) Are Drosophila-^ax^\i\c Howardula
monophyletic, or can interspecific colonization transcend boundaries of dipteran
families?
Obligate parasitism of insects has evolved multiple times in nematodes (Blaxter
et al. 1998), including at least once in the largely plant-parasitic order Tylenchida.
Arthropod-parasitic tylenchids infect insects from at least six orders, including beetles,
flies, wasps and bees, fleas, thrips. and true bugs, as well as mites (Siddiqi 2000). Two
patterns appear to characterize host associations in this group, although their taxonomy
and systematics are largely understudied: host switches to taxonomically and ecologically
diverse hosts, and subsequent specialization and close association with certain host
clades. Nematodes of the genus Howardula infect diverse beetles and flies, with the
greatest number of described species infecting chrysomelid leaf beetles (Elsey 1977;
Poinar et al. 1998). There are currently 18 valid described species of Howardula
(Zakharenkova 1996; Poinar et al. 1998), although this must be a great underestimate, as
parasites are discovered and their host associations determined only by dissecting adult
insects.
Mushroom-feeding Diptera also represent a large source of Howardula diversity,
with nematodes reported from the families Drosophilidae (Gillis and Hardy 1998),
Phoridae (Richardson et al. 1977), Sphaeroceridae, and Sepsidae (unpublished data).
Drosophila flies, particularly from the the closely related quinaria and testacea species 40
groups, are some of the most abundant insect visitors to decaying fleshy mushrooms in temperate and boreal forests (Grimaldi and Jaenike 1984; Kimura and Toda 1989;
Wertheim et al. 2000). These Drosophila are commonly infected by Howardula, and
their associations are well characterized in North America, Europe and Japan (Kimura
and Toda 1989; Jaenike 1992; Gillis and Hardy 1997). There are currently only two
described species of Drosophila-paTHsilic Howardula (Welch 1959; Poinar et al. 1998).
but. as we show below, this is an underestimate (see also Jaenike 1996).
Dro^op/j/Za-parasitic Howardula are direct parasites (i.e. no intermediate hosts),
and can often have severe effects on host fimess. including complete sterility of females
of some species (Jaenike 1992). Inseminated female nematodes infect fly larvae by
piercing through their cuticle (Welch 1959). When the adult fly emerges, the nematode
motherworm begins releasing juveniles into the hemocoel of the host. These are passed
from the anus and ovipositor of the host as it visits mushrooms, where the nematodes
subsequently mate and continue the cycle. Females that are not sterilized by Howardula
disperse both nematodes and offspring into mushrooms, and, therefore, a small fraction
of parasite transmission is potentially vertical (Jaenike 2000). However, because
individual mushrooms are often oviposited on by multiple adult Drosophila, belonging to
several species (Jaenike and James 1991), there are generally ample opportunities for
horizontal transmission of parasites both within and among host species. We therefore
predict specialization of parasites with certain lineages of hosts, as expected by
constraints of parasite adaptation, but not parallel cladogenesis of Drosophila and
Howardula. 41
MATERIALS AND METHODS
Taxon sampling and DNA extraction.
Howardida nematodes - Our study includes all Howardula (both described and undescribed species) that have been reported to infect Drosophila, except for two undescribed species, one that infects the cactophilic repleta group species D. nigrospiracula in the Sonoran desert (Polak 1993). and one that infects the quadrivittata species group and D. hisirio in Japan (Kimuraand Toda 1989) (Table I). We also consider three Howardula species that parasitize insects other than Drosophila, including two species obtained from mycophagous Leptocera sp. (Diptera: Sphaeroceridae) and H. dominicki. a parasite of the tobacco flea beetle, Epitrix hirtipennis (Coleoptera:
Chrysomelidae). Nematodes were collected by dissecting wild-caught adults, collected from North America, Europe, and Japan. Single motherworms were frozen upon dissection from hosts, and DNA was subsequently extracted using the DNeasy TVf' Tissue
Kit from Qiagen Inc. (protocol for animal tissues).
Drosophila - We include all known Drosophila hosts of Howardida (Table 2), except for the cactophilic species D. nigrospiracula (Polak 1993), and three Palearctic species in the quadrivittata species group {Hirtodrosophila radiation), D. sexvittata, D. trivittata and D. trilineata (Kimura and Toda 1989). All host Drosophila in our study breed primarily on mushrooms, except D. pseudoobscura. The breeding habits of D.
pseudoobscura are not well known, but it has been reported to breed in sap fluxes
(Carson 1951) and acorns (Spieth 1987). We also include nine quinaria group species 42
that are not known to harbor Howardula, including five non-mycophagous species, for a total of 20 (out of 28 described) qiiinaria group species. All four members of the testacea group are included.
Sequencing andphylogenetic analysis
General - Nematode DNA sequencing was carried out on an ABS 377 sequencer at the
Genomic Analysis and Technology Core (GATC) at the University of Arizona. Fly DNA sequencing was carried out at San Francisco State University on a Catalyst 800
Molecular Biology Lab Station. For all species, we sequenced DNA in both directions, and checked for contamination by performing BLAST searches (Altschul et al. 1997)
(http://www.ncbi.nlm.nih.gov/BLAST/). Sequences were first aligned in CLUSTALW
(Thompson et al. 1994) (http://www2.ebi.ac.uk/clustalw) using default settings, and then manually aligned in MacClade 4.0.1. (Maddison and Maddison 2001). We used
PAUP*4.0b6 (Swofford 2001) for all phylogenetic analyses.
Nematode ITS I - For initial molecular typing of nematodes, we sequenced the internal
transcribed spacer (ITSl) region of rDNA, which has been advocated as a useful marker
for closely related nematode species (Powers et al. 1997). We used the rDNA2 and
rDNA1.58s primers described in Powers et al. (1997) and the following PCR conditions:
2.5 mM dNTPs (0.4|il/reaction), 10 primers (0.6 |il), 50 mM MgClj (0.6-1 ^1), lOX
buffer (2(il), Taq polymerase (O.l^il), genomic DNA (l^il), water (14.3-I4.7nl).
Amplification proceeded for 35 cycles, with 1 minute each of denaturation (94°C), 43
annealing (56° or 57°C) and extension (72°C). Due to rapid sequence evolution, we were only able to align ITS I sequence of a subset of nematodes. We constructed a neighbor- joining (NJ) tree for these taxa, but did no further phylogenetic analysis for this region.
Nematode I8S • We sequenced the 18S small subunit of ribosomal DNA, using the
primers described in Blaxter et al. (1998)
(http://nema.cap.ed.ac.uk/biodiversity/sourhope/nemoprimers.html). PCR conditions
were as above. As outgroups, we used the tylenchid nematodes Subangiiina radicicola
(Genbank Accession No. AF202164) and Pratylenchoides magnicauda (AF202157).
which were sequenced by Felix et al. (2000) and had high similarity to Howardiila in
BLAST searches. Under maximum parsimony (MP), we performed heuristic searches
with TBR branch swapping and 1,000 random addition replicates. Maxtrees were set to
increase without limit. Gaps were treated as a new state, and there were no gaps larger
than 4 bases. All characters were weighted equally. We assessed clade robusmess by
bootstrap analysis (Felsenstein 1985), using heuristic searches with 5,000 replicates and a
random addition sequence of n = I. We also estimated the 18S phylogeny using
maximum likelihood analysis. We used a general time reversible model of nucleotide
substitution, with rate heterogeneity between sites (GTR+F+I). We used NJ and MP
trees to estimate the six nucleotide transition parameters, the gamma shape parameter for
rate heterogeneity (r)» and the proportion of invariable sites (I). We performed a
heuristic search with TBR branch swapping, a stepwise addition starting tree, and the asis
stepwise addition option. 44
We used SH tests (Shimodaira and Hasegawa 1999; Goldman et al. 2000) to ask whether tree topologies in which Howardula that parasitize Drosophila were constrained to be monophyletic were significantly different (i.e. less likely) than the ML topology.
We compared the ML tree with the 30 highest scoring constraint trees obtained in a search using the parameters estimated for the ML tree. SH tests were implemented in
P.AUP*. The test compares the difference in log-likelihoods between the best (ML) and alternate trees with a distribution of test statistics generated from 1000 nonparametric bootstrap replicates, using the resampling estimated log-likelihood (RELL) technique
(Goldman et al. 2000).
Nematode miDNA - In order to better resolve one clade of closely-related Drosophila
parasites (the'Drosophila-pnTasiie ITSl' clade), and because we did not have a suitable
outgroup for the rapidly evolving ITS I sequence, we sequenced a portion of
mitochondrial cytochrome c oxidase subunit L using primers developed by Folmer et al.
(1994) and described in Sukhdeo et al. (1997). We used the PGR protocol described
above, but with an annealing temperature of 50°C. We were unable to amplify mtDNA
from Howardula infecting D. macroptera (MA) and from one Howardula species
infecting Leptocera (SPB). We used the Howardula from D. pseudoobscura (PS) and
from one Leptocera parasite (SPA) as outgroups. Phylogenetic analyses were carried out
as above, except that there were no gaps. Also, for ML analysis, we used a GTR model
of nucleotide substitution with codon-position-specific rate variation. Because mtDNA
evolves rapidly in nematodes and is only recommended for phylogenetic reconstruction 45
of closely-related taxa (Blouin et al. 1998), we tested for DNA saturation at first, second and third position transitions and transversions. We compared genetic distances
(estimated from our ML model) with the number of changes between pairs of taxa, with
nonlinear relationships suggesting saturation.
Drosophila miDNA - Drosophila DNA extraction methods are described in Spicer
(1995). We obtained DNA sequences from the mitochondrial cytochrome oxidase 1, II.
and III subunits (COl-IlI). The corresponding mtDNA sequences from D. yakiiba and D.
meUinogasier, which were used as outgroups in this study, came from Clary and
Wolstenholme (1985) and de Bruijn (1983), respectively. We also used D. qffmis. D.
subobscura and D. husckii as additional outgroups. The amplification primers for COI.
Cl-N-2191 and CI-J-1751, were made specific to Drosophila, and can be found in Spicer
(1995). Most of the COII primers appeared in Liu and Beckenbach (1992), although
some (TK-N-3785 and TL2-J-3037) have been modified from the original compilation
(Spicer 1995). The amplification primers for COIII. C3-J-5014 and C3-N-5460,
appeared in Simon et al (1994). Most of the internal sequencing primers were designed
independently and can be found in Spicer (1995). PCR conditions are as in Spicer
(1995). Phylogenetic analyses were carried out as above (see nematode 18S), except that
any gaps were coded as missing data. The few gaps in this dataset were due to regions at
the ends of subunits for which we were not able to obtain sequence. 46
Determination of parasite host range - The presence of cryptic Howardula species complicates determinations of host range. Host associations for all Howardula in this study were determined molecular characterization of isolates obtained from wild-caught flies, except for the generalist H. aoronymphium. We obtained sequence from H. aoronymphium collected from three of its nine known host species: D. falleni, D. neotestacea and D. phalerata. In experimental lab infections (Perlman, unpublished data), we confirmed that H. aoronymphium can successfully infect all other reported hosts: D. immigrans, D. kuntzei. D. piitrida, D. recens, D. testacea, and D. transversa. It was previously shovsTi that in North America, the generalist H. aoronymphium consists of a single epidemiological unit and is not comprised of host races (Jaenike and Dombeck
1998).
To illustrate the general suitability of the quinaria and testacea groups to
Howardula parasitism, we mapped nematode parasitism in the wild onto host phylogeny. using MacClade 4.0.1.
Congruence of host and parasite phylogenies - We visualized host and parasite
phylogenies and used the method of reconciled trees in Treemap I.Ob (Page 1995) to test
for incongruence. This method aligns host and parasite cladograms such that the number
of cospeciation events (matching nodes) is maximized. For perfect congruence, the
number of cospeciation events is the number of internal nodes minus one (Page 1994).
We then created 1000 random associations by reshuffling host and parasite taxa,
calculating the maximum number of cospeciation events for each reshuffled association 47
and comparing their distribution. Random trees were generated in Treemap using the proportional-to-distinguishable model option. Treemap requires fully resolved trees; we therefore performed analyses for all possible resolutions of clades with low bootstrap values. We included only Drosophila and their parasites in the analysis.
RESULTS
Howardula phytogeny
Nematode ITS I • ITSl sequence revealed a total of eight distinct lineages of
Dro5op/7/7a-parasitic Howardula. and two distinct Leproct?ra-parasitic species. ITS 1 was highly .AT-biased (80%). Sequence length for all nematodes was 290 to 345 base pairs, except for one sphaerocerid parasite (SPA: 5I4bp) and H. dominicki (C: 435 bp). We were only able to align ITSl for a subset of nematodes. This group consisted of all
Drosophila-paxasitic Howardula except for the two species infecting D. macroptera
(MA) and D. pseudoobscura (PS). A neighbor-joining tree of the 'Dro5op/i/7a-parasite
ITSr clade reveals three distinct groupings (Fig. 1): \) H. aoronymphium from Europe
and America, which are identical, and a closely-related Japanese nematode (the AORO
group). 2) H. neocosntis from North America and a very close sister species (the NEO
group), and 3) two undescribed species, one from North America and one from Japan (the
B/F group). This NJ tree was constructed after removal of one 45-70 bp AT-rich region
of ambiguous alignment. We were also able to align the D. pseudoobscura (PS) parasite
sequence with that of one sphaerocerid parasite (SPB) after removing a 50-66 and an 8-
18 bp region, but we could not align this pair with any other sequences. 48
Nematode 18S - Sequence lengths per nematode species ranged from 1733 to 1791 bp.
We were unable to sequence 530 bases at the 5' end for one of the sphaerocerid parasites
CSPB). Only 1630 bases of 18S sequence were available for our two outgroups. Our complete aligned data set was therefore 1125 characters long. MP, NJ and ML analyses produced similar tree topologies (Fig. 2). MP analysis produced 4 most parsimonious trees (treelength = 348. CI = 0.80, 140 parsimony informative characters). These differed in their resolution of the' Drosophila-parvisile ITS I' clade. ML analysis produced a tree of score -In 3112.5, which was identical in topology to one of the most parsimonious trees. The following parameters of nucleotide substitution were used in the ML analysis:
A-C = 0.9. A-G = 3.96. A-T = 2.28. C-G = 0.21. C-T = 7.43. G-T = 1.1 = 0.55. T = 0.9.
The ML tree was significantly different from the 30 best trees having the constraint that
Drosophila parasites are monophyletic (p < 0.02). The best constrained tree had a score of -In 3130.69. for a difference in likelihood scores of 18.19 (SH test: p = 0.016). Thus, the Drosophila-pvLTasitic species of Howardula are not monophyletic.
Nematode mtDNA - Our complete aligned data set consisted of 303 bp. MtDNA was
.A.T-rich (65%), and only third position transitions appear saturated, as evidenced by the
lack of association between genetic distance and number of differences (Fig. 3). This data set does not resolve the relationships between the three main groupings of the
'Drosophila-pzx^s\\& ITSl' clade (AORO, NEO, B/F). MP analysis yielded a single MPR
(treelength = 176, CI = 0.78, 66 parsimony informative characters), with AORO and
NEO as sister groups. This tree was only one step shorter than NJ or ML topologies. ML 49
analysis produced a tree of score -In 1101.18 (Fig. 4), with the following parameters of
nucleotide substitution: A-C = 2.99, A-G = 9.01, A-T = 1.47, C-G = 1.20, C-T = 6.42, G-
T = 1, codon rates = 0.39, 0.074,2.54, and with NEO and B/F as sister groups. This tree
score was not significantly different from the NJ topology (-In 1102.21, SH test: p =
.058) or the MP topology (-In 1105.4, p = 0.15).
Nematode species delineation - We use a combination of pairwise sequence divergence
(using ML distances), experimental infections, and morphological differences to
delineate nematode species. At 18S. Howardula isolates/species MA. PS, SPA, and SPB
exhibit 2-5% sequence divergence from their nearest neighbors; these distances are
greater than or within the range of interspecific 18S divergence found within the
nematode genera Caenorhabditis (0.8-1.8%; Fitch et al. 1995). Heterorhabditis (0-1%;
Liu el al. 1997) and Steinernema (1-7%, Liu et al. 1997).
Within the closely related 'Dro5op/i//a-parasitic ITSl' clade, mtDNA sequence
divergence between the three species groups 'AORO', 'B/F', and "NEO", ranges from 16-
25%. These species groups also show morphological and potential host range differences
(Perlmari, unpublished data). Howardula sp. B and F, and H. aoronymphium and H. cf.
aoronymphium exhibit 20% and 10% mtDNA sequence divergence, respectively. In a
survey of nematode mtDNA divergence, Blouin et al. (1998) found that interspecific
divergences range from 10-20%, and intraspecific divergences firom 0-7%. In
experimental lab infections, H. aoronymphium and H. cf. aoronymphium also show
significantly different potential host ranges (Perlman, unpublished data). Finally, H. 50
neocosmis and H. cf. neocosmis exhibit 5% mtDNA divergence, and show quantitative morphological differences (Poinar et al. 1998). In lab infections, H. neocosmis infected its native host D. acutilabella at significantly higher rates than D. munda, the host of H. cf neocosmis (Perlman, unpublished data). In sum, these analyses indicate the presence of eight Drosophila-parasilic species of Howardula among the isolates we have examined: H. aoronymphium. H. cf. aoronymphiiim. H. neocosmis, H. cf neocosmis. and
Howardula species 'B'. 'F'. 'MA', and 'PS'.
Drosophila mtDNA phytogeny - The sequenced regions of the mitochondrial cytochrome oxidase gene encompass a 413 base pair (bp) segment of subunit I (D. yakuba positions 1778-2190; total length of subunit is 1535 bp), the entire subunit II (D. yakuba positions 3083-3766) comprising 688 bp, and a 416 bp segment of subunit III (D. yakuba positions 5015-5430; total length of subunit is 788). The D. yakuba positions
refer to the Clary and Wolstenholme (1985) sequence.
ML analysis of the COI, II and III dataset produced a tree of score -In 12619.50
(Fig. 5). with the following parameters of nucleotide substitution: A-C = 3.48, .A.-G =
41.98. A-T = 24.33. C-G = 8.02, C-T= 110.25, G-T= 1,1 = 0.59, r= 1.12. MP analysis
produced 12 most parsimonious trees (treelength = 2421, CI = 0.33,467 out of 1518
parsimony informative characters) (trees not shown). These trees differ in the following
places: 1) in the grouping of the closely related quinaria group species D. munda, D.
quinaria, D. recens. D. transversa, D. suboccidentalis and D. subquinaria, 2) in the
ordering of D. nigromaculata and D. guttifera, and 3) in the relationship between the 51
testacea, quinaria and (histrio + macroptera) species groups. None of the nodes where the MP trees differ from the ML tree have strong bootstrap support. In all analyses, D. siiboccidentalis appears paraphyletic with respect to D. occidentalis; these are almost
indistinguishable morphologically and might not be reproductively isolated. With respect
to species group relationships, only the (cardini group + tripunctata group) and the
(hisfrio group + macroptera group) pairings are consistently supported. Branches joining
these lineages with the quinaria and testacea groups are short, and no combinations are
significantly different than the species group topology obtained in the ML tree (SH tests:
p > 0.05).
Nematode parasitism is distributed throughout the quinaria and testacea groups.
Most species (9 out of 10) for which there are no records of Howardula parasitism belong
to one clade of the quinaria group (Fig. 6). However, most of the uninfected species
have not been sampled adequately to rule out the presence of parasites.
Congruence of Drosophila and Howardula phylogenies - Host and parasite phylogenies
are not congruent (Fig. 7). The maximum number of matching nodes (cospeciation
events) inferred from the reconciliation analysis was 3 (perfect congruence would be 8).
All resolutions of clades with low bootstrap support yielded the same number of
cospeciation events. The inferred number of cospeciation events was not significantly
different from that obtained by randomizing parasite taxa across Drosophila species,
which yielded 1.68 ± 0.03 s.e. (p = 0.14) cospeciation events. However, this number was
significantly different from one obtained by randomizing host taxa, which yielded 0.51 ± 52
0.02 (p = 0.006) cospeciation events. The reconstruction with the fewest steps yielded 3 cospeciation events. 5 host switches, 0 duplication events and 25 sorting events.
DISCUSSION
Our study reveals little congruence between Howardula and Drosophila phylogenies, indicating frequent colonizations of new host species. Such colonization requires at least two conditions. First, there must be opportunities for o-ansmission of nematodes between species. Most of the Drosophila in our study are generalists. utilizing many of the same species of mushrooms as breeding sites (Lacy 1984: Kimura and Toda 1989; Wertheim et al. 2000). More importantly, interspecific aggregation can be pronounced, and as many as four different Drosophila species can emerge from a single mushroom (Grimaldi and Jaenike 1984; Jaenike and James 1991). As mushrooms are the site of host infection by nematodes, these parasites will often encounter multiple potential host species.
The second requirement for a host shift is that the parasite be capable of infecting, developing, and reproducing in the new host. Thus, the new host must be similar, in certain critical respects, to the ancestral host. The present findings of widespread parasite colonization of phylogenetically distinct Drosophila species indicates that these flies are, to some extent, similar environments with respect to parasite adaptation. Such conditions
- opportunities for interspecific transmission and host similarity - can favor host generalism by the parasites. H. aoronymphium is a good example; it successfully infects 53
at least four species in North America (Jaenike 1992), and five in Europe (Gillis and
Hardy 1997).
The degree of host switching that has occurred between distantly related families
(specifically, sphaerocerids and drosophilids) was unexpected. As a consequence of such
host shifts, Dro5op/i//a-parasitic Howardula are paraphyletic. The parasite of D.
pseudoobscura (PS) is most closely related to one of the sphaerocerid parasites (SPB).
The 'Dr050/7/i/7a-parasitic ITSl' clade is distantly related to the two other Drosophila-
parasitic species (MA and PS). Virtually nothing is known about the biology of these
latter two Howardula species. Even the two sphaerocerid parasite species (SPA and
SPB) are distantly related, even though both species can occur in the same individual fly.
In addition to Drosophilidae and Sphaeroceridae. Howardula have been found in
mushroom-feeding Phoridae (Richardson et al. 1977) and Sepsidae (unpublished data).
The occurrence of Howardula in several families of mushroom-feeding flies, in
conjunction with our phylogenetic data, highlights the ecological potential for host-
switching between diverse hosts. It is important to emphasize that the Howardula in our
study are not fly generalists. In lab experiments, we were unable to infect sphaerocerids
with Drosophila parasites or Drosophila with sphaerocerid parasites (Jaenike 1992;
Perlman, unpublished data).
The potential for rapid host switching and incorporation of novel hosts by
Howardula is further suggested by the striking lack of genetic variation among American
and European H. aoronymphium, even at the rapidly evolving ITS1 sequence. ITS1 and
mitochondrial sequences of North American and European samples were found to be 54
identical, suggesting that one (or both) of these populations are evolutionarily very young. It is likely that H. aoronymphium recently invaded North America and/or Europe and, in the process, must have incorporated an entirely new set of Drosophila into its host range. H. neocosmis and its very closely related sister species, although very similar at the molecular level, parasitize hosts belonging to different species groups in eastern and western North America. As with H. aoronymphium, this suggests a recent host shift.
Despite the great potential for horizontal transmission and ecological conditions favoring generalism, our study uncovered a number of Howardula that are host specialists. A few of these are cryptic species that were previously mistaken for a more common host-generalist nematode. For example, Howardula sp. F, the parasite of D. falleni. is unable to infect D. neotestacea. D. puirida or D. recens (Jaenike 1996;
Perlman, unpublished data); these four species share mushrooms and all are parasitized by the generalist nematode H. aoronymphium. Indeed, Howardula sp. F was initially thought to be a host-restricted variant of H aoronymphium (Jaenike 1996). In addition, at least two Howardula species from Japan, the 'orientacea specialist' (//. cf. aoronymphium J) and the nematode infecting D. brachynephros {Howardula sp. B) were previously identified as H. aoronymphium (Kimura and Toda 1989). In lab infections, the 'orientacea specialist' was unable to infect Japanese quinaria group species (Perlman, unpublished data). In total, our molecular analysis identified 6 new Drosophila-^zizsiixc
Howardula species, and 2 new sphaerocerid-parasitic Howardula species.
There is some association between phylogenies of parasites and hosts in our analysis. This is demonstrated by the fact that the maximum number of inferred 55
cospeciation events is significantly more than that obtained by randomizing host taxa across parasite species (but not by randomizing parasite taxa across host species). This
pattern could result from actual cospeciation of host and parasite lineages or from
phylogenetic limitations on host shifts, so that parasites could only colonize closely
related species of hosts (Ricklefs and Fallon 2002). This indicates that certain parasite
clades tend to be associated with particular host lineages. For example, H.
aoronymphium, which occurs in both Europe and North America, and its Japanese sister
species are associated with all four species of the lestacea group. In addition, the
parasites of D. brachynephros (B) and D. falleni (F) are sister species, and their hosts are
close relatives. We predict that the nematodes that infect D. curvispina and D. unispina
(Kimura and Toda 1989) are the same species as infects D. brachynephros, since these
hosts are all closely related members of the D. quinaria group.
What determines nematode host range in the wild? While Howardula parasitism
is distributed throughout the testacea and quinaria groups, nine of the ten species for
which there are no records of parasitism occur in the 'recens-guiiifera' clade of the
quinaria group (Fig. 6). Howardula can infect and grow in seven of these ten species in
the lab (the other hosts were not tested), demonstrating that these species are intrinsically
suitable as hosts for Howardula (Perlman, unpublished data). It is possible that these
species are infected at low rates in the wild, but that limited sampling has not been
sufficient to find parasitized flies.
Alternatively, the lack of infection of species may be related to their breeding
sites. For example, five of the ten uninfected species, D. deflecta, D. quinaria, D. 56
limbata, D. subpalustris and D. paliistris, breed on decaying vegetation instead of mushrooms. It is not known why decaying vegetation breeders might not be infected in the wild. In a field experiment, it was shown that H. aoronymphium from lab-reared flies could be transmitted to D. quinaria breeding in decaying vegetation (Jaenike, unpublished data). It is possible that decaying vegetation breeders are not infected because they occur at densities too low to support a parasite population (Jaenike and
Perlman 2002). Because breeding in decaying vegetation is clearly a derived condition among these flies (Spicer and Jaenike 1996), the loss of parasitism associated with such a breeding site shift would represent a sorting event, thus bringing about incongruence of
host and parasite phylogenies.
In conclusion, our data reveal high levels of host switching and rapid
incorporation of novel hosts across North America. Europe and Asia. These patterns are
probably driven by the great potential for colonization of new hosts, due to shared host
breeding sites, in combination with large potential host ranges (Perlman, unpublished
data), even for parasites that currently utilize only a few host species. Thus, Drosophila-
Howardula associations are highly dynamic over evolutionary time scales. We suspect
that evolutionarily dynamic host ranges and lack of phylogenetic congruence are
common, if not the rule, in associations characterized by horizontal transmission of
parasites and ecological mingling of different host species, i.e., most host-parasite
associations. 57
ACKNOWLEDGMENTS
We thank Andy Beckenbach, Irene Dombeck, Irene Eijs, Ray Huey, Masahito Kimura,
Clyde Sorenson and Chuck Taylor for collecting insects. Kelly Dyer and Andrew
Holyoake for assistance in the lab, and Jake Russell for conunents. This work was supported by National Science Foundation grants DEB-9629546 to GSS and DEB-
0074141 to JJ and funds from the Center for Insect Science (U. of Arizona) and the NSF
Research Training Grant in the Analysis of Biological Diversification (U. of Arizona) to
SP. 58
LITERATURE CITED
Altschul, S. F., T. M. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller and D. J.
Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database
search programs. Nucleic Acids Res. 25:3389-3402.
Blaxter, M. L. et al. 1998. A molecular evolutionary framework for the phylum
Nematoda. Nature 392:71-75.
Blouin. M. S., C. A. Yowell. C. H. Courtney and J. B. Dame. 1998. Substitution bias,
rapid saturation, and the use of mtDNA for nematode systematics. Mol. Biol. Evol.
15:1719-1727.
Carson, H. L. 1951. Breeding sites of Drosophila pseudoobscura and D. persimilis in the
transition zone of the Sierra Nevada. Evolution 5:91-96.
Clary, D. O., and Wolstenholme, D. R. 1985. The mitochondrial DNA molecule of
Drosophila yakuba: nucleotide sequence, gene organization and genetic code. J. Mol.
Evol. 22:252-271.
Daszak, P.. A. A. Cunningham and A. D. Hyatt. 2000. Wildlife ecology - emerging
infectious diseases of wildlife - threats to diversity and human health. Science 287:443-
449. de Bruijn, M. H. L. 1983. Drosophila melanogaster mitochondrial DNA, a novel gene
organization and genetic code. Nature 304:234-241.
Elsey, K. D. 1977. Parasitism of some economically important species of Chrysomelidae
by nematodes of the genus Howardula. J. Invert. Path. 29:394-385.
Farrell, B. and C. Mitter. 1990. Phylogenesis of insect/plant interactions: have 59
Phyllobrotica and the Lamiales diversified in parallel? Evolution 44:1389-1403.
Felix, M.A., P. De Ley, R. J. Sommer, L. Frisse, S. A. Nadler, W. K.. Thomas, J.
Vanfleteren and P. W. Sternberg. 2000. Evolution of vulva development in the
Cephalobina (Nematoda). Dev. Biol. 221:68-86.
Felsenstein, J. 1985. Confidence limits on phylogenies: An approach using the bootstrap.
Evolution 39:783-791.
Fitch. D. H. A.. B. Bugaj-Gaweda and S. W. Emmons. 1995. 18S ribosomal RNA gene
phylogeny for some rhabditidae related to Caenorhabditis. .Vfol. Biol. Evol. 12:346-
358.
Folmer. O.. M. Black, W. Hoeh. R. Lutzand R. Vrijenhoek. 1994. DNA primers for
amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan
invertebrates. Xfol. Mar. Biol. Technol. 3:294-299.
Goldman, N., J. P. Anderson and A. G. Rodrigo. 2000. Likelihood-based tests of
topologies in phylogenetics. Syst. Biol. 49:652-670.
Gillis. J. E. M. and I. C. W. Hardy. 1997. Nematode parasitism in a northern European
drosophilid community. Ent. Exp. et Appl. 84:275-91.
Grimaldi, D. and J. Jaenike. 1984. Competition in natural populations of mycophagous
Drosophila. Ecology 65:1113-1120.
Grimaldi, D. A.. A. C. James, and J. Jaenike. 1992. Systematics and modes of
reproductive isolation in the Drosophila testacea group (Diptera: Drosophilidae/ Ann.
Ent. Soc. Amer. 85: 671-685.
Hafner, M. S. and S. A. Nadler. 1988. Phylogenetic trees support the coevolution of 60
parasites and their hosts. Nature 322:258-60.
Herre, E. A., N. Knowlton, U. G. Mueller and S. A. Rehner. 1999. The evolution of
mutualisms: exploring the paths bet\\'een conflict and cooperation. Trends. Ecol. Evol.
14:49-53.
Huelsenbeck, J. P.. B. Rannala and B. Larget. 2000. A Bayesian framework for the
analysis of cospeciation. Evolution 54:352-364.
Huelsenbeck, J. P.. B. Rannala and Z. Yang. 1997. Statistical tests of host-parasite
cospeciation. Evolution 51:410-9.
Jaenike. J. 1992. Mycophagous Drosophila and their nematode parasites. Am. .S'at.
139:893-906.
Jaenike. J. 1996. Rapid evolution of parasitic nematodes: not. Evol. Ecol. 10:565.
Jaenike, J. and A. C. James. 1991. Aggregation and the coexistence of mycophagous
Drosophila. J. Anim. Ecol. 60:913-928.
Jaenike. J. and I. Dombeck. 1998. General-purpose genotypes for host utilization in a
Dro5op/i//a-parasitic nematode. Evolution 52:832-840.
Jaenike, J. and S. J. Perlman. 2002. Ecology and evolution of host-parasite associations:
mycophagous Drosophila and their parasitic nematodes. Am. Nat. in press.
K-imura, M. T. and M. J. Toda. 1989. Food preferences and nematode parasitism in
mycophagous Drosophila. Ecol. Res. 4:209-18.
Lacy, R. C. 1984. Predictability, toxicity, and trophic niche breadth in fungus-feeding
Drosophilidae (Diptera). Ecol. EntomoL 9:43-54.
Liu, H., and A. T. Beckenbach. 1992. Evolution of the mitochondrial cytochrome oxidase 61
II gene among 10 orders of insects. Mol. Phylogen. Evol. 1: 41-52.
Liu, J., R. E. Berry and A. F. Moldenke. 1997. Phylogenetic relationships of
entomopathogenic nematodes (Heterorhabditidae and Steinemematidae) inferred from
partial 18S rRNA gene sequences. J. Invert. Path. 69:246-252.
Maddison, D. R. and W. P. Maddison. 2001. MacClade version 4.0.1. Sinauer
Associates, Sunderland, Massachusetts.
Moran N. A., C. D. vonDohlen and P. Baumann. 1995. Faster evolutionary rates in
endosymbiotic bacteria than in cospeciating insect hosts. J. Mol. Evol. 41:727-731.
Nishiguchi, M. K.. E. G. Ruby and M. J. McFall-Ngai. 1998. Competitive dominance
among strains of luminous bacteria provides an unusual form of evidence for parallel
evolution in sepiolid squid-Vibrio symbioses. Appl. Env. Microbiol. 64:3209-3213.
Page. R. D. M. 1994. Maps between trees and cladistic analysis of historical associations
among genes, organisms and areas. Syst. Biol. 43:58-77.
Page, R. D. M. 1995. TreeMap for Macintosh version 1.0b.
http://ta.xonomy.zoology.gla.ac.uk/rod/treemap.himl
Paterson. A. M.. G. P. Wallis, L. J. Wallis and R. D. Gray. 2000. Seabird and louse
coevolution: Complex histories revealed by 12S rDNA sequences and reconciliation
analyses. Syst. Biol. 49:383-399.
Peek. A. S.. R. A. Feldman, R. A. Lutz and R. C. Vrijenhoek. 1998. Cospeciation of
chemoautotrophic bacteria and deep sea clams. Proc. Nat. Acad. Sci, USA, 95:9962-
9966.
Poinar, G. O. Jr., J. Jaenike and D. D. Shoemaker. 1998. Howardula neocosmis sp. n. 62
(Tylenchida: Allantonematidae) parasitizing North American Drosophila (Diptera:
Drosophilidae) with a key to the species of Howardula. Fundam. Appl. Nematol.
21:547-552.
Polak, M. 1993. Parasites increase fluctuating asymmetry of male Drosophila
nigrospiracula - implications for sexual selection. Genetica 89:255-265.
Powers. T. O., T. C. Todd, M. Bumell, P. C. B. Murray, C. C. Fleming, A. L.
Szalanski. B. A. .'Kdams and T. S. Harris. 1997. The rDNA internal transcribed spacer
region as a taxonomic marker for nematodes. J. Nematol. 29:441-450.
Richardson. P. N.. Hesling, J. J. and I. L. Riding. 1977. Life-cycle and description of
Howardula husseyi n.sp. (Tylenchida: Allantonematidae), a nematode parasite of
mushroom phorid. X/egaselia halierata (Diptera: Phoridae). Nemaiologica 23:217.
Ricklefs, R. E. and S. M. Fallon. 2002. Diversification and host switching in avian
malaria parasites. Proc. R. Soc. Lond. B 269:885-892.
Shimodaira, H. and M. Hasegawa. 1999. Multiple comparisons of log-likelihoods with
applications to phylogenetic inference. Xfol. Biol. Evol. 16:1114-1116.
Siddiqi, M. R. 2000. Tylenchida: Parasites of Plants and Insects. Second Edition. CABI
Publishing, CAB International, Wallingford UK.
Simon, C., F. Frati. A. Beckenbach, B. Crespi, H. Liu, and P. Flook. 1994. Evolution,
weighting and phylogenetic utility of mitochondrial gene sequences and a compilation
of conserved polymerase chain reaction primers. Ann. Em. Soc. Amer. 87: 651-701.
Spicer, G. S. 1995. Phylogenetic utility of the mitochondrial cytochrome oxidase gene:
Molecular evolution of the Drosophila buzzatii species complex. J. Mol. Evol. 41:749- 63
759.
Spicer, G. S. and J. Jaenike. 1996. Phylogenetic analysis of breeding site use and a-
amanitin tolerance within the Drosophila quinaria group. Evolution 50:2328-37.
Spieth, H.T. 1987. The Drosophila fauna of a native California forest (Diptera:
Drosophilidae). Pan-Pacif. Entomol. 63:247-255.
Sukhdeo, S. C., M. V. K. Sukhdeo, M. B. Black and R. C. Vrijenhoek. 1997. The
evolution of tissue migration in parasitic nematodes (Nematoda: Strongylida) inferred
from a protein-coding mitochondrial gene. Biol. J. Linn. Soc. 61:281-298.
Swofford, D. L. 2001. PAUP*. Phylogenetic Analysis Using Parsimony (*and Other
Methods). Version 4. Sinauer Associates, Sunderland. Massachusetts.
Thompson. J. D.. D. G. Higgins and T. J. Gibson. 1994. CLUSTAL W: Improving the
sensitivity of progressive multiple sequence alignment through sequence weighting,
positions-specific gap penalties and weight matri.x choice. Nucleic Acids Res. 22:4673-
4680.
Weiblen, G. D. 2001. Phylogenetic relationships of fig wasps pollinating functionally
dioecious Ficiis based on mitochondrial DNA sequences and morphology. Syst. Biol.
50:243-267.
Welch, H. E. 1959. Taxonomy, life cycle, development, and habits of two new species of
Allantonematidae (Nematoda) parasitic in drosophilid flies. Parasitology 49:83-103.
Werren, J. H., W. Zhang and L. R. Guo. 1995. Evolution and phylogeny of Wolbachia:
reproductive parasites of arthropods. Proc. R. Soc. Lond. B 251:55-71.
Wertheim, B., J. G. Sevenster, I. E. M. Eijs and J. J. M. van Alphen. 2000. Species 64
diversity in a mycophagous insect community: the case of spatial aggregation vs.
resource partitioning. J. Anim. Ecol. 69:335-351.
Woolhouse, M. E. J., L. H. Taylor and D. T. Haydon. 2001. Population biology of
multihost pathogens. Science 292:1109-1112.
Zakharenkova, N. N. 1996. Howardula phyllotretae (Tylenchida: Allantonematidae) -
parasite of Phyllotreta tlea beetles (Coleoptera: Chrysomelidae). Russ. J. Nemaiol.
4:1-6. 65
Table 1. Nematodes included in phylogenetic analysis.
Nematode Known host Sample number Collection Sample host species
designation species locale
Howardula D. phalerata E329 Denmark D. phalerata
aoronymphium E334, E336, E2F Holland D. phalerata
D. falleni N66. N124. N399 New York D. falleni
D neotestacea N90 Virginia D. neotestacea
NIOI Pennsylvania D. neotestacea
D. inmigrans
D. kuntzet
D. pulrida
D. recens
D. lestacea
D. transversa
Howardula cf. D. orientacea J308. J316. J344 Japan D. orientacea
aoronymphium
Howardula ci. D. munda MU397. MU398 Arizona D. munda
neocosmis D. suboccidentalis SU32I,SU322 Washington D. suboccidentalis
Howardula sp. F D. falleni F125. F165, F167 New York D. falleni
Howardula sp. Q D.brachynephros B30I, B331, B332 Japan D. brachynephros 66
Table 1 (continued).
Nematode Known host Sample number Collection Sample host species
designation species locale
Howardulasp. D. macroptera MA38l,MA382 Arizona D. macroptera
MA
Howardula sp. D. pseudoobscura PS99 California D. pseudoobscura
PS
Howardula sp. Leptocera sp. SP392 Washington Leptocera sp.
SPA (Sphaeroceridae) SP2,SP36l,SP363 New York Leptocera sp.
Howardula sp. Leptocera sp. SP391 Washington Leptocera sp.
SPB (Sphaeroceridae)
Howardula Epitrix hirtipennis C348, C350 N. Carolina E. hirtipennts
dominicki (Coleoptera) 67
Table 2. Drosophila species included in the study and their Howardula parasites.
species group species collection locale' nematode species
leslacea D. neolestacea New York H. aoronymphium*'
D. orientacea Japan H. cf aoronymphium'
D. putrida New York H. aoronymphiun^
D. leslacea Germany H. aoronymphium'
quinaria D. brachynephros Japan Howardula sp.'
D. curvispina Japan Howardula sp.'
D. falleni New York. H. aoronymphium''
(15130-1961.0) Howardula sp."
D. gultifera Texas. Florida
(15130-1971.0. 1971.1)
D. kuntzei Holland H. aoronymphium'
D. limbata Holland
D. niunda Arizona H. cf. neocosmis"^
D. nigromaculata Japan
D. occidentalis California
D. pal usiris New York
D. phalerata Holland H. aoronymphium*
D. quinaria New York
D. recens New York H. aoronymphium^
D. suboccidentalis Oregon, California H. cf. neocosmis^
D. subpalustris South Carolina
(15130-2071.0,2071.1)
D. subquinaria Washington 68
Table 2 (continued).
species group species collection locale nematode species
D. transversa Holland H. aoronymphium'
D. unispina Japan Howardula sp.'
cardini D. actitilabella Florida H. neocosmis^
D. cardini Florida
tripunctata D. tripunctata Tennessee
immigrans D. immigrans New York H. aoronymphium^
histrio D. histrio Japan Howarduta sp.'
macroptera D. macroptera Arizona Howardula sp.'^
quacirivitlata D. histrioides Japan Howardula sp.'
b use kit D. biisckii Costa Rica
(13000-0081.0)
obscura D. affinis Nebraska
(14012-0141.0)
D. pseudoobscura Arizona Howardula sp.
(14011.0212.0)
D. subobscura Washington
a) Gillis and Hardy (1997); b) Jaenike (1992); c) Jaenike (1996); d) Jaenike and Perlman
(unpublished); e) Kimura and Toda (1989); f) Poinar et al. (1998); g) numbers refer to
strains from Drosophila species stock center (http://stockcenter.ari.arizona.edu/) 69
FIGURE LEGENDS
Fig. 1. Neigiibor-joining tree, using ITSl sequences for the Howardula ciade termed
'Drosophila-painsile ITSl', with midpoint rooting. This ciade comprises three species
complexes, denoted AORO, B/F and NEO. Sequences are 303 bp long. We were
unable to align the other Howardula ITS 1 sequences.
Fig. 2. Ma.\imum likelihood tree, using Howardula 18S sequences, rooted with the
nematodes Pratylenchoides magnicauda and Subanguina radicicola. Aligned data set
is 1125 characters. Topology is identical to one of 4 most parsimonious trees.
Numbers above branches indicate parsimony percent bootstrap values. Asterisks
indicate Howardula that do not infect Drosophila.
Fig. 3. Number of first, second and third position transitions and U^ansversions as a
function of pairwise maximum likelihood genetic distances for mtDNA Howardula
sequences. Note that only third position transitions appear saturated.
Fig. 4. Maximum likelihood miDNA trees for the 'Drojop/i/7a-parasite ITS 1' Howardula
ciade, rooted with Howardula species PS99 and SPA392. Numbers above branches
indicate parsimony percent bootstrap values. Sequences are 303 bp long.
Fig. 5. Maximum likelihood tree using Drosophila COI, II and III sequences. Numbers
above branches indicate parsimony percent bootstrap values. (None of the nodes where
MP trees differ from the ML tree have strong bootstrap support.) Sequences are 1517
bp long.
Fig. 6. Distribution of known nematode parasitism in the quinaria and testacea species
groups. Species that are known to be infected in the wild are labeled P. Note that 70
some uninfected species have not been sampled intensively enough to exclude the
possibility of parasitism.
Fig. 7. Associations of Howardula nematodes and Drosophila species, as determined by
host records in natural populations. AC340 H. neocosmis (D. acutilabella)
MU397 H. cf. neocosmis (D. munda) " NEO " MU398 H. cf. neocosmis (D. munda) SU321 H. cf. neocosmis (D. suboccidentalis)
J344 H. of. aoronymphium {D. orientacea) J316 H. ci. aoronymphium {D. orientacea)
E329 H. aoronymphium (D. phalerata) E334 H. aoronymphium (D. phalerata) "AORO "
E336 H. aoronymphium (D. phalerata)
E330 H. aoronymphium (D. phalerata)
E2F H. aoronymphium {D. phalerata) N399 H. aoronymphium {D. neotestacea)
N66 H. aoronymphium {D. neotestacea)
F167 Howardula sp. (D. falleni)
" B/F" B301 Howardula sp. {D. brachynephros)
B332 Howardula sp. {D. brachynephros)
B331 Howardula sp. (O. brachynephros) 0.005 substitutions/site 72
,AC3M H. neocosmis (D. acutilabella}
"NEO" H. cf. neocosmis (D. mmda, \ 0. subocddentalis)
100 I E3M H. aoronymphium (D. phalerata, D. neotestacea) "AOHO" too IJ344 H. cf.aoronymphium (D. orientacea
9S
92 — Ft65 Howardula sp. (D. falleni) B/F" - BMi Howardula sp. (D. brachynephros)
S3 ; SPA392 Howardula sp. (Leptocera sp.) *
.f>s99 Howardula sp. (D. pseudoobscura) 96 87
. spa39i Howardula sp. (Leptocera sp. ]
.MA382 Howardulasp.(D. macroptera)
• cwa H. dominicki {Co\eopXera)
Pratylenchoides *
Subanguina *
— 0.01 substitutions/site a) first and second position transitions c) third position transitions
15-
20-
• •
15-
10-
T -r 5i 1 1 1 r .2 .3 0 .1 .2 .3 .4
b) first and second position transversions d) third position transversions
20-
15
10
-i —I r -I 1 1 r .1 .2 .3 .1 .2 .3 .4 pairwise distances pairwise distances ^ 74
SP2 Howardula sp. {Leptocera sp.)
PS99 Howardula sp. (O. pseudoobscura)
J308 cf-aoronymphium (D. orientacea)
92 j , E2F aoronymphium j j (D. phalerata) "AORO" i I I N101 H. aoronymphium < nn (D. neotestacea)
'• fyjgQ H. aoronymphium ' (0. neotestacea)
.,. _ . H. aoronymphium N124 (D.Men/)
AC314 H. neocosmis 97 (0. acutilabella)
SU321 ctneocosmis {D. suboccidentalis)
Howardula sp. F125 (D. falleni) Howardula sp. (D. brachynephros) B301 100
B331 0.05 substitutions/site Howardula sp. (D. brachynephros) 75
98 acutilabella cardini group cardini tnpunctata tnpunctata group brachynephros r 100 phalerata 96 unispina 100 I curvispina 99 curvispina r fallen! _toori fallen! 97 L fallen!faL innubila 78 deflecta palustris 100 quinana group Li 001 subpalustris subpalustris 100 I limbata limbata — munda 6(1 quinana f- occidentalis 76 J suboccidentalis L sutjoccidentalis — suboccidentalis 100IUU p-r— recens 88 I 1- recens I— subquinariasubauinaria transversa nigromaculata 100 I guttifera w guttiferaI kuntzei 75 - histrio histrio group macroptera macroptera group 100 neotestacea 86 orientacea testacea group testacea putrida immigrans busckii - histrioides 98 subobscura affinis .pseudoobscura 99 melanogaster yakuba . 0.05 substitutions/site 76
— brachynephros p — phalerata P — unispina P — curvispina
falleni
innubila
deflecta
palustris
subpalustris
limbata
— munda
— quinana
— occidentalis p — suboccidentalis p — recens p -i- transversa
— subquinaria
nigromaculata
guttifera
kuntzei
neotestacea
orientacea
testacea
putrida pseudoobscuta MA
' immigrans
< macroptera •
' acutilabella •
falleni •
p brachynephros •
B ^ phalerata
' kuntzei- neocosmis AC — munda-
• suboccldentalis •
-SU/ MU — •transversa- cf. neocosmis
aoronymphium • recens- (Europe) E — • put rid a-
testaoea-
aoronymphium ^ neotestacea (America)
^ orientacea cf. aoronymphium J Drosophila Howardula 78
APPENDIX TWO:
POTENTIAL HOST RANGES OF DROSOPHILA - PARASITIC NEMATODES:
AN EXPERIMENTAL PHYLOGENETIC ANALYSIS 79
POTENTIAL HOST RANGES OF DROSOPHILA - PARASITIC NEMATODES:
AN EXPERIMENTAL PHYLOGENETIC ANALYSIS
Steve J. Perlman'* and John Jaenike'
' Department of Ecology and Evolutionary Biology
University of Arizona
Tucson. Arizona 85721
"Department of Biology
University of Rochester
Rochester. New York 14627
* corresponding author email; [email protected]
Tel: 520-626-8455
Fax:520-621-9190 80
Abstract. - Surprisingly little is known about what determines a parasite's host range, which is essential in enabling us to predict the fate of novel infections. In this study, we evaluate the importance of both host and parasite phylogeny in determining the ability of parasites to infect novel host species. Using experimental lab assays, we infected 24 taxonomically diverse species of Drosophila flies (Diptera: Drosophilidae) with five different nematode species (Tylenchida: Allantonematidae: Howardula.
Parasitylenchiis), and measured parasite infection success, growth, and effects on female host fecundity (i.e. virulence). These nematodes are obligate and direct parasites of mushroom-feeding Drosophila, particularly quinaria and testacea group species, often with severe fitness consequences on their hosts. We show that the potential host ranges of the nematodes are much larger than their actual ranges, even for parasites with only one known host species in nature. Novel hosts that are distantly related from the native host are much less likely to be infected, but among more closely related hosts, there is much variation in susceptibility. Potential host ranges differ greatly between the related parasite species. .A.11 nematode species that successfully infected novel hosts produced infective juveniles in these hosts. Most novel infections did not result in significant reductions in the fecundity of female hosts, with one exception: the host specialist P. nearcticus sterilized all quinaria group hosts, only one of which is a host in nature. The large potential host ranges of these parasites, in combination with the high potential for host colonization due to shared mushroom breeding sites, explain the widespread host switching observed in comparisons of nematode and Drosophila phylogenies. 81
Key words. - Coevolution, host-parasite interactions, insect-parasitic nematodes, mycophagy, parasitism, phylogeny. 82
We still understand surprisingly little about what determines a parasite's host range in the wild. This has important implications, for example, in enabling us to predict the outcome of novel infections. This is particularly timely considering the increasing number of emerging diseases observed in recent years (Daszak et al. 2000).
Understanding host range determinants is also crucial in the biological control of pest species, where pathogens or parasitoids for e.xample, are introduced with the hope that they will suppress a target species without affecting other non-pest species (Simberloff and Stiling 1996).
A parasite's actual host range is determined by two components. First, a host must be intrinsically suitable for parasite infection, development and transmission (Solter and Maddox 1998). This intrinsic suitability defines a parasite's potential host range, and is determined by physiological and biochemical properties of the host. Because of the genetic basis of these properties, intrinsic suitability is likely to have an important phylogenetic component, depending on rates of host and parasite evolution. A parasite's actual host range, however, is a subset of its potential host range and is affected by a
number of ecological factors which can exclude what would otherwise be a perfectly suitable host, such as geography, temperature (Jaenike 1995), host breeding site (Jaenike
1985), other competing parasite species (Dobson 1985), and a minimum host threshold
density required for parasite population persistence (Anderson and May 1978). The
recent, widespread movement of organisms among and between continents is providing
many opportunities for the establishment of new host-parasite associations. Such
associations are due to parasites with broad potential host ranges but which previously 83
lacked the ecological opportunities to colonize novel hosts. Identifying the factors other than sympatry that both permit and prevent the successful establishment of novel associations presents a clear challenge to biologists.
The goal of this study is to assess the importance of both host and parasite phylogeny in determining a parasite's potential host range. The few experimental studies of the phylogenetic determinants of host range have generally found that parasites are better able to infect species closely related to their natural hosts than distantly related species (Futuyma et al. 1995: Moore and Gotelli 1997; Reed and Hafner 1997;
Nishigushi et al. 1999; Morehead and Feener 2000). This is not surprising, as distantly related hosts are likely to represent an environment very different from that to which the parasites are adapted. These studies, however, have considered only a small number of potential host species and have been usually restricted to only one or two parasite species. Thus, the nature and generality of the decline in host suitability with genetic distance from the natural host remains unkown in virtually all host-parasite associations.
The importance of phylogeny as a determinant of infection success depends on
the mechanisms and evolutionary rates of host resistance and counter-resistance by the
parasites. If there is rapid biochemical and physiological evolution by parasites and
counter-evolution by hosts, then closely related host species may show significant
variation in susceptibility to specific parasites. For example, the ability of Leishmania
trypanosomes to successfully infect their phlebotomine sand fly hosts appears to be
largely determined by rapidly evolving and variable cell surface phosphoglycan
molecules (Sacks 2001). Among closely related phlebotomine sand flies that harbor 84
Leishmania, phylogeny would therefore not be a good predictor of host switching potential.
Another approach to understanding the potential for host switching by parasites is to compare the phylogenies of hosts and parasites. Such a comparison can be used to estimate the minimum frequency of host switches and to identify host lineages that are prone to colonization, as well as the degree to which such host shifts are constrained by phylogeny (Huelsenbeck et al. 2000; Ricklefs and Fallon 2002). Phylogenies that are highly congruent with each other indicate that little or no host-switching has occurred over extended periods of time. Phylogenetic analysis is also useful in identifying factors that influence host switching. For example, Becerra (1997) showed that plant chemistry was a better predictor of host switching by specialist herbivorous chrysomelid beetles than plant phylogeny. Tompkins and Clayton (1999) recently showed that barb size in the feathers of swiftlets was a better predictor of host suitability for louse parasites than was host phylogeny. Thus, in these cases, specific attributes of the hosts, rather than overall genetic similarity, govern a parasite's host range.
In this study we use an experimental approach in a phylogenetic context to examine the potential host ranges of parasitic nematodes of mycophagous Drosophila.
These parasites are horizontally transmitted by their host Drosophila when they visit
mushroom breeding sites. Because individual mushrooms are often oviposited on by
multiple Drosophila individuals from several species (Jaenike and James 1991), there is
substantial potential for colonization of novel host species by nematodes over
evolutionary time scales. Indeed, a comparisca of Drosophila and nematode phylogenies 85
reveals widespread host switching and apparently rapid incorporation of novel hosts into the parasites' host ranges (Perlman et al. 2002). We experimentally exposed 24 species of Drosophila to five different nematode species, and asked the following specific questions. 1) Is a nematode more likely to infect a new host species that is closely related to that parasite's current host? 2) Is a nematode more likely to infect a new host species that is already infected by a related parasite? 3) If a parasite is highly virulent to one particular host, does it tend to be especially virulent to closely related hosts?
Natural History of Drosophila-Nematode Associations - Mushroom-feeding Drosophila flies, particularly those from the closely related quinaria and testacea species groups, are some of the most abundant and diverse visitors to decaying mushrooms in temperate and boreal forests (Grimaldi and Jaenike 1984; Kimura and Toda 1989; Wertheim et al.
2000). These flies are commonly infected by parasitic nematodes (Tylenchida:
Allantonematidae: Howardula, Parasitylenchus) (reviewed in Jaenike and Perlman
2002). At least 25 mycophagous Drosophila species are known to be infected by various allantonematid nematodes, including at least eight species of Howardula and two of
Parasitylenchiis. .<\ctual nematode host ranges vary widely, from host specialists known only from one host species in nature (e.g. P. nearcticus), to host generalists. such as H. aoronymphium, which infects at least nine species on two continents (Poinar et al. 1997;
Perlman et al. 2002). These parasites often have severe consequences on host fitness,
including female sterility, reduced male fertility and mating success, and reduced survival. 86
The life cycle of these nematodes is as follows. An inseminated female nematode penetrates a fly larva by piercing through its cuticle with a stylet (Welch 1959). When the adult fly emerges, the female nematode has developed to a characteristic motherworm stage, and shortly begins shedding infective juveniles into the hemocoel of the fly. These infectives are then released through the ovipositor and anus of flies as they visit mushrooms, where the nematodes mate and continue the cycle. The life cycle of
Parasitylenchus differs from that of Howarclula in having one e.xtra sexual generation inside the host (Welch 1959). Motherworms produce offspring (Fl), which mate inside the fly and then release infectives (F2). Therefore, unlike most parasitoids and many herbivorous insects, in which host acceptance and development in that host are carried out by the mother and her offspring, respectively (e.g. van Kiinken 2000; Morehead and
Feener 2000), in Howaniula and Parasitylenchus, the same individuals (inseminated females) are responsible for both infection of the host and successful development inside it. This correlation between preference and performance and the lack of any intermediate hosts in the life cycle greatly simplify investigating the evolution of host range in these nematodes.
MATERIALS AND METHODS
Drosophila - We performed controlled laboratory infections of 24 Drosophila species
(Table I) with five different nematode species. We focused on the commonly infected, closely related testacea and qiiinaria species groups, including all four described species in the testacea group, and 13 out of 28 known species in the quinaria group. All of the 87
flies breed in mushrooms, except for three quinaria group species — D. limbata, D. palustris and D. quinaria — that breed in decaying vegetation. All flies are descended from multiple recently collected wild-caught flies, and were maintained in the lab on
Instant Drosophila Medium (Carolina Biological Supply, Burlington, North Carolina) supplemented with Agaricus bisponis mushroom. The three non-mycophagous species were maintained on medium supplemented with cucumber. The use of fly and nematode cultures established from multiple wild-caught individuals reduces the risk of unusual, strain-specific results. Flies were maintained at 22 °C on a 12:12 lightidark cycle.
Nematodes - Nematode stocks were established from multiple nematodes obtained from
infected wild-caught adults. We used five different nematode species, and two different
populations (European and North American) of H. aoronymphium (Table 2). Each
nematode species was maintained on a suitable host species in mason jars containing
Instant Drosophila Medium and mushrooms as breeding sites. Because infected adult
hosts shed nematodes into the mushrooms, many flies that develop as larvae in these
mushrooms become infected.
All of the nematodes in our study except for H. aoronymphium are known from
only one host species in the wild; these host specialists were collected from and
maintained on their native host in the lab. In contrast, H. aoronymphium infects at least
nine species on two continents. European H. aoronymphium was collected from and
maintained on D. phalerata. North American H. aoronymphium was collected from D.
falleni, D. neotestacea and D. ptitrida, and maintained on D. neotestacea. Previous 88
studies have shown that H. aoronymphium does not comprise host races or cryptic species (Jaenike and Dombeck 1998; Perlman et al. 2002). D. phalerata and D. neotestacea are the native European and North American hosts that are most commonly infected and in which motherworm size is typically greatest (Jaenike 1992; Gillis and
Hardy 1997). We ruled out the possibility that there were any cryptic nematode species
in our cultures by molecular characterization of motherworms, using PCR amplification
of species-specific DNA sequences (ITSl rDNA) (Perlman et al. 2002). Our infection
results were also consistent throughout the duration of the study.
Phylogenetic relationships of Drosophila and their nematodes - As measures of genetic
distance, we used branch lengths from Drosophila and Howardula phylogenies obtained
from mtDNA cytochrome c o.xidase sequences and estimated using maximum likelihood
methods (Perlman et al. 2002). In order to measure the genetic distance between
Parasitylenchus and Howardula. we sequenced a portion of P. nearcticiis mtDNA
cytochrome c oxidase subunit I, using primers developed by Folmer et al. (1994). DNA
extraction procedures and PCR conditions are described in Perlman et al. (2002).
Experimental infections - All 24 Drosophila hosts were experimentally exposed to all
nematode species except one. Because we were unable to maintain Howardula sp. F in
culture for the duration of the experiment, we tested only 12 Drosophila species for this
nematode. 89
To collect fly eggs for infection, uninfected females were placed in petri dishes with food plugs made from blended mushrooms, agar, and water. Nematodes were obtained by grinding one to two-week-old infected hosts in saline solution. Slurry containing ~200 larval nematodes was pipetted onto a 0.4g piece of A. bisporus mushroom. On the following day, 20 host eggs were placed on the mushroom pieces in vials with moistened cheesecloth. Mushroom was added to the vial when necessary to prevent starvation of fly larvae. Upon emergence, adult flies were collected and transferred to vials containing Instant Drosophila Medium and fresh A. bisporus. For the three non-mycophagous species, emergent adults were put in vials containing Instant
Drosophila Medium and fresh cucumber. Flies were frozen 7 days after adult emergence.
For Howardida infections, we recorded for each fly the number and size of motherworms, and the number of mature eggs (stage 10 and later) in fly ovaries.
Motherworm size was determined by tracing with a camera lucida and then measuring their longitudinal section area with a planimeter. For P. nearcticus infections, we did not measure motherworm size or number, but instead recorded the number of F1 worms.
This is because Parasitylenchus motherworms are much smaller than those of Howardida and often disintegrate soon after host adult emergence. The number of 20-egg replicate
vials per treatment ranged from 3 to 35, because some host species were more resistant to
infection and required more cultures to obtain an adequate sample size for determinations
of motherworm size and host fecimdity. 90
In order to gauge the relative suitability of a potential host species, parasite performance was compared to that in a standard host species. The standard host species were D. phalerata for European H. aoronymphium, D. neotestacea for North American
H. aoronymphium, D. orientacea for H. cf. aoronymphium, D. acutilabella for H. neocosmis, D.falleni for Howardula sp. F, and D. recens for P. nearcticus. These are the hosts that are most commonly (or exclusively) used in the wild and in which parasite performance is typically greatest.
Data were analyzed using the statistics packages JMP 4.0.4 (1989-2001) and SAS
8.0.2(1999-2001).
Infection success - Nematode infection success in a given host species was estimated as the least square mean number of Howardula motherworms per fly (or mean
Parasitylenchus F1 per fly), obtained from an analysis of variance, with host species and vials nested within hosts as effects in the model (worms per fly = host species + vial [host species]) (LSMEANS statement in PROC GLM in SAS).
Because the host species are not independent (due to phylogeny), we could not quantify the effect of host genetic distance on infection success using simple regression.
We instead took a conservative approach and performed a regression using clade averages. For each parasite species, we started with the standard host, and then determined the average relative infection success (i.e. the mean number of motherworms per fly in the test species relative to the standard species) for subsequent, more distantly related clades. For example, for European H. aoronymphium, we began with 1) Z). 91
phalerata (standard host, genetic distance = 0), followed by 2) D. brachynephros, 3) D. falleni, 4) the average of the remaining quinaria group species, 5) the cardini + iripimctata group clade, 6) the testacea group, 7) D. immigrans, 8) D. busckii and 9) the obscura group. While these clades share some short branches and are therefore not completely independent, this test is conservative because the performance data for large clades are collapsed into one average score. Our regression model took the form of a negative exponential, Y(d) = Y(0) * exp(-ad), where Y(d) is relative infection success for a clade whose genetic distance is'd' from the standard host, Y(0) is the infection success on the standard host, and 'a' describes the decline in infection success with genetic distance. We used a weighted least-squares regression, with Y(d) weighted by the reciprocal of the error variances, i.e. by the number of species per clade, to obtain the best linear unbiased estimators (SAS Instioite 1988). Y(d) is never negative, as relative infection success can never be less than zero. Regressions were plotted in SigmaPlot
5.00(1986-1999).
To visualize the effect of parasite phylogeny on potential host range, relative infection success (for a certain host species infected by the different nematodes in the study) is plotted against the genetic distance between the test parasite and the native parasite of that host species.
Parasite growth and reduction in host fecundity - We performed analyses of covariance to test whether motherworm size depended on host species, host sex, and the number of motherworms per fly. For each parasite species, we compared motherworm size in novel host species with that in the standard host species, using Duimett's test, which is designed 92
to make multiple comparisons against a single control (Kuehl 2000). We also performed analyses of covariance to test whether female fecundity depended on infection status and the number of motherworms per fly. For each host species, we compared the number of eggs in uninfected females with that in hosts infected with different parasite species.
We asked whether nematodes grow larger in species which they infect at higher rates, by testing for correlations between infection success and nematode motherworm size, using phylogenetic independent contrasts (Felsenstein 1985). implemented in
COMPARE 4.4 (Martins 2001). This correlation was determined only for H. aoronymphium. as the other nematodes infected too few host species. For this analysis, data were pooled for the European and North American H. aoronymphium infections.
For each host species, we regressed motherworm size as a function of motherworm
number per fly, and used the predicted values for hosts infected with a single
motherworm.
We also asked whether Drosophila species in which nematodes are larger have
reduced fecundity, by testing for correlations between motherworm size and the reduction
in host fecundity due to nematode parasitism. This was estimated as the difference in the
number of mature eggs between infected and uninfected females. As above, we only
performed this analysis for H. aoronymphium, and pooled European and North American
infections. For each host species, we regressed host fecundity as a function of
motherworm number per fly, and used the predicted values for hosts infected with a
single motherworm. 93
RESULTS
Potential host ranges and Howardula motherworm size in novel hosts - Fig 1. reports the infection rates (total Howardula motherworms per fly and total Parasitylenchiis F1 per fly) of the different infections. Nematode potential host ranges fall into two categories
(Fig. 2). The four species that are known from only one host in nature {P. nearcticus, H. cf. aoronymphiitm. H. neocosmis, and Howardula sp. F) successfully infected between one-fifth and one-third of the Drosophila species tested. In contrast, host ranges of
European and North American H. aoronymphium were much larger, being able to infect, respectively, 19 and 20 of 24 host species in our study. The potential host ranges of the two populations of//, aoronymphium were identical, with the exception that the
European H. aoronymphium did not infect D. orientacea.
For all nematodes tested, there was a clear negative relationship between relative
infection success and host genetic distance from the standard host (Fig 2). Infection
success, in general, declines asymptotically towards zero with increasing genetic distance
from the standard host. Fig. 3 shows the relationship between infection success of
various parasites in five specific host species - D. acutilabella, D. neotestacea, D.
phalerata, D. testacea and D. transversa. There is no consistent relationship between
infection success and parasite phylogeny (Fig. 3). That is, parasites distantly related to the
natural parasite of a given host species may be just as successful as more closely related
parasite species.
Howardula motherworms grew sufficiently to produce infective juveniles in all
host species that were successfully infected (Fig. 4). For the specialist Howardula 94
species (//. neocosmis, H. cf. aoronymphium, and H. sp. F), there was no clear pattern in the size of motherworms in novel hosts, being significantly smaller than in the native host in 3 out of 12 successfully infected novel hosts, and significantly larger in 2. Of the novel hosts successfully infected by the generalist H. aoronymphium, only D. orientacea produced worms that were significantly smaller than any native host species (p < 0.05).
In no novel host species were H. aoronymphium motherworms larger than in the "best" native host species. Motherworms of European and North American H. aoronymphium did not differ significantly in size, and we therefore present size data only for the North
American sample. For H. aoronymphium, motherworm size across host Drosophila was positively correlated with infection success (Fi.n = 16.77, p< 0.001; r- = 0.49).
Drosophila female fecundity in novel infected hosts - Only P. nearcticus significantly reduced female fecundity in all of the hosts that it successfully infected (Fig. 5d), sterilizing all quinaria group hosts, including its only known host in the wild, D. recens.
Species in the testacea group that were successfully infected by P. nearcticus were not rendered sterile, but did have significantly reduced fecundity. The other nematode species caused significant reductions in the fecundity of only 6 out of 22 novel hosts (Fig.
5). The North American and European samples of H. aoronymphium reduced the fecundity of their hosts similarly; we therefore only present the data for fecundity reduction due to the North American motherworms (Fig. 5a).
For H. aoronymphium, motherworm size was positively correlated with the reduction in female host fecundity across host Drosophila (Fi.i? = 5.78, p = 0.028; r^ = 95
0.25). This effect however, was due largely to two species, D. orientacea (which produces small H. aoronymphium motherworms with no reduction in host fecundity) and
D. neotestacea (which produces large motherworms with high fecundity reduction).
DISCUSSION
Our experimental infections reveal that the Z)ro5op/i/7a-parasitic nematodes have potential host ranges that are substantially greater than their actual host ranges. For example, the generalist H. aoronymphium was able to infect all species but one (20/21) tested in the subgenus Drosophila. More strikingly, parasites that are host specialists in nature, being known from only one species, are able to infect several species. For example. P. nearcticus successfully infected and grew in 7 of 23 novel host species tested, including two species in the testacea group, which diverged from the native host
D. recens -15-20 million years ago (extrapolating from genetic distances obtained in
Perlman et al. [2002] and estimated quinaria group divergences [Spicer and Jaenike
1996; Shoemaker et al. 1999]). Thus, P. nearcticus has the potential to utilize additional host species, should Drosophila or P. nearcticus distributions change. A large potential host range, coupled with interspecific aggregation of Drosophila within individual
mushrooms (Jaenike and James 1991), provides the basis both for colonization of novel
hosts and subsequent host switching. These two factors are likely responsible for the
frequent host switching and lack of congruence observed between Drosophila and
Howardula phylogenies (Perlman et al. 2002). 96
Despite their broad potential host ranges, our data do reveal adaptation to specific host lineages by these parasites, as shown by the finding that host phylogeny is an important determinant of infection success. With exceptions discussed below,
Drosophila species closely related to the natural host were themselves intrinsically suitable hosts. Distantly related host species were unsuitable hosts for any of the parasites we tested. Even the generalist H. aoronymphium, which successfully infected
20 of 21 species tested in the subgenus Drosophila, had extremely limited ability to infect any species outside this subgenus, including D. qffinis, D. busckii. and D. subobscura
(this study), and D. melanogaster (Jaenike 1992). While the nematodes we tested are restricted to species of the subgenus Drosophila. other Howardula and Parasitylenchus species have been reported from more distantly-related Drosophila (Jaenike and Perlman
2002), as well as other Diptera and Coleoptera (Siddiqi 2000), showing that these hosts are not intrinsically unsuitable to parasitism by these nematodes.
Although host phylogeny is an important determinant of suitability, closely related Drosophila can differ substantially in their susceptibility to particular parasites.
For example, D. neoresiacea, D. orientacea and D. testacea differ widely in their susceptibility to different nematodes. These three species are estimated to have diverged
-1-1.5 million years ago (Spicer and Jaenike 1996; Perlman et al. 2002); D. testacea and
D. orientacea can produce fertile hybrid offspring in the lab (Grimaldi et al. 1992). D. orientacea is the only testacea group species resistant to infection by European H. aoronymphiiim. yet it is much more susceptible to Japanese H. cf. aoronymphium than either D. testacea or D. neotestacea. Of the three host species, only D. testacea is 97
susceptible to P. nearcticus. As a second example, D. recens was heavily infected with
P. nearciicus in our lab assays and is the only known host for this nematode in the wild.
In contrast, D. subquinaria, which can produce fertile hybrids with D. recens and diverged from it -1.2 million years ago (Shoemaker et al. 1999), is almost completely resistant to P. nearcticus infection. These results show that levels of resistance to parasites can evolve quite rapidly, thus blurring the effect of phylogeny on host range.
Whereas related species of hosts tended to be similar in their susceptibility to particular parasites, related species of parasites often differed in their ability to infect various host species. For example, Japanese H. cf. aoronymphium infected only 6
Drosophila species at an infection rate >10% that of the standard species, in contrast to
20 species for its close relative H. aoronymphium. Parasite phylogeny (i.e. natural infection by a parasite that is related to the novel parasite) was not a good predictor of infection success in novel host-parasite combinations in this study. For e.xample, D. subobscura was not infected by P. nearcticus, despite being the only host in our study other than D. recens known to be infected with Parasitylenchus {P. diplogenus) in nature
(Gillis and Hardy 1997).
The potential host range of the generalist H. aoronymphium differed greatly from the other nematodes. This indicates that the broad host range of this species in nature is due at least in part to a broad potential host range, rather than a greater rate of encounter with potential host species. The potential host ranges of the European and North
American populations of//, aoronymphium, as well as motherworm size and fecundity reduction in novel hosts, were nearly identical, with the only difference being that 98
European H. aoronymphium did not infect D. orientacea. Besides having nearly identical potential host ranges, the European and North American populations of H. aoronymphium are genetically identical at sequences obtained from rapidly evolving ribosomal ITSl and mitochondrial loci (Perlman et al. 2002). Furthermore, the North
American population does not appear to harbor any genetic variation for host specialization (Jaenike and Dombeck 1998). These observations suggest that H. aoronymphium recently experienced a rapid expansion in geographic range, in the process incorporating novel host species into its host range.
One concern about this e.xperiment is whether maintaining nematodes in the lab on one fly species may have compromised their ability to infect other hosts. We think this is unlikely to be the case, for several reasons. First, all of the infections were consistent through time and with known infections in the wild. Second, many novel species were infected at rates that were similar to those for the native host, demonstrating that the parasites did not lose the ability to infect novel hosts as a result of lab
maintenance on the native host. For instance, experimental maintenance of North
American H. aoronymphium on either D. putrida or D. falleni for 25 generations did not
bring about changes in performance on these alternative hosts (Jaenike and Dombeck
1998). Similarly, in the present experiment, the H. aoronymphium from North America
and Europe were maintained in culture on D. neotestacea and D. phalerata, respectively,
yet they had essentially identical host ranges in our experimental assays. With respect to
hosts, none of the Drosophila species was infected at high rates by all of the parasites,
demonstrating that infections were not due to general loss of resistance as a result of 99
laboratory culture. Nevertheless, an important future direction would be to assess intraspecific variation in resistance to both native and novel nematode parasitism, as this may uncover a source of some of the residual, non-phylogenetic variation in resistance.
Our experimental infections also provided data on virulence, in terms of reduced host fecundity, of novel associations. While some novel infections in nature are highly virulent (Bull 1994; Fooden 1994), it is unclear whether this pattern is general, because benign novel infections are unlikely to be reported, or even noticed. Other than infections due to P. nearcticus, most of the novel infections in our study resulted in modest or no reductions in host fecundity. Furthermore, parasite-induced fecundity reduction was rarely greater in the novel host than in the native host (but see Perlman and
Jaenike 2002). It is important to stress that we only measured the effect of parasites on one component of host fimess. For example, two of the four species in the testacea species group - D. neotestacea and D. puirida, but not D. testacea or D. orientacea, experienced severe mortality when infected with a novel Howardiila (Perlman and
Jaenike 2002).
Our data show that there can be a host phylogenetic component to the parasite- induced reductions in fecundity. For instance, P. nearcticus effectively sterilized all quinaria group hosts, but neither of the testacea group species it was able to infect. Host species belonging to the cardini group were much more adversely affected by H. neocosmis and H. aoronymphium than hosts from the quinaria group.
Previous phylogenetic analyses of hosts and parasites have revealed that
associations resulting from host switching are common (Futuyma and McCafferty 1990; 100
Werren et al. 1995), particularly switches to hosts within a restricted phylogenetic range
(Ricklefs and Fallon 2002). Thus, particular clades of parasites may be limited to particular clades of hosts for extended evolutionary periods, but with new host-parasite associations continually being generated, as is the case with Drosophila and their nematode parasites. Indeed, parallel cladogenesis appears to be rare, being limited to associations where parasite transmission and dispersal are highly restricted (Hafner and
Nadler 1988; Moran et al. 1995). While host switching over evolutionary time scales has been demonstrated repeatedly, attempts to quantify the present day potential for host switching within parasite species have met with limited success, suggesting that successful host sw itches are rare events and that most parasites are locked into their current host association (Futuyma et al. 1995; van Klinken 2000). What is particularly striking about the Dro5op/i//a-nematode associations is the concordance between the widespread host switching that has occurred over evolutionary time scales (Perlman et al.
2002). and the relatively large potential host ranges exhibited by both host-generalist and host-specialist species of nematodes.
ACKNOWLEDGMENTS
We thank Andy Beckenbach, Irene Eijs, Ray Huey, Masahito Kimura, and Chuck Taylor for collecting insects, Irene Dombeck and Jake Russell for assistance in the lab, and
Wayne Maddison, Teri Markow, Nancy Moran and Howard Ochman for comments.
This work was supported by National Science Foundation grants to JJ (most recently
DEB-0074141). 101
LITERATURE CITED
Anderson, R. M. and May, R. M. 1978. Regulation and stability of host-parasite
population interactions. I. Regulatory processes. J. Animal. Ecol. 47:219-247.
Becerra, J. X. 1997. Insects on plants: Macroevolutionary chemical trends in host use.
Science 276:253-256.
Bull. J. J. 1994. Virulence. Evolution 48:1423-1437.
Daszak, P.. .A.. A. Cunningham and A. D. Hyatt. 2000. Wildlife ecology - emerging
infectious diseases of wildlife - threats to diversity and human health. Science 287:443-
449.
Dobson, A. P. 1985. The population dynamics of competition between parasites.
Parasitology 91;317-47.
Felsenstein, J. 1985. Phylogenies and the comparative method. Am. Nat. 125:1-15.
Folmer. O., M. Black. W. Hoeh, R. Lutz and R. Vrijenhoek. 1994. DNA primers for
amplification of mitochondrial cytochrome c oxidase subunit I from diverse meiazoan
invertebrates. Mol. Mar. Biol. Technol. 3:294-299.
Fooden. J. 1994. Malaria in macaques. Int. J. Primatol. 15:573-596.
Futuyma, D. J.. M. C. K.eese and D. J. Funk. 1995. Genetic constraints on
macroevolution: the evolution of host affiliation in the leaf beetle genus Ophraella.
Evolution 49:797-809.
Futuyma D. J and S. S. McCafferty. 1990. Phylogeny and the evolution of host plant
associations in the leaf beetle genus Ophraella (Coleoptera, Chrysomelidae). Evolution
44:1885-1913 102
Gillis, J. E. M. and I. C. W. Hardy. 1997. Nematode parasitism in a northern European
drosophilid community. Ent. Exp. et App. 84:275-91.
Grimaldi, D. and J. Jaenike. 1984. Competition in natural populations of mycophagous
Drosophila. Ecology 65:1113-1120.
Grimaldi, D. A., A. C. James, and J. Jaenike. 1992. Systematics and modes of
reproductive isolation in the Drosophila tesiacea group (Diptera: Drosophilidae). Ann.
Ent. Soc. Amer. 85:671-685.
Hafner. M. S. and S. A. Nadler. 1988. Phylogenetic trees support the coevolution of
parasites and their hosts. Nature 322:258-60.
Huelsenbeck, J. P.. B. Rannala and B. Larget. 2000. A Bayesian framework for the
analysis of cospeciation. Evolution 54:352-364.
Jaenike. J. 1985. Parasite pressure and the evolution of amanitin tolerance in Drosophila.
Evolution 39:1295-1301.
Jaenike, J. 1992. Mycophagous Drosophila and their nematode parasites. Am. Nat.
139:893-906.
Jaenike, J. 1995. Interactions between mycophagous Drosophila and their nematode
parasites: from physiological to community ecology. Oikos 72:235-44.
Jaenike. J. 1996. Rapid evolution of parasitic nematodes: not. Evol. Ecol. 10:565.
Jaenike, J. and I. Dombeck. 1998. General-purpose genotypes for host utilization in a
Dro5op/i//a-parasitic nematode. Evolution 52:832-840.
Jaenike, J. and A. C. James. 1991. Aggregation and the coexistence of mycophagous
Drosophila. J. Anim. Ecol. 60:913-928. 103
Jaenike, J. and S. J. Perlman. 2002. Ecology and evolution of host-parasite associations:
mycophagous Drosophila and their nematode parasites. Ant. Nat. in press.
JMP 4.0.4. 1989-2001. SAS Institute. Gary, N. Carolina, USA.
Kimura, M. T. and M. J. Toda. 1989. Food preferences and nematode parasitism in
mycophagous Drosophila. Ecol. Res. 4:209-18. van Klinken. R. D. 2000. Host-specificity constrains evolutionary host change in the
psyllid Prosopidopsylla Jlava. Ecol. Enl. 25:413-422.
Kuehl, R. O. 2000. Design of experiments: Statistical principles of research design and
analysis. Second edition. Duxbury Press, Pacific Grove. CA.
Manins, E. P. 2001. COMPARE, version 4.4. Computer programs for the statistical
analysis of comparative data. Distributed by the author via the WWW at
http://compare.bio.indiana.edu Department of Biology, Indiana University,
Bloomington IN.
Moore, J. and N. J. Gotelli. 1996. Evolutionary patterns of altered behavior and
susceptibility in parasitized hosts. Evolution 50:807-19.
Moran N. A.. C. D. vonDohlen and P. Baumann. 1995. Faster evolutionary rates in
endosymbiotic bacteria than in cospeciating insect hosts. J. Mol. Evol. 41:727-731.
Morehead, S. A. and D. H. Feener. 2000. An experimental test of potential host range in
the ant paiasiioid Apocephalus paraponerae. Ecol. Ent. 25:332-340.
Nishiguchi, M. K., E. G. Ruby and M. J. McFall-Ngai. 1998. Competitive dominance
among strains of luminous bacteria provides an unusual form of evidence for parallel
evolution in sepiolid squid-Vibrio symbioses. i4pp/. Env. Microbiol. 64:3209-3213. 104
Perltnan, S. J. and J. Jaenike. 2002. Evolutionary dynamics of virulence in Drosophila
and their parasitic nematodes. Unpublished ms.
Perlman, S. J., G. S. Spicer, D. D. Shoemaker and J. Jaenike. 2002. Associations between
mycophagous Drosophila and their Howardula nematode parasites: a worldwide
phylogenetic shuffle. Unpublished ms.
Poinar, G. O. Jr.. 1. Dombeck and J. Jaenike. 1997. Parasitylenchus nearciicus sp. n.
(Allantonematidae; Tylenchida) parasitizing Drosophila (Drosophilidae: Diptera) in
North America. Fund. Appl. Nemai. 20:187-90.
Poinar. G. O. Jr.. J. Jaenike and D. D. Shoemaker. 1998. Howardula neocosmis sp. n.
(Tylenchida: Allantonematidae) parasitizing North American Drosophila (Diptera:
Drosophilidae) with a key to the species of Howardula. Fundam. Appl. Nematol.
21:547-552.
Reed. D. L. and M. S. Hafner. 1997. Host specificity of chewing lice on pocket gophers:
a potential mechanism for cospeciation. J. Mammal. 78:655-660.
Ricklefs. R. E. and S. M. Fallon. 2002. Diversification and host switching in avian
malaria parasites. Proc. R. Sac. Lond. B 269:885-892.
Sacks. D. L. 2001. Leishmania-saxvd fly interactions controlling species-specific vector
competence. Cell. Mcroft/o/. 3:189-196.
SAS Institute. 1988. SAS/ST AT User's Guide. SAS Institute Inc., Gary NC, USA.
SAS 1999-2001. Windows version 8.02. SAS Institute Inc., Gary NC, USA.
Shoemaker, D. D., V. Katju and J. Jaenike. 1999. Wolbachia and the evolution of
reproductive isolation between Drosophila recens and Drosophila subquinaria. 105
Evolution 53:1157-1164.
Siddiqi, M. R. 2000. Tylenchida: Parasites of Plants and Insects. Second Edition. CABI
Publishing, CAB International, Wallingford UK.
SigmaPlot 5.00. 1986-1999. SPSS Inc. Richmond CA.
Simberloff. D. and P. Stiling. 1996. How risky is biological control? Ecology 77:1965-74.
Solter, L. F. and J. V. Maddox. 1998. Physiological host specificity of microsporidia as
an indicator of ecological host specificity. J. Invert. Path. 71:207-16.
Spicer. G. S. and J. Jaenike. 1996. Phylogenetic analysis of breeding site use and
amanitin tolerance within the Drosophila quinaria species group. Evolution 50:2328-
2337.
Tompkins. D. M. and D. H. Clayton. 1999. Host resources govern the specificit\' of
swiftlet lice: size matters. J. Anim. Ecol. 68:489-500.
Welch, H. E. 1959. Ta.xonomy, life cycle, development, and habits of two new species of
Allantonematidae (Nematoda) parasitic in drosophilid flies. Parasitology 49:83-103.
Werren, J. H., W. Zhang and L. R. Guo. 1995. Evolution and phylogeny of Wolbachia:
reproductive parasites of arthropods. Proc. R. Soc. London, B. 251:55-71.
Wertheim. B.. J. G. Sevenster, I. E. M. Eijs and J. J. M. van Alphen. 2000. Species
diversity in a mycophagous insect community: the case of spatial aggregation vs.
resource partitioning, y. Anim. Ecol. 69:335-351. 106
Table I. Nematodes included in experimental infections and phylogenetic analysis.
Nematode designation Known host species Collection locale and host
Howarduta aoronymphium D. falleni New York (D. falleni, D. neotesiacea,
(North American) D. neotesiacea D. putrida)
D. putrida
D. recens
Howardula aoronymphium D. phalerata Holland (D. phalerata)
(European) D. immigrans
D. kuntzei
D. testacea
D. transversa
Howardula cf. D. orientacea Japan (D. orientacea)
aoronymphium
Howardula neocosmis D. acutilabella Florida (D. acutilabella)
Howardula sp. F D. falleni New York {D. falleni)
Parasitvlenchus nearcticus D. recens New York (D. recens) 107
Table 2. Drosophila species included in the study and their known nematode parasites.
species group species collection locale'^ nematode species
testacea D. neotestacea New York H. aoronymphium^
D. orientacea Japan H. cf aoronymphium'
D. putrida New York H. aoronymphium^
D. tesiacea Germany H. aoronymphium^
qumanu D. brachynephros Japan Howardula sp.'
D. falleni New York H. aoronymphium^
Howardula sp."
D. guitifera Florida
D. kuntzei Holland H. aoronymphium^
D. limbaia Holland
D. nigromaculata Japan
D. occtdentalis California
D. palustris New York
D. phalerata Holland H. aoronymphium^
D. quinaha New York
D. recens New York H. aoronymphium^
P. nearcticus
D. subquinaria Washington
D. transversa Holland H. aoronymphium' 108
Table 2 (continued).
species group species collection locale nematode species
cardini D. acuiilabella Florida H. neocosmis^
D cardini Florida
tripunctata D. tripunctata Tennessee
immigrans D. immigrans New York H. aoronymphium'
busckii D. biisckii New York
obscura D u/finis New York
D subobscura Washington P diplogeniis'
a) Gillis and Hardy (1997); b) Jaenike (1992); c) Jaenike (1996); d) Poinar et ai. (1997); e) Kimura and Toda (1989); f) Poinar et al. (1998) 109
FIGURE LEGENDS
Fig. L Infection success (total Howardula motherworms per fly and total
Parasitylenchus F1 worms per fly ± s.e, correcting for vial effects) for European and
North American H. aoronymphium, Japanese H. cf. aoronymphium, P. nearcticus, H.
neocosmis, and Howardula sp. F. Sample sizes (number of flies examined) are in
parentheses. Shaded bo.xes represent infection rates that are >10% those of the
standard hosts, and circled boxes denote native hosts. Letters signify infection rates
that differ significantly from the standard host (p < 0.05). Fly phylogeny is based on
mtDNA (COL II and III) (Perlman et al. 2002).
Fig. 2. Infection success (relative to that in the standard host species), plotted against the
genetic distance between the standard and experimental clades. Each plot represents
one nematode species. Each point represents the infection success averaged over all
species in a given clade. Points in the nonlinear regression are weighted by the number
of species they represent. Note that the regression for H. aoronymphium is pulled up
by one point representing 10 host species (indicated with an asterisk).
Fig. 3. Host suitability to infection by a parasite as a function of the genetic distance
between the natural parasite of the host species and the test parasites. Each plot
represents the results of all parasites tested on a single host species. Results are shown
for five representative host species - D. acutilabella. D. neotestacea. D. phalerata, D.
testacea and D. transversa. 110
Fig. 4. Nematode motherworm sizes corrected for number of motherworms per fly (mm"
± s.e), for A) North American H. aoronymphium, B) Japanese H. cf. aoronymphiiim,
C) H. neocosmis. and D) Howardula sp. F. Sample sizes are in parentheses. Letters
signify motherworm sizes that differ significantly from those in the standard host (p <
0.05). Standard hosts are denoted with an arrow. Note that these are all the host
species that were successfully infected by the various nematodes.
Fig. 5. Fecundity of females infected with nematodes (black bars), compared to
uninfected controls (white bars), for A) North American H. aoronymphium. B)
Japanese H. cf. aoronymphium, C) H. neocosmis. D) P. nearcticus. E) Howardula sp.
F. Sample sizes are in parentheses. Values are mean number of mature eggs ± s.e per
female. .Asterisks signify infected females differing significantly from uninfected ones
(p < 0.05). Standard hosts are denoted with an arrow. Note that these are all the host
species that were successfully infected by the various nematodes. North American Ejropean Japanese H aoronymphium H aoronymphium H cl aoronymphium Howardula sp. H. neocosmis P. nearclicus
acultabolla 1.791 0.17(60)/ 0,221 0.04(143)* 0.2510.05(70)* 0.007 ±0.013 (78). S210.23 (961> 0(79) . — cunkn 2.24 10.23 (45)r^ 1,1810.14 (78) 0(54). N/A 2.571 0.85(44) 0(81) , — liipuncMa 0.098 ± 0.039 (60). 0.052 ± 0.013 (103^ 0(66). N/A 0.086 ±0.58 (103)* 0(61). bractiynephros 0.1910.048(83)* 0.231 0.064(62)* 0(52). N/A 0(57) . 0(62) . phaletaia 1.661 0.13(77) Ipll 0.073 (2SS> 0(61). 0.271 0.059(71)* 0(56) . 0(94) . iallanl 0.8110.13 (68) 0(88). I 0(59) • 0(58) . — guiMaa 2.16±P;i4(84) w 0.831 35 (56) 0.15 ± 0.045 (48). N/A 1.1710.0W(5?). 4.8111.02 (70) limttala 1.29-l-0,20(7iyirK 0,^10,17(71) 0.065 ± 0.028 (98). N/A 0.8910,12(7))* i ocadenMis 3,00* 0.23 fl.9ip.i:S(123)« 0.621 0.10(46). N/A 1.0510.1^91(48)« 26.(^it^^l(S4)ir fecens 05310,097(87)* 0.022 ± 0.027 (52). 0(91) . 0(56) . iTsi it 1.07(2^ stdiqutntria i.39±p;i6^f^ 1,401 0,38 (102) 0(62). 0(62). 0(34). 0.75 ±0.58 (59). Irvavorsa a83iO.W^88)«V 0(81). N/A 0(51) . 25,6711;88(88)'« qiMtaiia 2,O710vl7(«);i^ OSStQJOM (88)* 0(59)« 0.3310.09(40) • 0(90) . 0.33 ±0.18 (95). - pahistns 2,0210.19 (?0)U| 0,6810,069(138)* 0,4710,081 (89). 0(19) . 0(12) . 13,88 1,52 (56)*. —nigranaculala 2,2piO.|9(52)'f 0:7510.085 (92) 0.01 ±0.089(50). N/A 2.6110.52(30) 0.80 ±0.55(60) . kunUet 2,05*0,14(1»),; 0(149). 0(35). 0.069 ± 0.037 (7). 0(78) . neolaslacea 0,7510.075 (89) 0.6810.084(79)* 0(102). 0.04 ±0.02 (82) . 0 (77). onenlaooa 0,84 10,18 (48)* 0(139) • ^810.19(12^ 0(85) . 0(19) . 0(70). leslacea 1.1810,1.1 (^s 0.1910.042(98)* 0.421 0.083(116)* 0.016 ± 0.027 (44). 0.5410.08(54) * 12,031 0.82(99)* — putnda 0.7510.075(89) 0.13 ±0.051 (58)* 0(30). 0.11 ±0.049 (47). 5,7511.18(88)* Imm^ana 0.7610,16(37) • ^710.038(11^ 0 12 ±0051 (61)* N/A 0 033 ±0.019 (54). 0.29 ±0.24 (84) . ttusdui 0.10 ±0.044 (55). 0.013 ±0.012 (117). 0(54)« 0(61) . 0.013 ± 0.012 (1I7)« 0.03 ±0.02 (108). aubobscua 0 (50). 0(123). 0(105) . N/A 0.052 ± 0.034 (41). 0(119). alfnis 0.022 ±0.017 (101 > 0(156). 0(41) . N/A 0.421 0.12(35) . 0(78) . 19/24 hosts infected 6/24 3/12 612A 8/24 (r'= 0.30, p< 0,01) (r^ = 0.92, p< 0.001) (r'= 0.96, p< 0.001) (r'= 0.67, p< 0.001) (r' = 0.51. p < 0.001)
V> c.0 19 1•D 2 n t o 10" 2 o 5 ® OS ° I !i 3 0 Lti II 0 2 0] 04 OS 04 ~ 0 0.1 0.2 0.1 04 01 02 01 04 0.2 OS 0.4 genetic distance from standard host (substitutions per site)
H aoronymphium H cf. aoronymphium Howardula sp. F. H neocosmis P. nearcticus (Europe - D. phalerata) {D. orientacea) ( D. falleni) ( D. acutilabella ) ( D. recens) -f——1 1 r f I I » -1~T—IJ guffim t60 a iintiata Huncei putitaa immtgrans atnns 0.05 suostitutions per site Drosophila motherworm size (mm^) 115 suocumana Qumana putnaa 0.05 substitutions per site 0.1 0.3 0.5 Drosophila motherworm size (mtn^) 116 icuaiateMa 226 eartiun • mpunctaea D/acfjynepfiros phsietaia fattem gumf&a limoata ocadentais recens suOqutnana ti9nsvers3 Qutnana — paiustns — mgromxuiata kunaei neotestacea oftentacea tastacea — putnaa . imm^ans _ tkiscka suoooseua atfns 0.05 substitutions per site Drosophila motherworm size (mm^) ' minoa 0.05 substitutions per site 0.2 0.4 Drosophila motherworm size (mm^) 118 guttiBra Quinana Kunaet purnaa mmgrans .05 substitutions per site Drosophila number of eggs cardni taUen gutbfara transversa '60 0.05 substitutions per site 30 50 Drosophila number of eggs 120 acutiiateaa canimi . uiQunaata oractr^nephros pnaJerata fallen guaf&a iimtata ocadentaia recens sutJQwnana transversa Quinana paiustns mgromaculata kurtza naotestacm onencacea testacea FT putiHJa ifTfTiigfans OuSdai suoooscu/a atfims —I— —1— —I 0.05 substitutions per site 15 30 45 Drosophila number of eggs 121 aeutiaMia camtni - tnpunctata oracfiynepftms pnaierata fatient gutbfaa luroata =4^ ocaOentaUs —17« recens •_|l54 suOQuinana transversa ziiL Quinana — paiustns — nigmmactMata kuntzBi neoteetaeaa onantacm testacea — outnda 'mmigrans tnjscku suoooscura affins 35 0.05 substitutions per site 15 25 Drosophila number of eggs 122 acut/aoeia faffen pnaieraa paJustrs quinana neoi^tacea - punca 0.05 substitutions per site 10 20 30 Drosophila number of eggs 123 APPENDIX THREE: EVOLUTIONARY DYNAMICS OF VIRULENCE IN DROSOPHILA AND THEIR PARASITIC NEMATODES EVOLUTIONARY DYNAMICS OF VIRULENCE IN DROSOPHILA AND THEIR PARASITIC NEMATODES Steve J. Perlman'* and John Jaenike" ' Department of Ecology and Evolutionar>' Biology University of Arizona Tucson. Arizona 85721 "Department of Biology University of Rochester Rochester. New York 14627 Email: [email protected] Phone: 520-626-8455 Fax: 520-621-9190 LRH: S. PERLMAN AND J. JAENIKE RRH: EVOLUTION OF VIRULENCE 125 Abstract. - Virulence is of central importance in host-parasite interactions, yet little is known about how it changes over extended evolutionary periods. In this study, all four species in the testacea species group of Drosophila were experimentally infected with sympatric and allopatric nematodes in the Howardula aoronymphiiim species complex, and the effect of parasite infection on three components of host fitness was determined. The Drosophila species show striking differences in their responses to infection, with reductions reaching 80% in adult lifespan. 100% in female fertility, and 90% in male fertility. Female sterility appears to be determined by the host; species that are sterilized by their local nematodes are also sterilized by the other nematodes in the H. aoronymphiiim complex. Host species that are not sterilized by their local parasite are not sterilized by other nematodes in the complex. In contrast, reductions in host adult lifespan and male fertility depend on both the host and the parasite. Whereas all nematodes reduced the survival of their local host species equally (-40-45%), survival of two host species was drastically reduced (-80%) when infected with an allopauic parasite. Thus, virulence is evolutionarily labile in associations between Drosophila testacea group species and their Howardula parasites. The high level of virulence manifested in some host-parasite combinations is due to a lack of resistance in the hosts, perhaps as a result of recent host colonization by Howardula. These results provide evidence for a continuum of host-parasite interaction, with initial host susceptibility and high parasite virulence followed by responses in both the parasite, resulting in decreased host mortality, and the host, resulting in resistance to parasite-induced sterility. 126 Key words. - Coevolution, host-parasite interactions, insect-parasitic nematodes, local adaptation, parasite-induced mortality, parasite-induced sterility. 127 Empirical observations of naturally occurring host-parasite associations have provided conflicting evidence over whether virulence is a transitory stage or an equilibrium condition in the coevolution of hosts and parasites. The observation that parasites can be highly virulent in novel hosts, and that this virulence can decrease over time (Fermer &. Radcliffe 1965). indicates that high levels of virulence can be transitory phenomena (Bull 1994). However, many long-standing host-parasite associations display significant levels of virulence (Herre 1993). indicative of an evolutionar\' equilibrium (Bull 1994). Based on the assumption that virulence is correlated with parasite fitness, and therefore not transitory, much theoretical and empirical work has attempted to e.xplore how the optimal level of virulence is affected by such factors as mode of parasite transmission, population structure of hosts and parasites, within-host competition between parasite strains, level of host resistance, and the use of multiple host species (Frank 1996; Kaltz et al. 1999; Sasaki and Godfray 1999; Taylor et al. 1998; Davies et al. 2001). To reconcile these alternative views on the evolution of virulence, it is important to recognize that for some components of host fitness, the effects of virulence on host and parasite fitness are similar, whereas for others they are not. While reductions in host survival adversely affect both host and parasite fitness, reductions in host fertility typically take a much greater toll on the hosts. The evolution of virulence should thus be considered in terms of specific fitness components affected, and the degree to which these components are correlated (Koella and Agnew 1999). For e.xample, Herre (1993) showed that vertically transmitted nematode species reduce the fecundity of their fig 128 wasp hosts less than those that have some potential for horizontal transmission. This makes adaptive sense, as reproduction of vertically transmitted parasites depends on host reproduction. However. Herre (1995) subsequently suggested that these nematodes might also cause elevated host mortality. Whether or not they are fertile, wasps that die before colonizing figs do not transmit any nematodes. Thus, host survival is also important in molding the evolution of parasite virulence. Empirical studies of natural host-parasite associations reveal considerable variation in the effect that fertility-reducing parasites have on host mortality (Baudoin 1975; .Vlinchella 1985: Hard et al. 2001). .A^lthough virulence is often modeled as a parasite attribute, its e.xpression will also depend on the host in which a parasite occurs. Yet ver\' little is known about the relative contributions of the interacting host and parasite species to the manifestation of virulence. Cross-infection experiments provide an opportunity to explore these relative contributions. Such cross-infections have been used to determine whether parasites are adapted to local populations of their hosts (Ebert 1994; Lively and Jokela 1996; Imhoof and Schmid-Hempel 1998; Kaltz and Shykoff 1998; Altizer 2001). For example. Ebert (1994) showed that microsporidian parasites were more successful at infecting and killing their local crustacean hosts than more geographically distant hosts, and he argued that these parasites were locally adapted to be virulent to their hosts. Previous cross-infection studies have focused on geographic variation within a particular host species-parasite species association. Our study explores the host and parasite contributions to virulence at a higher taxonomic level - among closely related species of hosts and parasites. 129 In this study, we use cross-infections to explore the determinants of virulence and resistance in multiple components of host fitness - female fecundity, male fertility, and adult survival - manifested in natural associations between parasitic nematodes and Drosophila hosts. We focus on all known members of the Drosophila testacea species group, all of which are infected by nematodes in the Howardula aoronymphium species complex (see Fig. 1; Kimura and Toda 1989; Jaenike 1992; Gillis and Hardy 1997; .A.. J. Klarenburg, pers. comm.). This group is also ideal for study because it e.xhibits interesting variation in parasite-induced fecundity reduction; infected females of the North American species D. neotestacea and D. puirida are rendered completely sterile by their local nematodes, whereas the European species D. testacea is not. We also explore whether virulence is correlated with parasite fitness by examining the relationship between parasite reproduction (as indicated by motherworm size and number) and observed levels of virulence. Natural History of Drosophila/Howardula Associations Mushroom-feeding Drosophila flies (Diptera: Drosophilidae) are some of the most abundant insect visitors to decaying fleshy mushrooms on the forest floor and are commonly infected by parasitic nematodes (Tylenchida: Allantonematidae: Howardula, Parasitylenchus) (Jaenike 1992; Poinaret al. 1997). Inseminated female nematodes infect fly larvae by piercing through their cuticle, inside mushrooms (Welch 1959). When the adult fly emerges, the nematode motherworm releases infective nematodes (Fl) into the hemocoel of the host. These juvenile nematodes are passed from the host's 130 ovipositor and anus to new mushrooms, where they mate and continue the cycle. Thus, parasite transmission requires survival of the host. Because individual mushrooms are often oviposited on by multiple adult Drosophila, belonging to several species (Jaenike and James 1991), there are generally ample opportunities for horizontal transmission of parasites both within and among host species. If parasite reproduction increases in proportion to the diversion of resources away from host reproduction, then one might expect these parasites to cause a substantial reduction in host fecundity (Herre 1993; Jaenike 1996). Thus, to the extent that virulence depends solely on parasite evolution. Howanhila nematodes should sterilize their Drosophila hosts, but have little effect on their survival. If parasites express optimal levels of virulence, then we would expect different Drosophila/Howardiila associations to e.xhibit similar patterns of parasite virulence. We would then predict Drosophila to have lower fecundity and higher survival when infected with sympatric nematodes than with allopatric ones. MATERIALS AND METHODS Drosophila and Howardula Stocks Fly stocks were descended from multiple wild-caught females: D. neotestacea and D. putrida from Monroe County, New York (collected by J. Jaenike in 1992), D. tesiacea from Regensburg, Germany (J. Jaenike. 1990), and D. orientacea from Sapporo. Japan (M. Kimura, 1991). Flies were maintained and all experiments were carried out at 22°C on a 12:12 light:dark cycle. Stocks were maintained on Instant Drosophila 131 Medium (Carolina Biological Supply) and a piece of store-bought Agaricus bisporus mushroom. European H. aoronymphium was established from multiple nematodes obtained from infected D. phalerata collected in Holland by 1. Eijs in 1996. These were maintained in the laboratory on D. phalerata hosts. North American H. aoronymphium was established from multiple nematodes obtained from D. falleni. D. recens. D. neoiestacea. and D. puirida collected in Monroe Count>'. New York in 1992, and again in 1999. for the survival and male fertility experiment. These nematodes were maintained in the laboratory on D. neoiestacea hosts. Japanese H. aoronymphium was descended from multiple nematodes collected along with D. orientacea (collected by M. Kimura in Sapporo. Japan. 1996 and again in 1999. for the survival and male fertility experiment). These were maintained in the laboratory on D. orientacea hosts. The use of fly and nematode cultures established from multiple wild-caught individuals reduces the risk that our results would reflect unusual, strain-specific interactions. Nematodes were maintained with suitable host species in mason jars containing Instant Drosophila Medium and mushrooms as breeding sites. Because infected adult flies pass nematodes to the mushrooms, many flies that develop as larvae in these mushrooms become infected. When the prevalence of infection in culture jars was low. we placed 10-20 infected males plus 2-3 uninfected mated females of the appropriate Drosophila species in a vial with Instant Drosophila Medium plus mushroom. 132 Effect of Nematodes on Female Fecundity We measured the effect of Japanese, European and North American H. aoronymphium on the fecundity of D. testacea group females. To collect fly eggs for infection, uninfected females were placed in petri dishes with food plugs made from blended mushrooms, agar, and water. Nematodes were obtained by grinding one to two week old infected hosts in saline solution. Slurry containing -200 larval nematodes was pipetted onto a 0.4g piece of .-1. bisporus mushroom. On the following day. 20 host eggs were placed on the mushroom pieces in vials with moistened cheesecloth. Mushroom was added to the vial when necessary to prevent starvation of fly larvae. Upon emergence, adult flies were collected and transferred to vials containing Instant Drosophila Medium and fresh A. bisporus. Flies were frozen 7 days after adult emergence. For each fly we recorded the number and size of//, aoronymphium motherworms. and the number of mature eggs (stage 10 and later) in fly ovaries. Size was determined by tracing motherworms with a camera lucida and then measuring their longitudinal section area with a planimeter. Numbers of replicates per treatment varied because some host species were more resistant to infection and required more cultures to obtain an adequate sample size; numbers of vials per treatment ranged from 5 (for uninfected controls) to 35 (for infections of D. orientacea with European nematodes). An analysis of variance was performed to test whether the number of mature eggs per fly depended on host species (D. neotestacea, D. orientacea. D. putrida and D. testacea), experimental treatment (uninfected, or infection with North American, European or Japanese nematodes), and a host species by treatment interaction. Because 133 fecundity data departed significantly from normality, even after transformation (due to many females having no offspring), we assessed the significance of the effects in our model by using randomization tests (Manly 1998). We compared F ratios to a distribution of F ratios obtained from 1000 reshuffled data sets, in which the y variable (host egg number) was fully randomized. We used least significant difference tests to compare fecundity between host classes. We also examined the effect of motherworm number and total motherworm size on host egg number, using regression. All statistics were obtained using the package JMP 4.0.4 (1989-2001). and data were reshuffled in MINIT.AB (version 10. 1994). Ejfect of Mematodes on Adult Survival .A.dult survival of all four tesiacea group species was examined as a function of infection with either Japanese and North American H. aoronymphium. European H. aoronymphium were not included in this experiment, due to loss of stocks. Infections were established as above, but with nematode slurries containing either -400 or -800 larval nematodes per culture, and each culture starting with 25 host eggs. Numbers of culture vials per treatment ranged from 9 to 22. Twenty uninfected control vials were also established for each host. Upon emergence of adults, males and females were aspirated into separate vials to avoid any effects of mating on survival (Chapman et al. 1995). Flies were transferred weekly to new vials containing 8 ml of a mushroom/sucrose/agar medium. Vials were checked daily for dead flies, which were frozen for later dissection. Lifespan, infection status, and number of H. aoronymphium 134 motherworms were recorded for each fly. We used log-rank tests and Cox proportional hazard regressions to test whether adult mortality was affected by host species (D. neotestacea, D. orieniacea, D. putrida and D. testacea), infection status (uninfected. North American or Japanese H. aoronymphium), and a host species by infection status interaction. We also used log-rank tests to examine the effect of motherworm number on adult lifespan. Finally, we used least significant difference tests to compare lifespan between classes of infected hosts, with lifespan square-root transformed to more closely fit a normal distribution. Effect of Nemalodes on Male Fertility Male fertility was examined as a function of infection with either Japanese or North American H. aoronymphium. We assayed those fly-nematode combinations that had a sufficiently high rate of infection in the lab. and for which infected adult males were expected to survive for at least two weeks. Combinations assayed were: D. orieniacea/Japanese H. aoronymphium. D. putridafHot^h American H. aoronymphium. D. neotestaceafHox^h American H. aoronymphium. D. testacea!Japanese H. aoronymphium. and D. testaceafbiot\h American H. aoronymphium. Infections were established as above with slurry containing —400 larval nematodes and 25 host eggs per mushroom (n > 20 vials per combination). We also included uninfected control treatments (n = 15 vials). Newly emerged, virgin males were collected by aspiration and aged for 8 to 12 days in vials containing 8 ml of mushroom/sucrose/agar medium. These were then mated to an uninfected 8 to 12 day old virgin female that had been maintained 135 on similar medium. After copulation, males were removed from the vials, dissected, and scored for nematode infection. Females were transferred every four to five days (until they died) to new vials with Instant Drosophila Medium and a piece of A. bisporiis. All emergent adult offspring were recorded. Because many males sired no offspring, the distribution of offspring numbers departed significantly from normality, even after transformation. We therefore used nonparametric Wilco.xon rank sums tests to compare the fertility of infected versus uninfected males. In addition, contingency tests (G* likelihood ratio test) were used to test whether parasitism affected the probability that a male would sire no progeny, despite having copulated. The effects of H. aoronymphium on the fertility of D. neotestacea males in the wild were determined by sweep netting males over mushrooms at Mendon Ponds Park, .Monroe County, New York. These were immediately placed in a dark container to prevent mating, returned to the laborator\', and mated within one hour of collection to an uninfected seven day old virgin female that had been reared in laboratory culture. Pairs were separated after copulation, and females were transferred to new vials every three days for the duration of their egg laying period. RESULTS Effect of Nematodes on Female Fecundity The cross-infections yielded infected flies in ail combinations except D. orientacea with European H. aoronymphium (Table I), thus precluding study of 136 virulence in this particular host-parasite combination. Host species, infection treatment, and a host by treatment interaction all contribute significantly to the fecundity of female Drosophila (Fig. 2; Table 2). Females of both North American species, D. neoiesiacea and D. piitrida, are rendered sterile both by their local nematode and by all other nematodes in the H. aoronymphium comple.x. The average number of mature eggs (± s.e.) for D. neoiesiacea and D. puirida infected with any nematode in the H. aoronymphium complex was 1.15 ± 0.24 (n = 132). In contrast, the fecundity of D. lesiacea and D. orieniacea is only modestly affected by any of the three nematodes in the H. aoronymphium comple.x. For those fly-nematode combinations that do not result in host sterility (i.e.. those involving D. lesiacea and D. orieniacea), host female fecundity was negatively correlated with motherworm number (Fig. 3: for flies with less than five motherworms. r" = .08. P = .015). This effect was lost when flies with many motherworms were included (r" = .01. P = .38). because these motherworms were small. Motherworm size is negatively correlated with the number of motherworms per host (all host species, r" = .12, P < .001). There was no relationship between female fecundity and total motherworm size, defined as the sum of the areas of all motherworms inside a host (D. lesiacea and D. orieniacea-. r~ < .001. P = .79). North American motherworms infecting Japanese D. orieniacea were smaller than nematodes in any other lesiacea group hosts (t429= 13.99, p <.0001). 137 Effect of Nematodes on Adult Survival Host species, infection status, and a host by infection status interaction all contribute significantly to adult mortality (Table 3). For all host-parasite combinations, average survival was significantly lower than for uninfected adults (Fig. 4). In all but two cases, including all sympatric combinations (i.e.. a host species infected with its local nematode), infection resulted in similar (-40-45%) reductions in host lifespan relative to uninfected hosts. .As notable e.xceptions. when the North American species D. putrida and D. neoiestacea were infected with Japanese nematodes, the adults lived an average of only 9 (± 0.88 s.e) and 10.95 (± 2.31 s.e) days, respectively, representing an -80% reduction in lifespan. This high level of mortality was not due to infection with high numbers of motherworms. as most (50/67) infected flies harbored only one or two motherworms. Motherworm number also affected adult survival (Fig. 5). Flies with more motherworms had higher mortality (North American nematodes, all host species pooled: log-rank test. x~9 121.55. P < .0001; Japanese nematodes, all hosts pooled: log-rank test. 191.06. P< .0001). Only D. neotestacea and D. putrida exhibited significant effects due to host sex or sex by infection status interaction. Uninfected D. neotestacea males lived longer than uninfected females (log-rank test, x'l = 9.15, P = .0025). and D. neotestacea females infected with North American nematodes lived longer than infected males (x"i = 8.43, P = .0037). D. putrida females lived longer than males, both when uninfected (x"i = 29.26, P < .0001), and when infected with North American nematodes (x"i = 15.78. P < .0001). 138 Effect of Nematodes on Male Fertility Except for D. testacea, experimentally infected males sired fewer offspring than uninfected ones (Fig. 6). although the difference was statistically significant only for D. neotestacea (Wilcoxon rank-sums test: x* 19.53, P < .0001), in which a large proportion of males did not sire any offspring, despite copulating (Table 4). Infected D. putrida males also showed higher levels of sterility - a greater proportion of infected than uninfected males sired no offspring (12/35 versus 7/45. G" likelihood ratio test. P = .05). Wild-caught males of D. neotestacea that were naturally parasitized by H. aoronymphium sired only about 30% as many offspring as did uninfected males (Table 5; P = 0.002). paralleling results found in the laboratory assays. DISCUSSION Virulence is Evolutionarily Labile in Drosophila/Howardula Associations All major components of fimess e.xamined - adult survival, female fertility, and male fertility - can be very adversely affected by infection with H. aoronymphium. These effects range up to an 80% reduction in adult lifespan, a 100% reduction in female fertility, and a 90% reduction in male fertility. These large fitness reductions were not due to the flies having been infected with abnormally high numbers of nematodes. In fact, most of the infected flies in our study were parzisitized by only a single motherworm. Equally striking is the finding that virulence varies greatly among natural associations between species of the D. testacea group and their H. aoronymphium 139 parasites. With respect to female fertility, D. putrida and D. neoiestacea are completely sterilized by North American H. aoronymphium, D. orientacea experiences a modest decline in fertility as a result of Japanese H. aoronymphiitm infection, while European H. aoronymphium has no apparent effect on the fertility of D. testacea (Fig. 2). Male fertility is affected much more severely in D. neoiestacea than in either D. putrida or D. orientacea when these species are infected with sympatric H. aoronymphium. Such patterns show that virulence is evolutionarily labile in associations between closely related species of the D. testacea group and parasites of the H. aoronymphium comple.x. These large changes in virulence are likely to have arisen in less than 260,000 years; this date is based on R1 transposable element sequence divergence in D. testacea, D. neotestacea, and D. orientacea. which is similar to that in D. simutans and D. mauritiana (Kliman et al. 2000; Gentile et al. 2001). The patterns of virulence and infection evident in our laboratory assays match previous observations of testacea/doronymphium associations in the wild. The number of motherworms per host in the experimental treatments is similar to those in wild-caught infected flies, i.e.. mostly one or two (Jaenike 1992). Infected D. neotestacea and D. putrida females are always sterile, both in the wild (Jaenike 1992) and in the laboratory (Jaenike 1996; this study), even when infected with only one motherworm. Infected D. testacea females are not sterilized in the field (Gillis and Hardy 1997; A. J. Klarenberg, pers. comm.) or in the laboratory (this study). This is the first study to document effects of nematode parasitism on fecundity of female D. orientacea. These consistencies 140 indicate that patterns of parasite virulence can be realistically examined in controlled laboratory infections. Sterility is Due to Host Susceptibility Among combinations between D. testacea group host species and H. aoronymphium complex parasites, the degree to which female fertility is reduced by infection is determined almost exclusively by the host species. Females of D. putrida and D. neotestacea are rendered sterile not only by the parasites that normally infect them (North .American H. aoronymphium), but also by the European and Japanese nematodes. In contrast, infected females of both D. testacea and D. orientacea are nearly as fertile as uninfected females, regardless of the nematodes with which they are infected. This suggests that there are two classes of host in the testacea group: species that are susceptible to parasite-induced sterility (D. putrida and D. neotestacea), and species that are resistant (£). orientacea and D. testacea). The susceptible species are even sterilized by nematodes that infect them at low rates in the lab (£). putrida and Japanese nematode), indicating that susceptibility to infection and to parasite-induced sterility are independent characters in these tlies. In contrast to female sterility, reductions in the lifespan of adult flies depend strongly on the nematodes with which they are infected. However, these patterns further highlight the vulnerability of D. putrida and D. neotestacea. While all other host-parasite combinations result in a 40-45% reduction in lifespan relative to uninfected flies, these two North American species suffer very high levels of parasite-induced mortality when 141 infected with Japanese nematodes (Fig. 4). The fact that all sympatric infections show similar levels of parasite-induced mortality suggests that there may be a minimal unavoidable level of mortality due to infection, and is consistent with the hypothesis that this component of virulence has reached evolutionary equilibrium, perhaps via matching of intrinsic parasite virulence with intrinsic host resistance or tolerance to infection (Sasaki and Godfray 1999). Yet this does not explain the variation we obser\'e in parasite-induced reductions in fertility. In the final section, we present a hypothesis for the evolution of virulence in Drosophila-Howardiila associations. A Model for the Evolution of Virulence in Drosophila/Howardula Associations Our results demonstrate a continuum of virulence effects in testacea- aoronymphium infections. Most notably, D. lestacea and D. orientacea are resistant to the potentially sterilizing effects of the Howardula with which they are associated, but D. putrida and D. neotestacea are not. We hypothesize that such variation results from differences in the evolutionary ages of these associations. We have found that North .American populations of H. aoronymphium harbor no sequence variation at both mitochondrial and nuclear (ribosomal ITS 1) loci, indicative of a recent population bottleneck (Appendix 1). Furthermore, the North American mtDNA and ITSl sequences are identical to those in European H. aoronymphium. supporting the idea that the North .American nematode population was established recently and is closely related to the European nematodes. If H. aoronymphium only recently became established in North 142 America, then D. putrida and D. neotestacea may simply not have had enough time to evolve resistance to the sterilizing effects of these infections. An alternative possibility is that resistance to the sterilizing effects of nematodes may be unnecessarily costly if there is a low probability of infection in the wild. However, the prevalence of infection of D. putrida and D. neotestacea by North American H. aoronymphium can be substantial, up to 30-50% (Jaenike 1992). Furthermore, D. testacea. which is resistant to the sterilizing effects of H. aoronymphium. is infected at low rates in the wild (<5%) (Klarenberg and Kersten 1994; Gillis and Hardy 1997). Given that the North American associations may be recent, we hypothesize that virulence and resistance evolve as follows (Fig. 7). When a susceptible and naive host is infected with a novel parasite, both the survival and fecundity of this host may be severely reduced. For example, imagine the colonization of parasite-free populations of D. putrida and D. neotestacea by Japanese H. aoronymphium. Because Howardula cause greater mortality of adult Drosophila in the field than in the laboratory (Jaenike et al. 1995), associations between North American flies and Japanese nematodes would bring about extremely high mortality of infected flies. This situation would impose immediate and intense selection on the nematodes to reduce their virulence with respect to host surv ival, as less virulent nematodes would have greater reproductive success. Failure to evolve reduced virulence could lead to parasite extinction. Consequently, we do not expect to find established Drosophila/Howardula associations characterized by high levels of parasite-induced host mortality. 143 The decreased host mortality evident in established associations is more likely due to parasite evolution than to host evolution. This is because selection on the host is much less intense in the early stages of an association, as only a miniscule fraction of the host population would be infected. In addition, because infected Drosophila females are often sterile (and infected males can also have low fertility), host resistance to parasite- induced mortality would confer little or no fitness benefits to the tlies. If successful establishment of the parasites in a new host species requires reduction in the rate of parasite-induced mortality, then virulence will subsequently be manifested via reductions in host reproduction. .Associations between H. aoronymphiiim and D. putrida and D. neutesiacea currently exhibit such virulence. .As argued above, these North American associations probably are evolutionarily young. Established parasites that infect a significant fraction of the host population will impose strong selection on the hosts to resist infection (Kraajeveid et al. 1998) or to resist the sterilizing effects of the parasites. Thus, evolution of the hosts (but not parasites) is expected to ameliorate the fertility-reducing effects of these parasites. The European and Japanese associations between D. lestacea and D. orientacea and their nematodes appear to be at this stage, as the flies are highly resistant to the potentially sterilizing effects of sympatric Howardiila. Whether this is a stable evolutionary endpoint, or whether the parasites can periodically gain the upper hand, remains to be seen. While many insect-parasitic nematodes completely sterilize their hosts (Siddiqi 2000), the ages of these associations, and thus the evolutionary stability of the expressed virulence, remain largely unknown. In any case, our comparative studies clearly show that virulence is evolutionarily labile. 144 even vvithin closely related sets of host and parasite species. Consequently, the potential for parasites to regulate host populations and the intensity of selection imposed by parasites may vary substantially over the evolutionary course of a given host-parasite association. .ACKNOWLEDGMENTS We thank M. T. Kimura and I. Eijs for sending us flies and nematodes. H. Mallorv' for assistance with experiments, and L. ICnovvIes, T. Markow. N. Moran and H. Ochman for comments. This research was supported by National Science Foundation (NSF) grant DEB-0074I41 to JJ. and fellowships to SJP from the Flinn Foundation and the NSF Research Training Grant in the Analysis of Biological Diversification to the University of .Arizona. 145 LITERATURE CITED Altizer, S. M. 2001. Migratory behaviour and host-parasite co-evolution in natural populations of monarch butterflies infected with a protozoan parasite. Evol. Ecol. Res. 3:611-632. Baudoin, M. 1975. Host castration as a parasitic strategy. Evolution 29:335-352. Bull. J. J. 1994. Virulence. Evolution 48:1423-1437. Chapman, T., L. F. Liddle. J. M. Kals. M. Wolfner and L. Partridge. 1995. Cost of mating in Drosophila melanogaster females is mediated by male accessory gland products. Nature 373:241-244. Davies. C. M., J. P. Webster, and M. E. J. Woolhouse. 2001. Trade-offs in the evolution of virulence in an indirectly transmitted macroparasite. Proc. R. Soc. Lond. B 268:251-257. Ebert. D. 1994. Virulence and local adaptation of a horizontally transmitted parasite. Science 265:1084-1086. Fermer. F. and F. N. Radcliffe. 1965. Myxomatosis. Cambridge University Press. Cambridge. Frank. S. A. 1996. Models of parasite virulence. O. Rev. Biol. 71:37-78. Gentile. K. L., W. D. Burke and T. H. Eickbush. 2001. Multiple lineages of R1 retrotransposable elements can coexist in the rDNA loci of Drosophila. Mol. Biol. Evol. 18:235-245. Gillis. J. E. M. and I. C. W. Hardy. 1997. Nematode parasitism in a northern European drosophilid community. Ent. Exp. etApp. 84:275-291. 146 Herre, E. A. 1993. Population structure and the evolution of virulence in nematode parasites of fig wasps. Science 59:1442-1445. —. 1995. Factors affecting the evolution of virulence: nematode parasites of fig wasps as a case study. Parasitology 111 :S 179-S191. Hurd, H.. E. Warr and A. Polwart.2001. A parasite that increases host lifespan. Proc. R. Soc. Land. 5 268:1749-1753. Imhoof. B. and P. Schmid-Hempel. 1998. Patterns of local adaptation of a protozoan parasite to its bumblebee host. Oikos 82:59-65. Jaenike. J. 1988. Parasitism and male mating success in Drosophila testacea. Am. Nat. 131:774-780. —. 1992. Mycophagous Drosophila and their nematode parasites. Am. Nat. 139:893-906. —. 1996. Sub-optimal virulence of an insect-parasitic nematode. Evolution 50:2241- 2247. Jaenike, J. and A. C. James. 1991. Aggregation and the coexistence of mycophagous Drosophila. J. Anim. Ecol. 60:913-928. Jaenike, J.. H. Benway and G. Stevens. 1995. Parasite-induced mortalitv' in mycophagous Drosophila. Ecology 76:383-391. James. .A.. C. and J. Jaenike. 1992. Determinants of mating success in wild Drosophila testacea. Animal Behaviour 44:168-170. JMP 4.0.4. 1989-2001. SAS Institute. Gary, N. Garolina, USA. 147 Kaltz, O., S. Gandon, Y. Michalakis and J. A. Shykoff. 1999. Local maladaptation in the anther-smut ftingus Microbotryum violaceum to its host plant Silene latifolia: evidence from a cross-inoculation e.\periment. Evolution 53:395-407. Kaltz. O. and J. A. Shykoff. 1998. Local adaptation in host-parasite systems. Heredity 81:361-370. Kimura. M. T. and M. J. Toda. 1989. Food preferences and nematode parasitism in mycophagous Drosophila. Ecol. Res. 4:209-218. Klarenberg. A. J. and L. Kersten. 1994. Nematode parasitism in Drosophila quinaria and D. testacea species groups. Drosophila Information Serx'ice 75:125-126. Kliman. R. M.. P. .Andolfatto, J. A. Coyne. F. Depaulis. M. Kreitman. A. J. Berrv'. J. McCarter. J. Wakeley. and J. Hey. 2000. The population genetics of the origin and divergence of the Drosophila simulans comple.x species. Genetics 156:1913- 1931. Koella. J. C. and P. .Agnew. 1999. A correlated response of a parasite's virulence and life cycle to selection on the host's life history. J. Evol. Biol. 12:70-79. Kraaijeveld. A. R.. J. J. M. Van Alphen, and H. C. J. Godfray. 1998. The coevolution of host resistance and parasitoid virulence. Parasitology 116:S29-S45. Lively. C.M. and J. Jokela. 1996. Clinal variation for local adaptation in a host-parasite interaction. Proc. R. Soc. London, 5 263:891-897. Manly, B. F. J. 1998. Randomization, bootstrap and Monte Carlo methods in biology. 2"'' Edition. Chapman & Hall. London, England. 148 Minchella, D. J. 1985. Host life-history variation in response to parasitism. Parasitology 90:205-216. MINITAB Release 10 for Windows. 1994. Minitab Inc. 3081 Enterprise Dr. State College, Pennsylvania, USA. Poinar. G. O. Jr., I. Dombeck and J. Jaenike. 1997. Parasitylenchus nearciicus sp. n. (.Allantonematidae: Tylenchida) parasitizing Drosophila (Drosophilidae: Diptera) in North America. Fund. Appl. Nemat. 20:187-190. Sasaki. .A. and H. C. J. Godfray. 1999. model for the coevolution of resistance and virulence in coupled host-parasitoid interactions. Proc. R. Soc. London. B 266:455-463. Siddiqi, M. R. 2000. Tylenchida: Parasites of Plants and Insects. Second Edition. CABl Publishing. CAB International. Wallingford UK. Taylor. L. H.. M. J. Mackinnon and A. F. Read. 1998. Virulence of mixed-clone and single-clone infections of the rodent malaria Plasmodium chabaudi. Evolution 52:583-591. Welch. H. E. 1959. Ta.\onomy. life cycle, development, and habits of two new species of Allantonematidae (Nematoda) parasitic in drosophilid flies. Parasitology 49:83- 103. 149 Table I. Number of motherworms (± s.e.) per fly in experimental infections (with and initial inoculum of 200 infective nematodes per sample). Sample sizes in parentheses. Infections with sympatric nematodes indicated in bold. Only European nematodes were unable to infect D. orientacea. Source of nematodes Host species North .•\merica Europe Japan D. neotestacea 1.85 ± .13 (79) .83 ±.076(101) .29 ±.08 (45) D. orientacea 1.27 ±.30 05) 0 (139) 1.15 ±.13 (106) D. piurida 2.28 ±.21 (66) .75 ± .075 (89) .15 ±.05 (55) D. testacea .84 ±.091 (100) .19 ±.042 (98) .22 ±.048 (106) 150 Table 2. Female fecundity as a function of host species (£>. neotestacea, D. orientacea, D. putrida and D. testacea), infection treatment (uninfected, American, European nematode, and Japanese nematode) and host species by treatment interaction. Source df SS F P Host species 3 35499.51 16.85 <.001 Treatment 3 12198.46 5.79 <.001 Host .\ treatment 9 44416.31 7.03 <.001 Error 514 360939.30 151 Table 3. Adult mortality (proportional hazards regression model) as a function of host species (D. neotestacea. D. orientacea, D. putrida and D. testacea). infection status (uninfected, American nematode and Japanese nematode), and a host species by infection status interaction. Source df X' P Host species 3 105.75 <.0001 Infection status 2 238.11 <.0001 Host X status 6 45.02 <.0001 152 Table 4. Fertility of D. testacea group males mated with one female, as a function of infection status. (NA = infection with North American H. aoronymphium; J = infection with Japanese H. aoronymphium.) Host species Infection status Number of males G" likelihood ratio test produced no x" P offspring offspring D. neotestacea uninfected 29 18 18.212 <0.0001 NA 4 23 D. puirida uninfected 38 7 3.805 0.0511 NA 23 12 D. orientacea uninfected 15 11 .236 0.627 8 8 D. testacea uninfected 56 9 1.53 0.465 NA 13 4 153 Table 5. Effect of H. aoronymphium nematodes on fertility of wild D. neotestacea males. Infection status N no. offspring sired (± s.e) F ratio P infected 6 52 ±23 10.3 0.002 uninfected 69 171 ± 12 154 FIGURE LEGENDS Fig. 1. Natural associations between the four D. lestacea group flies and their nematode parasites. Host phylogeny is from Gentile et al. (2001) and the parasite phylogeny is from S. Perlman (unpublished ITSl rDNA sequence data). Fig. 2. Mean (± s.e.) number of mature eggs carried by female flies as a function of infection status. Numbers above bars indicate sample sizes. .Arrows indicate infections with regionally sympatric hosts and parasites. Different letters above bars indicate pairs that are significantly different from each other (P < 0.05). (We do not include any pairwise comparisons with the D. lestacea infected with European nematodes, as there are only 2 such observations.) Fig 3. Effect of motherworm number on host female fecundit\'. Nematodes were pooled, as the different types (North American. European, and Japanese) do not differ in the effects on individual host species. Fig. 4. Survivorship of infected and uninfected flies. Individual trajectories indicate whether the flies were uninfected or infected with North American or Japanese H. aoronymphium. along with the number of flies each group was started with. Fig. 5. Effect of motherworm number on host adult survivorship, for two host-parasite combinations: A) D. neotestacea infected with its local North American nematode, and B) D. orieniacea infected with its local Japanese nematode. Individual trajectories are for flies infected with 1, 2, 3 or >3 motherworms. Fig. 6. Number of offspring (mean ± s.e.) sired by testacea group males mated with one uninfected female. Numbers above bars indicate sample sizes. Arrows indicate flies 155 infected with their local nematode. Fig. 7. Model of the evolution of virulence in testacea/aoronymphium associations. Initial host susceptibility to parasite-induced sterility and mortality is followed by parasite evolution to reduce the effects on host survival, which in turn may be followed by host evolution to resist the sterilizing effects of parasites. See te.xt for details. 156 HOSTS PARASITES D. orientacea Japan D. testacea Europe D. neotestacea America D. putrida Drosophila testacea group Howardula aoronymphium group ^ sterilized by parasite 157 2 160 n nO e 3 CO) oO) D.neotestacea D. putrida D. orientacea D.testacea infection status I I uninrected H American nematode I I Japanese nematode ^3 European nematode D orientacea D lesiacea D neotestacea D putrida motherworm number 1.0 10 D. neotestacea D. putrida (North America) 0.8 (North America) 08 g" 0.6 \ g> 06 > •> E E 0.2 V. 02 V A HWO... (Wl'j __ • 0.0 0.0 40 60 120 I 40 60 120 days days 1,0 1.0 B K D, orient aoea D. testacea (Europe) (Japan) 08\ 0.8 A \ ' >, I. g" 0.6 c \'I •> 'V '5 V £ \ E M 04 i»iTmiKan(B) w 04 ), \ \ \ \ (641 02 02 A, ^1. s::~i \ I l^^new(JUSI urwtfecled (M) I _ :\ 00 -p - - 00 40 80D 1 20 160 40 80 120 days days L/( vO 1.0 n D. neotestacea / North American nematode 0.8- ™ 0.6-1 0.4- 0.2- 0 40 80 100 days 0 40 80 120 days 26 16 D. neotestacea D. putrida D. orientacea D. testacea infection status I I uninrectcd •1 American nematode H Japanese nematode I. susceptible fly parasite evolution 2. susceptible fly with a novel parasite (decreased parasite-induced with its local parasite (e.g. D. putrida and mortality) (e.g. D. putrida and Japanese nematode) American nematode) STER LE & DEAD STERILE & ALIVE host evolution (resistance to sterility) 3. resistant fly with its local parasite (e.g. D. orientacea and Japanese nematode) FERTILE & ALIVE ON lO 163 APPENDIX FOUR: COMPETITIVE INTERACTIONS AND PERSISTENCE OF TWO NEMATODE SPECIES THAT PARASITIZE DROSOPHILA RECENS^ 'published in Ecology Letters. (2001) 4:577-584 164 COMPETITIVE INTERACTIONS AND PERSISTENCE OF TWO NEMATODE SPECIES THAT PARASITIZE DROSOPHILA RECENS S. Perlman and J. Jaenike Department of Ecology and Evolutionary' Biology University of Arizona Tucson. Arizona. U.S..A.. 85721 Correspondence: S. Perlman. Dept. of Ecology and Evolution. Biological Sciences West. University of Arizona. Tucson. AZ. 85721. U.S.A. Phone: 520-626-8455 Fa.\: 520-621-9190 Email: [email protected] Running title: HOST USE AND PARASITE COMPETITION 165 Abstract. - Drosophila recens is parasitized in the wild by two nematodes, Howardula aoronymphium, a host generalist, and Parasitylenchiis nearcticus, a host specialist known only from D. recens. In order to understand how these two parasite species coexist, we compared their ability to infect and grow in D. recens, their effects on host fecundity and survival, and whether one parasite species was competitively superior in double infections. The specialist nematode P. nearcticus had greater rates of infection and reproduction than the generalist H. aoronymphium. and completely sterilized females in single and mixed infections. The specialist was competitively superior in mi.xed infections, as generalist mothervvorms were significantly smaller than in single infections. These results suggest that P. nearcticus might competitively exclude H. aoronymphium if D. recens were the only host available. It is likely that H. aoronymphium persists in D. recens by transmission from other, more suitable host species. Key words. - Allantonematidae. host specificity, interspecific competition, mycophagous Drosophila. parasite coexistence, virulence. 166 Many host species typically harbor several species of parasites (Petney & Andrews 1998). But because hosts are finite resources, parasites that share the same host species may adversely affect each other's densities through interspecific competition, which can occur in two distinct ways (Dobson 1985). Exploitation competition does not require any direct interaction between parasite species. Instead, parasites affect each other by reducing host densities, possibly below thresholds necessary' for parasite population growth. The most important properties that atTect the nature of exploitation competition are a parasite's virulence and transmission rate, and the statistical distribution of parasites among hosts. Interference competition results from coinfection of host individuals. In addition to competing directly for a common pool of resources within coinfected hosts, parasites can also interact in a directly antagonistic manner, for example, by releasing toxic compounds that inhibit growth or causing displacement from preferred attachmentsites. Such interactions are often asymmetric (Poulin 1998). Interference between parasites canalso be indirect, mediated for example by host defensive responses. If a host species is considered a single limiting resource, then the competitive exclusion principle would suggest that only one parasite species could persist within a given host species. Given that individual host species are often infected by many different parasite species, this raises the question of how these parasites coexist. One possible mechanism is parasite aggregation (Ives & May 1985; Dobson 1985; Roberts & Dobson 1995; Comins & Hassell 1996). By analogy with species that utilize patchy ephemeral resources (e.g. Wertheim et al. 2000), it has been suggested that 167 if different parasite species exiiibit independent aggregated distributions, then several species can be stably maintained within a single host species. As parasites become more aggregated, they have less effect on the host population, while being subject to greater within-host competition. If the distributions of the different parasite species are independent, this lessens the probability of co-occurrence, and the consequent interspecific competition, within a single host individual. Many parasites e.xhibit highly aggregated distributions, so this mechanism is probably broadly relevant (Shaw & Dobson 1995). Parasites may also coe.xist if there is both within-host resource partitioning and little or no virulence by the parasite, so that host density is little affected by the different parasites. Within-host resource partitioning will reduce interference competition between parasites in the same host individual (Poulin 1998). Coe.xistence of parasite species can also be maintained through tradeoffs between exploitation and interference competition (Hochberg & Holt 1990; Amarasekare 2000). A parasite that is a good colonizer, with high infection and reproductive rates under non competitive conditions, can coexist with a species that is superior in interference competition but which has a higher host threshold density. For example, one parasite species may release toxins that negatively affect the growth of a second species that has a higher pathogenicity. The better interference competitor might even require the presence of the other parasite species within a host for successful infection (see Schall & Brumwich 1994 for an example of facilitation). 168 Finally, different parasite species can coexist within a single host through source- sink dynamics involving more than one host species. Parasites infecting a single host species would consist of both core and satellite species (Poulin 1998). The core parasite species in a certain host would be maintained even if this were the only host species in a community, whereas satellite species would be present as a result of input from other host species. Drosophila recens Wheeler and its parasitic nematodes Mushroom-feeding Drosophila flies (Diptera: Drosophilidae) are often the most common insect visitors to decaying fleshy mushrooms on the forest floor. .Associated with them are a number of parasitic nematodes that vary greatly in both host range and virulence. What contributes to the maintenance of this diverse host-parasite community? In this paper, we examine the factors involved in the coexistence of the two parasitic nematodes that infect Drosophila recens. Specifically, we ask which parasite utilizes D. recens better in terms of infection rate and reproduction? Which parasite is more virulent and thus likely to have a more adverse effect on host population density? Finally, which parasite species is competitively superior in doubly infected hosts? Drosophila recens (Diptera: Drosophilidae) is a mushroom-feeding fly found in deciduous forests of northeastern North America. It is known to be parasitized by two species of nematode in nature, Howardula aoronymphium Welch and Parasitylenchiis nearciicus Poinar, Jaenike and Dombeck (Tylenchida: Allantonematidae). In eastern North America, H. aoronymphium infects four species of Drosophila~D. neotestacea and 169 D. putrida of the testacea species group, and D.falleni Wheeler and D. recens of the quinaria group (Welch 1959; Jaenike 1992). Parasitylenchus nearcticus has been found only in D. recens (Poinar et al. 1997). Both nematode species have direct life cycles, involving no intermediate hosts, and this greatly simplifies understanding their population dynamics. For both Howardula and Parasitylenchus, an inseminated female nematode infects the host by piercing the cuticle of a tly lar\'a. When the adult fly emerges, the Howardula mothervvorm grows rapidly and then begins releasing juvenile nematodes into the hemocoel of the host. These juveniles are passed from the anus and ovipositor of the host as it visits mushrooms, and the nematodes subsequently mate inside these mushrooms and continue the cycle. The most apparent fitness consequence of Howardula parasitism is reduced fertility of infected hosts. Females of the testacea group are completely sterilized by H. aoronymphium. whereas females of the quinaria group e.xperience about a 50% reduction in fecundity (Jaenike 1992). Nematodes have also been shown to affect male mating success (Jaenike 1988) in D. neoiestacea. as well as adult survival in D. neotestacea and D. putrida (Jaenike et al. 1995). The infection rate of D. recens varies from 0 to 20%, but averages only about 5% (Jaenike 1992), which is substantially less than the other host species of H. aoronymphium in northeastern North America (Jaenike 1992). Nematodes of the genus Parasitylenchus have two obligate parasitic generations in their hosts. Inseminated motherworms infect fly larvae and then give birth to larval nematodes (Fl), which then mate inside the same host. It is their larval offspring (F2) 170 that are then passed to mushrooms. Parasitylenchns nearcticiis completely sterilizes its female host (Poinar et al. 1997). The preliminary collections from which P. nearcticiis was described in the Adirondack Mountains in New York reported a 5% infection rate in D. recens\ 2% of collected flies were found to be infected with both P. nearcticus and H. aoronymphium (Poinar et al. 1997). The following e.xperiments were conducted to determine the suitability of D. recens for these two parasite species, their virulence to this host, and the nature of competitive interactions between them in coinfected individuals. MATERIALS AND METHODS Stocks - The strain of Drosophila recens was established from multiple females collected in 1994 and 1995 from the Adirondack Mountains in New York. The Parasitylenchus nearcticus strain was descended from multiple nematodes collected along with the D. recens. These nematodes were maintained in the laboratory using D. recens as hosts. The Howardida aoronymphium strain was established from multiple nematodes collected from its four host species, which were collected in Rochester. New York in 1992. These nematodes were maintained in the laboratory using D. neotestacea as hosts. Experimental Infections - In order to collect fly eggs for infection, uninfected D. recens females were placed in petri dishes with food plugs made from blended mushrooms, agar, and water. Howardula aoronymphium were obtained by grinding one to two-week old 171 infected D. neotestacea flies in Drosophila Ringer's solution (Roberts 1986). After determining the density of larval nematodes in this slurry, slurry volumes containing either 200. 400 or 800 larval nematodes were pipetted onto a 0.4 g piece of Agan'cus bisporus mushroom. Larval P. nearcticus nematodes were obtained by grinding infected three-week old D. recens. Two e.vperimental trials were conducted to assay parasite infection, reproduction, and effects on host fertility. In the first, the following treatments were set up: I) no infection (n=4 vials), 2) 200 larval P. nearcticus (n=5). 3) 200 larval H. aoronymphium (n=4). and 4) 200 larval H. aoronymphium and 200 larval P. nearcticus (n=4). In the second trial, we set up 5 vials each at the following higher densities: I) no infection. 2) 400 larval P. nearcticus. 3) 800 larval P. nearcticus. 4) 400 larval H. aoronymphium. 5) 800 larval H. aoronymphium. 6) 400 larval P. nearcticus and 400 larval H. aoronymphium. and 7) 800 larval P. nearcticus and 800 larval H. aoronymphium. After addition of the larval nematodes, 20 D. recens eggs were placed on the mushroom pieces, which were then placed in a vial with moistened cheesecloth on the bottom. Pieces of moistened mushroom were added to the vial to prevent starvation when necessary. Upon their emergence, adult flies were collected and transferred to fresh vials containing Instant Drosophila Medium (Carolina Biological Supply) plus a piece of fresh A. bisporus mushroom. Flies were frozen after 7 days. The following data were later recorded for each fly: number and size of H. aoronymphium motherworms, number of P. nearcticus Ft larvae, and number of mature eggs (stage 10 and later) in fly ovaries. Nematodes of the two species are readily distinguished. The much larger H. 172 aoronymphium motherworms were traced with a camera lucida, and their longitudinal section area was then measured with a planimeter. Parasitylenchus nearcticus motherworms often disintegrate after a few days in their hosts and therefore caimot be counted or measured in week-old flies. Howardula aoronymphium motherworm size is a good predictor of parasite fecundity (Jaenike 1996a), and the number of P. nearcticus F1 offspring within a host is a measure of motherworm reproduction, although one needs to correct for the number of mothersvorms per host. A third e.\perimental infection was conducted to assess the effect of parasites on the surv ival of adult flies. This experiment also provided data on infection rates. Si.x replicate vials of each of the following treatments were set up: 1) uninfected, 2) 800 larval P. nearcticus. 3) 800 larval H. aoronymphium, 4) 800 larval P. nearcticus and 800 larval H. aoronymphium. After addition of the nematodes, 20 D. recens eggs per vial were added. Upon emergence of adult flies, males and females were put in separate vials, to avoid any effects of mating on survival (e.g. Chapman et al. 1995). Every other day. flies were transferred to vials containing 8 ml of a mushroom/sucrose/agar medium. Vials were checked daily for dead flies, which were frozen and later dissected. Lifespan, infection status, number of H. aoronymphium motherworms, and number of P. nearcticus larvae were recorded for each fly in the experiment. Survival data were square-root u^sformed to more closely approximate a normal distribution. Data were analyzed using the statistical package JMP 3.1.5. (SAS Institute 1989 - 1995). 173 RESULTS Host suitability for the two parasites - The rates of infection in the three experimental trials are presented in Table 1. The percent infection by P. nearcticus was much higher than that by H. aoronymphium in all three trials and at all densities of larval nematodes. In every case, at least 80% of the D. recens were infected by P. nearcticus. Males and females of D. recens did not differ in susceptibility to infection (Table 2). The relative density of Howardula aoronymphium, defined as the total number of motherworms divided by the total number of flies (Margolis et al. 1982). was consistently low, in all cases less than or equal to one motherworm per host fly. Because P. nearcticus motherworms disintegrate after a few days in their hosts, it is not possible to census them in week-old flies. The number of F1 larvae is therefore only an indirect measure of relative density, since we are not able to measure the number of offspring per motherworm. The reproductive rate of the different parasites can be compared indirectly, through their effects on host (female) fecundity, with greater reductions in host fecundity being correlated with greater parasite reproduction (Jaenike 1996a). Almost all females infected with P. nearcticus were completely sterile (average number of mature eggs = 0.28. n = 86 flies). For females infected only with H. aoronymphium, fecundity was reduced by about one quarter (average number of eggs for infected flies = 17.8 (n = 23 flies), and for uninfected flies = 24.6 (n = 95 flies)), a modestly significant difference (t=2.07. df=l 16, p=0.04) (Fig. 1). The greater reduction in host fertility suggests that P. nearcticus has a higher per capita rate of reproduction than does H. aoronymphium within 174 D. recens. Because all D. recens females were rendered completely sterile by P. nearcticus, this strongly suggests that infection by even a single P. nearcticiis motherworm is sufficient to bring about this sterility. Mean longevity of female flies was substantially reduced by infection with either nematode species (Figure 2; ANOVA: F= 5.72, df= 3,78, p<0.01). There was no significant difference in the survival of male tlies as a function of infection status [ANOVA: F= 0.80. df= 3,86. NS]. Although both nematodes increased the mortality of infected flies, there was no difference in the mean longevity of female tlies that were infected by H. aoronymphium only. P. nearcticus only, or coinfected with both nematodes simultaneously [ANOVA: F= 0.055. df= 2.47. NS]. Interference competition benveen the two parasites - There was no effect of mi.xed infection on infection rate for either nematode species (Table 2). That is. the percent infection by either was not affected by the presence of the other. There were however, significant effects of mixed infection on parasite reproduction. For H. aoronymphium, motherworm size was significantly less in flies that were also infected with P. nearcticus. for all larN'al densities tested [ANOVA: F= 17.78, df= 1,81, p<0.001] (Fig. 3). There was no correlation between H. aoronymphium motherworm size and the number of P. nearcticus Fl offspring (r" < 0.001). Since P. nearcticus F1 number increases with infection density (and presumably motherworm number), this indicates that the reduction in H. aoronymphium motherworm size is independent of the number of P. nearcticus motherworms in the same individual fly. The size of H. aoronymphium motherworms 175 was not affected by the number of conspecific motherworms per host [ANOVA: F= 0.24, df= 2,43, NS]. although this was rarely more than two in our e.xperiments. Other experiments have shown that reductions in motherworm size are evident at higher densities (Jaenike 1998). Parasitylenchus nearcticus did not e.xhibit any negative effects of coinfection by H. auronymphium (Fig. 4). For two of the e.xperimental treatments (200 and 800 larval nematodes per treatment), the number of P. nearcticus F1 was greater in the presence of H. tioronymphium in the same host than in single infections; this difference was only significant at the highest nematode concentration [ANOVA: effect of either single or mi.xed infection. F= 7.62. df=l.50. p= 0.02; nested effect of vial within treatment, F= 41.17. dt^S.SO, p=0.003]. In mixed infections, the nematodes do not adversely affect each other through increased host mortality. There was no difference in host mortality in mixed compared to single infections (Fig 2; see earlier section on mortality). In mixed infections, however, female hosts were completely sterile, most likely due to the presence of P. nearcticus (Fig. 1). DISCUSSION The results presented above show that the host specialist P. nearcticus utilizes D. recens more effectively than does the generalist H. aoronymphium. Prevalence of infection by P. nearcticus was consistently much higher than by H. aoronymphium using similar densities of infective nematodes. Female flies infected by P. nearcticus were almost 176 always completely sterile, compared to only about a 25% reduction in female fertility caused by infection with H. aoronymphium. The greater reduction in host fertility caused by P. nearcticus suggests that a greater proportion of host resources is allocated to reproduction of this parasite. Although parasites did adversely affect host survival, there was no difference in the survival of flies as a function of the parasite species with which they were infected. This reduction in survival under benign laboratory conditions suggests that these parasites also reduce fly survival in the wild. It was previously shown that H. aoronymphium has a greater adverse effect on host survival in the field than in the laboratory (Jaenike et al. 1995). Because both nematodes are transmitted by live flies, host mortality is detrimental to these parasites (e.g. Blower & Roughgarden 1987). One important concern that needs to be addressed is whether maintaining nematodes for many generations in the lab on one fly species may have affected their ability to infect other hosts. Experimental lab infections have shown that both nematode species can successfully infect and reproduce in hosts other than the fly used for maintenance in the lab. For example. H. aoronymphium successfully parasitized D. neotestacea and D. falleni after several generations in D. putrida (Jaenike & Dombeck 1998). This study also revealed that H.aoronymphium harbors little if any adaptive genetic variation in ability to utilize different host species (Jaenike & Dombeck 1998). Also. P. nearcticus can successfully infect and reproduce in species other than D. recens (Appendix 2). We therefore think it unlikely that the H. aoronymphium used in the present experiments became maladapted to D. recens during laboratory culture, or that P. 177 nearcticus has lost the ability to infect other hosts as a result of being brought into the lab. The poor performance of H. aoronymphium in D. recens is not due to a generally lo%v ability to utilize Drosophila. For instance, the decrease in fecundity of infected females was much less than that observed in the other Drosophila species infected by H. aoronymphium (Jaenike 1992). Of the four species of Drosophila infected by H. aoronymphium in northeastern North America, rates of infection in the wild and in the laboratory are lowest for D. recens (Jaenike 1992). Because these four species often occur on the same mushroom in the wild (Jaenike & James 1991). there are ample opportunities for interspecific transmission of H. aoronymphium. Thus. D. recens may be an incidental host for this parasite. Besides having a better capacity to infect and reproduce within D. recens, P. nearcticus is also the better competitor in mi.xed infections. The most notable effect is on H. aoronymphium motherworm reproduction, as motherworms of this species were significantly smaller, and thus probably less fecund, in flies that also harboured P. nearcticus. The reproduction of P. nearcticus. as measured by offspring (Fl) number, was not adversely affected by the presence of H. aoronymphium. Asymmetric competitive interactions are common in entomophagous nematodes (e.g., Koppenhofer et al. 1995), and in many other parasites (Dobson 1985; Sousa 1993; Poulin 1998). Inoculation of mushrooms with H. aoronymphium and P. nearcticus at the same time may have given us an underestimate of the competitive superiority of P. nearcticus within hosts, because this species is more likely to parasitize D. recens larvae prior to 178 infection by H. aoronymphium. This is because D. recens adults come to mushrooms before D. putrida and D. neotestacea, two of the principal hosts of H. aoronymphium. These latter two flies are preferentially attracted to more rotten (older) mushrooms (Grimaldi 1985). Thus, the ratio of P. nearcticus to H. aoronymphium is likely to decrease with mushroom age. However, because motherworms develop much later than the window of time in which host penetration occurs, the effect of priority in this host- parasite system is not clear. Infections by multiple parasite species can have a severe combined effect on host fitness (Petney and Andrews 1998), possibly because of the difficulty of resisting multiple parasites (Taylor ei al. 1998). For D. recens, however, coinfections by H. aoronymphium plus P. nearcticus were no more virulent than infections by just a single species. Mortality of singly and doubly infected flies was similar. Both doubly infected female flies and those infected with only P. nearcticus were sterile. Coexistence of H. aoronymphium and P. nearcticus How do infection rale, parasite reproductive rate, and virulence affect the population dynamics of these two parasite species? A macroparasite's host threshold density (Nj), or the minimum density of hosts required for positive parasite population growth (and parasite invasion of a host population), is inversely proportional to parasite transmission and reproductive rates (Anderson & May 1979; Jaenike 1998). Our experiments have shown that P. nearcticus has greater infection and reproductive rates (as inferred by their effects on host fertility) than H. aoronymphium in D. recens. In addition, NT will 179 increase with the rate of parasite-induced host mortality. We found that both H. aoronymphium and P. nearcticus have similar adverse effects on the survival of D. recens. Considering the combined effects of infection rate, parasite reproductive rate, and parasite-induced host mortality, we conclude that the threshold population density of D. recens is lower for P. nearcticus than for H. aoronymphium. Thus, in the absence of within-host interactions between the parasites, we would expect P. nearcticus to competitively exclude H. aoronymphium at the population level. The asymmetric competitive interactions within coinfected hosts - the greater adverse effect of P. nearcticus on H. aoronymphium than vice versa - further bolsters this conclusion. Thus, if D. recens were the only host species available, we predict that P. nearcticus would competitively exclude H. aoronymphium. both through within-host competitive interactions and through a greater reduction in host density, possibly below the threshold density for H. aoronymphium. How does H. aoronymphium persist in D. recens, despite its poor performance and competitive inferiority? Here we evaluate the possible mechanisms for parasite species coexistence. First, coexistence due to independently aggregated distributions appears unlikely. Our field data shows no evidence of aggregation by H. aoronymphium in D. recens. with 21 out of 25 infected flies harboring only a single motherworm. Both species of nematodes co-occur in the Adirondack Mountains. In a sample of 116 D. recens from the Adirondacks, 105 were uninfected. 5 were infected with H. aoronymphium only. 4 were infected with P. nearcticus only, and 2 were infected with 180 both parasites (Poinar et al. 1997). In this sample of flies, the tvvo parasites actually exhibit a positive association (p = 0.04; Fisher's exact test). Within-host resource partitioning is also unlikely as a mechanism for parasite coexistence in D. recens. Both H. aoronymphium and P. nearcticus are found in the hemocoel of the fly. and the present experiments revealed negative competitive interactions between them within co-infected hosts. Persistence of the two nematode species is not due to coexistence of a good colonizer and a good within-host competitor, because P. nearcticus is superior in both respects. We suggest that H. aoronymphium persists in populations of D. recens as a result of transmission from other host species, especially D. putrida and D. neotestacea. which are superior hosts for both infection and reproduction of //. aoronymphium (Jaenike 1992; Jaenike & Dombeck 1998). These Drosophila species share microhabitats and often breed in the same individual mushroom (Grimaldi & Jaenike 1984; Jaenike &. James 1991). There is therefore great opportunity for horizontal transmission of H. aoronymphium between host species. Thus, the extent to which this generalist nematode encounters D. recens will largely depend on how frequently larvae of D. recens inhabit the same mushrooms as the other hosts of H. aoronymphium. Indeed, the poor performance of H. aoronymphium in D. recens, particularly its low infection rate, suggest that D. recens may represent a demographic sink for H. aoronymphium. Host species that serve as demographic sinks for certain parasites can be profoundly affected by them. For example, endangered species can be sinks for parasites that are maintained in other hosts; parasitism may contribute to extinction of these 181 threatened species, even when they fall below threshold density for their "own' parasites (McCallum & Dobson 1995). The role of source-sink dynamics on the coexistence of parasites and the broader community-level effects of such dynamics warrant further examination in natural populations. ACKNOWLEDGMENTS We thank I. Dombeck and J. Russell for assistance with experiments. B. Walsh tor statistical advice, and K. Dyer and M. Hunter for comments on the manuscript. This research was supported by NSF grants DEB-9615065 and DEB-0074141 to JJ. and fellowships to SP from the Flirm Foundation and the NSF Research Training Grant in the Analysis of Biological Diversification to the University of Arizona. 182 LITERATURE CITED Amarasekare, P. 2000. Coexistence of competing parasitoids on a patchily distributed host: local vs. spatial mechanisms. £co/ogy 81:1286-1296. Anderson, R.M. and R. M. May. 1979. Population biology of infectious diseases : Part I. Nature 280:361-367. Blower. S.M. and J. Roughgarden. 1987. Population dynamics and parasitic castration: a mathematical model. Am. Nat. 129:730-754. Chapman. T., L. F. Liddle, J. M. K.als. M. Wolfner and L. Partridge. 1995. Cost of mating in Drosophila melanogaster females is mediated by male accessor\' gland products. Nature 373:241-244. Comins, H.N. and M. P. Hassell. 1996. Persistence of multispecies host-parasitoid interactions in spatially distributed models with local dispersal../. Theor. Biol. 183:19-28. Dobson, A. 1985. The population dynamics of competition between parasites. Parasitology 91:317-347. Grimaldi, D. 1985. Niche separation and competitive coexistence in mycophagous Drosophila (Diptera: Drosophilidae). Proc. Ent. Soc. PVash. 87: 498-511. Grimaldi, D. and J. Jaenike. 1984. Competition in natural populations of mycophagous Drosophila. Ecology 65:1113-1120. Hochberg, M.E. and R. D. Holt. 1990. The coexistence of competing parasites. I. The role of cross-species infection. A/flf. 136:517-541. Ives, A.R. and R. M. May. 1985. Competition within and between competing species in a 183 patchy environment: relations between microscopic and macroscopic models. J. Theor. Biol. 115:65-92. Jaenike, J. 1988. Parasitism and male mating success in Drosophila tesiacea. Am. Mat. 131:774-780. Jaenike, J. 1992. Mycophagous Drosophila and their nematode parasites. Am. Nat. 139:893-906. Jaenike. J. 1994. .Aggregation of nematode parasites within Drosophila: proximate causes. Parasitology 108:569-577. Jaenike. J. 1996a. Suboptimal virulence of an insect-parasitic nematode. Evolution 50: 2241-2247. Jaenike. J. 1996b. Rapid evolution of parasitic nematodes: not. Evol. Ecol. 10:565. Jaenike. J. 1998. On the capacity of macroparasites to control insect populations. Am. Nat. 151:84-96. Jaenike. J. and I. Dombeck. 1998. General-purpose genotypes for host species utilization in a nematode parasite of Drosophila. Evolution 52:832-40. Jaenike. J. and A. C. James. 1991. Aggregation and the coexistence of mycophagous Drosophila. J. Animal Ecol. 60:913-928. Jaenike. J.. H. Benway and G. Stevens. 1995. Parasite-induced mortality in mycophagous Drosophila. Ecology 76:383-391. JMP. 3.1.5. 1989 - 1995. SAS Institute, Inc. Koppenhofer, A.M.. H. K. Kaya, S. Shaiunugam and G. L. Wood. 1995. Interspecific competition between steinemematid nematodes within an insect host. J. 184 Invert. Path. 66: 99-103. Margolis, L., G. W. Esch, J. C. Holmes, A. M. Kuris and G. A. Schad. 1982. The use of ecological terms in parasitology (report of an ad hoc committee of the American Society of Parasitologists). J. Parasit. 68:131-133. McCallum, H. and A. Dobson. 1995. Detecting disease and parasite threats to endangered species and ecosystems. Trends Ecol. Evol. 10:90-94. Petney. T.N. and R. H. .Andrews. 1998. Multiparasite communities in animals and humans: frequency, structure and pathogenic significance. Int. J. Parasit. 28:377- 393. Poinar. G. O. Jr.. J. Jaenike and 1. Dombeck. 1997. Parasityienchus nearcticus sp. n. (Allantonematidae: Tyienchida) parasitizing Drosophila (Drosophilidae: Diptera) in North .A.merica. Fund. Appl. Nem. 20:187-190. Poulin, R. 1998. Evolutionary ecology of parasites: from individuals to communities. Chapman and Hall. Roberts. D.B. 1986. Drosophila: a practical approach. IRL Press. Oxford, UK. Roberts, M.G and A. P. Dobson. 1995. The population dynamics of communities of parasitic helminths. Math. Biosci. 126:191-214. Schall. J.J. and C. R. Brumwich. 1994. Interspecific interactions tested in two species of malarial parasite in a West African lizard. Oecologia 97:326-332. Shaw, D.J. and A. P. Dobson. 1995. Patterns of macroparasite abundance and aggregation in wildlife populations: a quantitative review. Parasitology 111:S111-S133. 185 Sousa, W.P. 1993. Interspecific antagonism and species coexistence in a diverse guild of larval trematode parasites. Ecol. Monogr. 63:103-128. Taylor. L.H., M. J. Mackinnon and A. F. Read. 1998. Virulence of mixed-clone and single-clone infections of the rodent malaria Plasmodium chabaiidi. Evolution 52:583-591. Welch. H.E. 1959. Ta.\onomy. life cycle, development, and habits of two new species of .A.ilantonematidae ("Nematoda. parasitic in drosophilid flies. Parasitology 49:83- 103. Wertheim. B.. J. G. Sevenster. I. E. M. Eijs. and J.J.M. van Alphen. 2000. Species diversity in a mycophagous insect communitN': the case of spatial aggregation vs. resource partitioning. J. Animal Ecol. 69: 335-351. 186 Table I. Percent infection by Howardula aoronymphium and Parasitylenchus nearcticus (with lowest and highest vials per treatment), and relative density of H. aoronymphium motherworms (total number of motherworms divided by the total number of flies (± SE)) in Drosophila recens. Trial Nematode species Infectives No. Parasitylenchus Howardula Howardula per vial flies % infection % infection relative density I H. aoronymphium 200 44 18 (0. 28) .23 ± .08 1 P. nearcticus 200 64 80 (62. 100) I both nematodes 400 48 88 (75. 100) 25(14.31) .33 ± .09 t H. aoronymphium 400 61 48 (27. 80) .66 ± .10 -) P. nearcticus 400 24 83 (75. 100) -> both nematodes 800 52 90 (85. 100) 38(17.67) .63 ±.15 H. aoronymphium 800 63 30(7.75) .46 ±.10 2 P. nearcticus 800 29 97 (83. 100) "> both nematodes 1600 29 100 41 (11.80) 1.00 ±.27 3 H. aoronymphium 800 50 50 (25. 100) .84 ±.15 3 P. nearcticus 800 46 98 (91. 100) 3 both nematodes 1600 42 98 (86.100) 43 (22. 58) .67 ±.15 187 Table 2. Infection rates of A) Howardula aoronymphium (relative density = total mothervvorms per total flies) and B) Parasitylenchus nearcticus (percent infection) as a function of host sex, infection density, infection with or without the other nematode species, and replicate vial. Flies were pooled from all experiments. (Note that because P. nearcticus motherworms disintegrate in hosts, we measure their % infection and not their relative density. Because P. nearcticus percent infection data is not normally distributed, even after arcsin sqrt transformation (due to very high infection rates), we assessed significance by comparing test statistics to a distribution obtained by randomizing data 500 times.) All effects are fi.xed. except for vials, which are nested. A) = == - Source df SS F P>F Se.\ I 0.06 0.07 0.78 Infection concentration 2 13.53 8.24 0.0003 Single vs. double infection I 0.03 0.03 0.85 Vial [Treatment] 10 18.86 2.30 0.01 Error 374 307.30 B) - Source df SS F F*>F Sex 1 0.015 1.16 0.29 Infection concentration 2 0.267 10.13 0.002 Single vs. double infection I 0.001 O.Il 0.71 Vial [Treatment] 10 0.119 0.90 0.54 Error 63 0.8 188 FIGURE LEGENDS Fig. 1. Number of mature eggs ± SE (stage 10 and up) in one-week old female Drosophila recens, of var\'ing infection status. Numbers above bars indicate sample sizes of flies. Fig. 2. Mean survival in days ± SE of adult Drosophila recens, of varying infection status. Numbers above bars indicate sample sizes of flies. Solid bars = females; open bars = males. Fig. 3. Mean size ± SE {mm\ longitudinal section area) of Howardula aoronymphiiim motherworms in one-week old Drosophila recens. in single and mi.xed infections. Open bars = motherworm size in tlies infected with only H. aoronymphiiim: solid bars = motherworm size in tlies infected with both H. aoronymphiiim and Parasiiyienchiis nearcliciis. Numbers above bars indicate sample sizes of tlies. Fig. 4. Number of Parasiiyienchiis nearcliciis F1 offspring ± SE. in single and mi.\ed infections. Solid bars = adults infected with P. nearcliciis only; open bars = flies infected with both H. aoronymphiiim and P. nearcliciis. Numbers above bars indicate sample sizes of flies. 189 uninfected H. aoronymphium P. nearcticus mixed infection status 190 uninfected H. aoronymphium P. nearcticus mixed infection status motherworm size 192 200 400 800 larval nematodes per sample treatment 193 APPENDIX FIVE: ACTUAL VERSUS POTENTIAL HOST RANGE OF A PARASITIC NEMATODE OF DROSOPHILA 194 ACTUAL VERSUS POTENTIAL HOST RANGE OF A PARASITIC NEMATODE OF DROSOPHILA Steve J. Perlman'* and John Jaenike" 'Department of Ecology and Evolutionary Biology Biological Sciences West. University of Arizona. Tucson, AZ, 85721 "Department of Biology Hutchison Hall. University' of Rochester. Rochester. NY. 14627 * corresponding author email: sperlman(2iu.arizona.edu Tel; 520-626-8455 Fax:520-621-9190 195 Abstract. - Parasites have been implicated as important forces in determining the geographic distribution of their hosts. This has been motivated by the observation that closely-related species often differ in their susceptibility to parasites, with one species being more severely affected. In the extreme case, a host species can be excluded from a particular habitat due to a virulent parasite. In this study we document differences between two closely related Drosophila flies (Diptera : Drosophilidae) in their ecological host suitability to a parasitic nematode, Howardula neocosmis (Tylenchida : Allantonematidae). We show that H. neocosmis infects D. acutilabella at significantly higher rates in nature than the closely related, common and microsympatric species. D. cardini. The nematode successfully infects and grows in both species in the laborator>'. Differences in parasite virulence do not explain why D. cardini is rarely infected in nature, as the nematode affects host survival of both species equally. Nor is higher prevalence in D. acutilabella due to competitive interactions between D. acutilabella and D. cardini within individual mushroom breeding sites, as equal numbers of flies emerged from mixed mushroom treatments. Instead, we suggest that D. cardini is rarely infected because the two host species do not share breeding sites in nature. Thus, differential resource use may drive differences in parasitism between the two host species, and not vice versa. Key words. - Host range, mycophagy, parasite ecology, parasite-mediated competition, virulence. 196 Two distinct sets of factors determine a parasite's host range. First, a host must be intrinsically suitable for parasite infection, development and transmission (Solter and Maddo.\ 1998). Intrinsic suitability is determined by physiological and biochemical properties of the host, and may have an important phylogenetic component. In addition to intrinsic host suitability however, a number of ecological conditions are required in order to permit the maintenance of the parasite on a specific host in nature. For e.xample, temperature (Jaenike 1995), host breeding site (Jaenike 1985), and the presence of other competing parasite species (Dobson 1985) can all e.\clude an otherwise perfectly intrinsically suitable host from a parasite's host range. Furthermore, factors which lower a parasite's host threshold density, or the minimum density of hosts required for positive parasite population growth (Anderson and May 1979), will also restrict a parasite's host range. These include high parasite virulence and low host density. A parasite with different host suitabilities can have important ecological consequences for its hosts. One example is parasite-mediated competition, where differences in the suitability of one host species over another can facilitate the coexistence of both (Park 1948; Price et al. 1986). This phenomenon has typically described the situation where one host species is more severely affected by a parasite, while the other host species is an inferior competitor. In the extreme case of parasite- mediated competition, a host can be excluded from a certain habitat because of a particularly virulent parasite (Kohler and Wiley 1997). In this study, we examine the host range of Howardula neocosmis (Tylenchida: Allantonematidae), a recently discovered parasitic nematode of Drosophila flies (Diptera: 197 Drosophilidae). This parasite shows interesting differences between its potential and actual host ranges, which yield clues about the ecology of its hosts. In preliminary surveys in the Southeastern United States (Poinar et al. 1998), H. neocosmis was only collected from Drosophila acutilabella and not D. cardini, which is also common and microsympatric with D. acutilabella. These species are close relatives in the Drosophila cardini species group, and will readily mate in the lab, producing few. sterile hybrids (Stalker 1953). We first determine the actual host range of H. neocosmis in the Southeastern United States through detailed sampling of potential hosts and confirm that D. cardini is rarely infected. We then assess the intrinsic host suitability of D. acutilabella and D. cardini by measuring infection success and parasite growth in multiple strains of the two host species. Finally, we test two hypotheses for why H. neocosmis may infect D. acutilabella at higher rates than D. cardini in nature. To determine if D. cardini is rarely infected in the wild because the nematode is highly virulent to it. we compare parasite- induced adult mortality in the two host species. We also examine competitive interactions between D. acutilabella and D. cardini within mushroom breeding sites, in order to evaluate the potential for interspecific transmission of the nematode. If D. cardini is severely outcompeted by D. acutilabella, such that few D. cardini emerge from mixed mushrooms, then it will seldom be infected by the nematode, as mushrooms are the site of nematode infection. Mycophagous Drosophila are commonly infected by parasitic Howardula nematodes (Kimura and Toda 1989; Jaenike 1992; Gillis and Hardy 1997). Inseminated 198 female nematodes infect fly larvae when they are feeding inside mushrooms, by piercing through their cuticle (Welch 1959). When the adult fly emerges, the nematode mothervvorm begins releasing juveniles (Fl) into the hemocoel of the host. These juvenile nematodes are passed from the anus and ovipositor of the host as it visits mushrooms, where the nematodes subsequently mate and continue the cycle. Because individual mushrooms are often oviposited on by multiple adult Drosophila, belonging to several species (Jaenike and James 1991), there are generally many opportunities for horizontal transmission of parasites both within and among host species. These nematodes can have severe effects on host fitness, including reduced female fecundity, male fertility, male mating success, and adult survival (Jaenike 1988. 1992: Jaenike et al. 1995). Through differential effects on different host species, Howardula may also affect the structure of Drosophila communities (Jaenike 1995). MATERIALS AND METHODS Collections of Howardula neocosmis in Florida In order to determine the actual host range of Florida H. neocosmis. we collected mushroom-feeding Drosophila in Florida. We used store-bought soaked Agaricus bisporus as baits, placing them in shady wooded areas. We collected flies in Englewood, Sarasota County, in February 1997 (collected by John Jaenike), and Ft. Lauderdale, Broward County, in March 2000 and September 2001 (collected by Steve Perlman). We also collected D. acutilabella and D. cardini that visited compost supplemented with mushroom, in Tallahassee, Leon County, in September and October 2001 (collected by 199 Steve Perlman and Jeff Birdsley). Flies were returned to the laboratory, dissected and scored for the presence of adult motherworm nematodes. Each motherworm represents an independent successful infection. We used contingency tests to ask whether there were significant differences in infection frequency between host species and between localities. Drosophila and Howardula Stocks Flies were maintained on Instant Drosophila Medium (Carolina Biological Supply) with store-bought A. bisporus mushroom. .All experiments were carried out at room temperature on a 12:12 lightrdark cycle. All H. neocosmis used in experiments were established from multiple nematodes infecting D. acutilabella. collected in Ft. Lauderdale. March 2000. These were maintained in the laborator>' on a stock of D. acutilabella collected on Sanibel I.. Lee County, in 1997. in vials containing Instant Drosophila Medium and A. bisporus mushrooms as breeding sites. Because infected adult flies pass nematodes to the mushrooms, many flies that develop as larvae in these mushrooms become infected. Infection assays of D. acutilabella and D. cardini To assess the intrinsic suitability of D. cardini and D. acutilabella as hosts for H. neocosmis, we infected multiple stocks with nematodes. The strains that were used are listed in Table 2. To collect fly eggs for infection, uninfected females were placed in petri dishes with food plugs made from blended mushrooms, agar, and water. Nematodes 200 were obtained by grinding one to two week old infected hosts in saline solution. Slurry containing ~200 juvenile infective nematodes was pipetted onto a 0.4g piece of A. bisporns mushroom. On the following day, 25 host eggs were placed on the mushroom pieces in vials with moistened cheesecloth (8 replicates). Mushroom was added to the vial when necessary to prevent starvation of fly larvae. Upon emergence, adult flies were collected and transferred to vials containing Instant Drosophila Medium and fresh mushroom. Flies were frozen 7 days after emergence, and dissected later for the presence of motherworms. We also measured the sizes of a subset of these motherworms, using National Institutes of Health IMAGE digital imaging software (http://rsb.info.nih.gov/nih-image/). Digital images were captured on an Olympus stereomicroscope with an attached digital camera. .An analysis of variance was performed to test whether the total number of motherworms per fly depended on host species (D. cardini or D. acutilabella). host strain, and se.\. with treatment vials nested in host strain and species. Because infection data departed significantly from normality, even after transformation, we assessed the significance of the effects in our model by using randomization tests (Manly 1998). We compared F ratios to a distribution of F ratios obtained from 1000 reshuffled data sets, in which the y variable (motherworm number) was fully randomized. We used least significant difference tests to compare motherworm number between host strains. We used analysis of covariance to test whether motherworm size was significantly affected by host species, host strain, host sex and by the number of motherworms developing 201 inside a host. All statistics were obtained using the package JMP 4.0.4 (1989-2001), and data were reshuffled in MINITAB (version 10, 1994). Effect of Nematodes on Adult Survival In order to determine whether D. cardini suffer high parasite-induced mortality relative to D. acuiilabella, we compared the survival of infected and uninfected flies of the two species in controlled laboratory infections. Flies were descended from recently collected females (>5 generations in the lab), collected in Ft. Lauderdale, in September 2001. Infections were set up as above, but with an initial nematode sluny containing -400 juvenile nematodes per culture. We set up 23 replicate vials of D. cardini. and 28 with D. acutilabella. We also set up 14 uninfected control vials of D. cardini and 14 of D. acutilabella. Upon emergence of adults, males and females were aspirated into separate vials to avoid any effects of mating on survival (Chapman et al. 1995). Flies were transferred weekly to new vials containing standard commeal and yeast medium. Vials were checked daily for dead flies, which were frozen for later dissection, with lifespan, infection status, and number of motherworms recorded for each fly. We used log-rank tests and Cox proportional hazard regressions to test whether adult mortality was affected by infection with H. neocosmis. and whether D. cardini and D. acutilabella were affected differently. 202 Competition between D. acutilabella and D. cardini within mushrooms In order to examine the potential for interspecific transmission of nematodes, and to determine whether D. cardini suffer from competition with D. acutilabella within mushrooms, we gave females of both species an opportunity to oviposit on a small mushroom and recorded the number of emergent offspring. Flies were descended from females collected in Ft. Lauderdale, in September 2001. Females were aged for a week before the experiment, and kept in vials with mushroom and Carolina Instant Drosophila medium. .After one week, four female D. cardini and four female D. acutilabella were placed in a mason jar with cheesecloth and a soaked 20g .4. bisporus mushroom piece (14 replicates). As controls, we set up jars with either four female D. cardini (seven replicates) or four female D. acutilabella (eight replicates). Females were kept on the mushroom for four days. .All emergent offspring were counted and identified to species. RESULTS Collections of Howardula neocosmis in Florida Over 95% of the Drosophila that visited mushrooms were D. acutilabella and D. cardini: no other Drosophila that visited baits (D. tripunctata and D. putrida) were infected with nematodes. Only D. acutilabella was infected by H. neocosmis (Table 1), with significant differences in infection prevalence over the three collection sites (likelihood ratio test: x2 = 23.51. p < .0001). The highest infection frequency was in Ft. Lauderdale - 5% of D. acutilabella collected there were infected. There were no sex differences in infection (Ft. Lauderdale only. Fisher's exact test: p = .38). No D. cardini were infected. 203 although over 1400 flies were dissected; this infection frequency differs significantly from D. acutilabella (Fisher's exact test: p = .001). Susceptibility of D. acutilabella and D. cardini In experimental lab infections. Howardula neocosmis successfully infected and developed on both host species (Table 2. 3). There were significant ditTerences in infection success between strains of D. acutilabella. but not between strains of D. cardini (Table 2. 3), with two strains highly susceptible (15181-2171.1, 2171.7). and two that appeared to be highly resistant to infection (15181-2171.2. 2171.9). Motherworm sizes differed significantly between strains, but not between species (Table 2. 4). Motherworms were larger in female hosts (Table 4). probably because female flies are larger. Worm size was negatively correlated with the number of motherworms per individual fly (r" = 0.21. p < 0.0001). Effect of Nematodes on Adult Survival Uninfected D. cardini survived significantly longer than uninfected D. acutilabella (Fig. 1). but there was no significant effect of nematode infection on sur\ ival (proportional hazards regression: host species: x"i = 9.65. p < 0.002; infection status: x"i ~ 0.73. p > 0-3: host species X infection status: x"i = 0.84, p > 0.3). Infected D. cardini and D. acutilabella survived an average (± s.e) 53.52 ± 2.64 and 43.34 ±7.1 days, respectively. There were no significant sex differences in survival, for either species or infection status, except that uninfected D. acutilabella females lived slightly longer than uninfected 204 males (log-rank test, x"i = 3.89, p = 0.05). There was no difference in the rate of infection (number of motherworms per fly) for D. cardini versus D. acutilabella (ANOVA: host species F1.186 = 0.25, p > 0.6; vial [host species] F54.i86= 2.27, p < 0.01). Competition between D. acutilabella and D. cardini within mushrooms D. cardini did not suffer in the presence of D. acutilabella (Fig. 2); equal numbers of D. cardini emerged from jars that had one or both species (ti9 = .91, p = .37). Significantly fewer D. acutilabella emerged from mi.xed than single species treatments jars (tjo = 2.07, p = .05). Equal numbers of the two species emerged from mixed jars (mean difference per jar = 2.14 i 15.12: matched pair t-test: ti3 = .14, p = .89). In 8 out of 14 mi.xed jars however, there was a clear asymmetry in species emergence. Fewer than ten D. acutilabella emerged in three of the mi.xed jars, and fewer than ten D. cardini emerged in five jars. DISCUSSION We have clearly demonstrated a difference between the potential and actual host ranges of Howardula neocosmis. While H. neocosmis can successfiilly infect and reproduce in D. cardini. we did not collect any infected D. cardini nature. This is despite the fact that D. cardini and D. acutilabella are microsympatric and can be readily collected fi-om the same mushroom baits. The nematode is however, relatively rare, having only been collected at appreciable frequencies at one of three collection sites in Florida. We therefore cannot conclude that D. cardini is never infected in the wild. Frequencies of other Drosophila-paiasixic Howardula in the wild vary w idely, and can 205 range from less than 1% to over 50% (Kimura and Toda 1989; Jaenike 1992; Gillis and Hardy 1997). We are confident that we have not excluded any other major hosts of H. neocosmis in the Southeastern United States. From preliminary experimental infections, the only other intrinsically suitable host for H. neocosmis which occurs in Florida is D. giittifera (Appendix 2). This mushroom-breeding species is rare and was not collected in this study. D. ccirdini is an intrinsically suitable host for H. neocosmis. All of the strains of D. cardini we tested were successfully infected by H. neocosmis. including tlies collected from the same locale as the nematode. There were no significant differences in the size of motherworms infecting either of the two host species. There was much greater variation in infection prevalence in the D. acutilabella strains we tested than in those of D. ccirdini. with some strains appearing resistant and others highly susceptible. It is unclear however, how to interpret these differences, since four out of five D. acutilabella strains tested have been maintained at the Drosophila species stock center for many generations and are potentially highly inbred. Why then are D. cardini rarely, if ever, infected in nature? This lower prevalence is not due to high parasite virulence, and there was no difference in the survival of infected D. cardini and D. acutilabella. Uninfected D. cardini live slightly longer than infected adults (67.79 ± 2.67 and 53.52 ± 2.64 days); this difference is not significant (log-rank test, x"i = 1-28. p > 0.2). While Howardula-mdwc^d mortality will be greater in the wild than in the laboratory (Jaenike et al. 1995), our results do not suggest that virulence restricts D. cardini from serving as a suitable host. We previously showed that 206 novel Howardula infections of naive host species can result in severe adult mortality (Appendix 3). Because Howardula are transmitted to new mushrooms by adult Drosophila, parasite-induced mortality is detrimental to the nematodes as well. For a Howardula infection to be successfully established and maintained in nature, we therefore expect parasite-induced mortality to be low. Nor does lower prevalence in D. cardini appear to be due to competition with D. acuiilabella within individual mushrooms. D. cardini must suffer an extreme competitive disadvantage for this mechanism to explain the host range differences we observe in nature. Even a few D. cardini emerging from mixed mushrooms, as was obser\ ed in our competition experiment, would permit a host switch. While other factors such as mushroom size, desiccation, temporal priority and temperature (Hodge et al. 1996; Worthen et al. 1998; Worthen and Haney 1999) may play a role in D. cardini - D. acuiilabella interactions, and may even tip the balance within a mushroom in favor of D. acuiilabella, this mechanism is probably still too restrictive to explain the host range differences. The most likely explanation for why D. cardini is seldom infected is that D. acuiilabella and D. cardini probably do not share breeding sites. Many studies have documented the importance of resource partitioning in promoting the coexistence of fungal-feeding Drosophila (Kimura and Toda 1989; Toda et al. 1999; Wertheim et al. 2000). However, little is known about the ecology of D. aciitilabella and D. cardini, or of the entire D. cardini species group for that matter, and it remains to be determined conclusively whether these species breed primarily in mushrooms. Multiple factors point 207 to the importance of fungi as breeding sites for D. acutilabella and D. cardini. Both species are attracted to and breed in mushroom baits, and have been maintained for over 50 generations in the laboratory on A. bisponts mushrooms. We have also collected D. acutilabella adults and larvae (but not D. cardini) on wild mushrooms in Florida (personal observation). D. cardini group species have also been collected on and observed breeding in decaying fruits and vegetation, although less so than strictly fruit- breeding species such as D. melanogaster (Martinez-Pico et al. 1965). They also appear to have lower ethanol tolerance than other fruit-breeding Drosophila (Colon-Parrilla and Perez-Chiesa 1999). Finally, the nematodes themselves offer clues to the breeding habits of their hosts. Howardiila neocosniis is found in a clade of nematodes that infect mycophagous Drosophila. further implicating mushrooms as hosts for D. acutilabella. .\ ver>' closely related nematode has also been collected from two mushroom-breeders in the D. quinaria species group ~ D. suboccidentalis. in British Columbia (Poinar et al. 1998) and D. munda. in Arizona (Appendix I). Quantitative morphological differences, as well as DNA sequence differences between the British Columbia and Florida strains of H. neocosmis suggest that they may be closely related species (Poinar et al. 1998; Appendix I). The different nematodes also appear to be specialized on their local hosts; in preliminar>' e.xperimental infections, H. neocosmis from D. acutilabella infected D. munda and D. suboccidentalis at much lower frequencies than D. acutilabella (Perlman. unpublished data). 208 Parasites have been implicated as important selective agents in determining the geographic distribution of their hosts (Price et al. 1986; Wullschleger and Jokela 1999). Indeed, closely related species often differ in their susceptibility to parasites (Park 1948; Price et al. 1986; Thomas et al. 1995), and it is this difference that drives parasite- mediated competition. In this study however, the reverse phenomenon may be operating. Host and parasite distribution patterns do not result from differences in susceptibility, as both D. cardini and D. acutilabella are equally susceptible to H. neocosmis. Instead, differences in parasitism between the two host species may be a consequence of differential resource use. and not the other way round. ACKNOWLEDGMENTS We thank D. Houle for use of his lab while in Florida. J. Birdsley for collecting flies in Tallahassee, and the staff at Pinewood Park, in Broward County, for permission to collect flies. We also thank W. Maddison for use of his digital camera, and L. Knowles for help with the imaging software. This research was supported by National Science Foundation (NSF) grant DEB-0074141 to JJ, and ftmds to SJP from the .American Museum of Natural History and the Center for Insect Science at the University of Arizona. 209 LITERATURE CITED Anderson, R. M. and May, R. M. 1978. Regulation and stability of host-parasite population interactions. I. Regulatory processes. J. Animal. Ecol. 47:219-247. Chapman, T.. Liddle, L. F., Kals, J. M., Woifner, M. and Partridge, L. 1995. Cost of mating in Drosophila melanogaster females is mediated by male accessory gland products. Nature 373:241-4. Colon-Parrilla. W. V. and 1. Perez-Chiesa. 1999. Ethanol tolerance and alcohol dehydrogenase (ADH; EC 1.1.1.1) activity in species of the cardini group of Drosophila. Biochemical Genetics 37:95-107. Dobson. A. P. 1985. The population dynamics of competition between parasites. Parasitology 91:317-47. Gillis. J. E. M. and I. C. W. Hardy. 1997. Nematode parasitism in a northern European drosophilid community. £m. Exp. et App. 84:275-91. Hodge, S., W. Arthur and P. Mitchell. 1996. Effects of temporal priority on interspecific interactions and community development. Oikos 76:350-358. Jaenike, J. 1985. Parasite pressure and the evolution of amanitin tolerance in Drosophila. Evolution 39:1295-1301. Jaenike, J. 1988. Parasitism and male mating success in Drosophila testacea. Am. Xat. 131:774-780. Jaenike, J. 1992. Mycophagous Drosophila and their nematode parasites. Am. Nat. 139:893-906. Jaenike, J. 1995. Interactions between mycophagous Drosophila and their nematode 210 parasites: from physiological to community ecology. Oikos 72:235-44. Jaenike, J., H. Benway and G. Stevens. 1995. Parasite-induced mortality in mycophagous Drosophila. Eco/ogy 76:383-91. Jaenike, J. and A. C. James. 1991. Aggregation and the coexistence of mycophagous Drosophila. J. Anim. Ecol. 60:913-928. JMP 4.0.4. 1989-2001. SAS Institute. Gary, N. Carolina. USA. Kimura, M. T. and M. J. Toda. 1989. Food preferences and nematode parasitism in mycophagous Drosophila. Ecol. Res. 4:209-18. Kohler. S. L. and M. J. Wiley. 1997. Pathogen outbreaks reveal large-scale effects of competition in stream communities. Ecology 78:2164-70. Manly. B. F. J. 1998. Randomization, bootstrap and Monte Carlo methods in biology. 2"'' Edition. Chapman & Hall. London, England. Martinez Pico. M.. C. Maldonado and R. Levins. 1965. Ecology and genetics of Puerto Rican Drosophila-. I. Food preferences of sympatric species. Carib. J. Sci. 5:29- 37. MINIT.4B Release 10 for Windows. 1994. Minitab Inc. 3081 Enterprise Dr. State College. Pennsylvania, USA. Park. T. 1948. E.xperimental studies of competition. I. Competition between populations of the flour beetles Tribolium confiisiim Duval and Tribolium castaneum Herbst. Ecol. Monogr. 18:265-308. Poinar, G. O. Jr., J. Jaenike and D. D. Shoemaker. 1998. Howardula neocosmis sp. n. (Tylenchida: Allantonematidae) parasitizing North American Drosophila 211 (Diptera: Drosophilidae) with a key to the species of Howardula. Fundam. Appl. Nematol. 21:547-552. Price. P. W., M. Westoby, B. Rice, P. T. Atsatt, R. S. Fritz, J. N. Thompson and K. Mobley. 1986. Parasite mediation in ecological interactions. Ann. Rev. Ecol. Syst. 17:487-505. Solter. L. F. and J. V. Maddox. 1998. Physiological host specificity of microsporidia as an indicator of ecological host specificity. J. Invert. Path. 71:207-16. Stalker. H. D. 1953. Ta.\onomy and hybridization in the cardini group o'L DrosophiUi. Ann. Ent. Sac. Amer. 46:343-358. Thomas. F.. F. Renaud, F. Rousset. F. Cezilly and T. De Meeus. 1995. Differential mortality of two closely related host species induced by one parasite. Proc. R. Soc. Lond B 260:349-352. Toda. M. J.. M. T. Kimura and N. Tuno. 1999. Coe.xistence mechanisms of mycophagous drosophilids on multispecies fungal hosts: aggregation and resource partitioning. J. Anim. Ecol. 68:794-803. Welch. H. E. 1959. Taxonomy, life cycle, development, and habits of two new species of Allantonematidae (Nematoda) parasitic in drosophilid flies. Parasitology 49:83- 103. Wertheim. B.. J. G. Sevenster. I. E. M. Eijs and J. J. M. van Alphen. 2000. Species diversity in a mycophagous insect community: the case of spatial aggregation vs. resource partitioning, y. ^mm. Ecol. 69:335-351. Worthen, W.B., M. T. Jones and R. M. Jetton. 1998. Community structure and 212 environmental stress: desiccation promotes nestedness in mycophagous fly communities. Oikos 81:45-54. Worthen, W. B. and D. C. Haney. 1999. Temperature tolerance m three mycophagous Drosophila species: relationships with community structure. Oikos 86:113-118. Wullschleger, E. and J. Jokela. 1999. Does habitat-specific variation in trematode infection risks influence habitat distribution of two closely related freshwater snails? Oecologia 121:32-38. 213 Table I. Prevalence of H. neocosmis in wild-caught flies from Florida . Collection location and Host species date D. acutilabella D. cardini D. puirida D. tripunciata Englewood, 1997 2/224 0/1048 0/52 Ft. Lauderdale, 2000 14/220" 0/80 0/49 Ft. Lauderdale. 2001 7/225 0/102 Tallahassee. 2001 0/261 0/216 Totals 23/930 0/1446 0/52 0/49 a. One of these tlies was infected with 2 moiherworms, and another with 3. 214 Table 2. Relative densities (total motherworms per fly ± s.e) and motherworm sizes (mm" ± s.e) of H. neocosmis in experimental infections of strains of D. aciitilabella and D cardini. SC refers to stock numbers of strains obtained from the Drosophila species stock center (http://stockcenter.arl.arizona.edu). Different letters indicate pairs that are significantly different from each other (P < 0.05). Host species and Strain locale relative density ± motherworm size ± strain s.e. s.e. D. acitiilabella SC- 15181-2171.1 Petionville. Haiti 0.95 ± 0.077 (65) a 0.41 ±0.097 (41) a SC - 15181-2171.2 San Vicente, Cuba 0.05 ± 0.035 (40) c 0.52 (l)ab SC - 15181-2171.7 Hermitage Reservoir, 0.93 ± 0.083 (84) b 0.41 ± 0.099 (46) a Jamaica SC - 15181-2171.9 Everglades. Rorida 0 (27) be n/a March 2000 Ft. Lauderdale. Rorida 0.24 ± 0.074 (42) be 0.57 ± 0.0083 (4) b D. cardini SC - 15181-2181.9 Contramaesu-e, Cuba 0.22 ± 0.043 (92) c 0.51 ±0.032 (5)bc SC - 15181-2181.11 Pisco, Peru 0.29 ±0.11 (7)c 0.59 ± 0.022 (ll)c SC - 15181-2181.13 Grand Cayman 0.062 ± 0.025 (97) c 0.41 ±0.024 (3) ac March 2000 Ft. Lauderdale, Rorida 0.083 ±0.083 (12) c 0.59 (l)ab 215 Table 3. Motherworm number as a function of host species (D. aciitilabella and D. cardini), strain, sex and vial. Source df SS F P Host species I 0.97 4.56 0.047 Sex I 0.83 3.92 0.051 Strain [Species] 7 4.70 3.16 0.014 Vial [Strain. Species) 55 21.24 1.82 0.003 Error 411 87.37 216 Table 4. Mothervvorm size as a function of host species (D. acutilabella and D. cardini), strain, sex and motherworm number per fly. Source df SS F P Host species I 0.005 0.87 0.35 Sex 1 0.17 29.52 <0.0001 Strain [Species] 6 0.094 2.78 0.015 Motlierworm Number I 0.25 44.73 <0.0001 Error 102 0.58 217 FIGURE LEGENDS 1. Survivorship of (A) infected and (B) uninfected D. cardini and D. acutilabella. Individual trajectories indicate whether the flies were uninfected or infected with H. neocosmis. along with the number of flies each group was started with. 2. Mean (± s.e.) number of D. cardini and D. acutilabella emerging from single and mixed jar treatments. Numbers above bars indicate sample sizes. We also show the percent D. cardini emerging from each of the 14 mixed jars, with a mean of 43%. ufintectedD. careitni {r\»t66) n( ecf e infected CXcaniini (n * 23) day 14 90 D. cardini •Zl D. acutilabella 60 o Q. ocn (0 o •a « X 30- 0) CL 0.5 "S O D. acutilabella both D. cardini alone flies alone