-SYMBIONT INTERACTIONS Ecology of Symbiotes of Larval Black (Diptera: Simuliidae): Distribution, Diversity, and Scale

1 2 2 JOHN W. MCCREADIE, PETER H. ADLER, AND CHARLES E. BEARD

Environ. Entomol. 40(2): 289Ð302 (2011); DOI: 10.1603/EN10258

ABSTRACT Symbioses are major drivers in ecology and evolution. Although nearly omnipresent in Downloaded from https://academic.oup.com/ee/article/40/2/289/409195 by guest on 24 September 2021 ßowing waters, they remain poorly studied in these systems. To examine fundamental aspects of the ecology of symbioses in ßowing-water systems, we use larval black ßies as hosts and various fungi, nematodes, and as symbiotes, focusing on aspects of distribution, diversity, and scale. Most symbiotes of larval black ßies are considered parasitic, although the dynamic nature of the relationship is becoming apparent for some systems in which it shifts along a continuum involving commensalism, mutualism, and . Perspective also is moving from a pairwise view of symbiotic associations to a multispecies network of interactions. Distributions of symbiotes are related to scale-dependent processes that inßuence the hosts and the stream habitats of the hosts; thus, characteristics of streams, as well as hosts, can be useful in predicting spatial patterns of symbiotes. As the of symbiotes improves, so too does the understanding of ecological relationships of symbiosis, such as host speciÞcity and patterns of diversity along spatial and temporal scales.

KEY WORDS freshwater, microsporidia, nematodes, parasitism, streams

The pursuit by ecologists to Þnd universal patterns of and diversity are the picture we wish to illustrate, scale diversity requires a broad understanding of species is the canvas on which they are drawn. interactions. The study of species interactions, how- Black ßies are ideal hosts for this exploration be- ever, has been biased, with predation and competition cause (1) the larvae play a critical role as ecosystem commanding a disproportionate amount of attention engineers in resource turnover and foodweb dynamics (Begon et al. 2006). However, multispecies microbial in lotic communities (Cummins 1988, Wotton et al. interactions are probably the prevailing form of spe- 1998, Malmqvist 2004); (2) they are taxonomically one cies interactions (Schmitt et al. 2007). Symbiosis is of the best-known groups of aquatic in many now recognized as a major force in the ecology and areas of the globe (Adler et al. 1999, 2004), facilitating evolution of organisms at both population and com- detection of host associations at the species level; and munity levels (Boucher 1985, Douglas 1994, Sapp 1994, (3) they are globally widespread in lotic systems Thompson 2005, Moran 2006) and as a driver of com- (Crosskey 1990), representing part of a prevailing munity structure and coevolutionary processes (Vo- symbiotic relationship. We emphasize North Ameri- gelsang et al. 2006, Moran 2007, Noda et al. 2007). can studies, but consult the world literature when Despite the ubiquitous nature and ecological im- relevant. We use our unpublished data when the lit- portance of symbiotic relationships, little is known erature lacks examples of particular patterns or pro- about the community structure and function of sym- cesses of symbiote ecology, and we reevaluate previ- bioses in freshwater habitats. To explore the ecology ously published data to provide insights into patterns of symbiosis in stream insects, we use the larval black of the symbiote assemblage. ßy host as a platform. To impose order on the review, we have selected a major theme of ecological thoughtÐ What Is Symbiosis? distribution. Indeed, ecology, broadly deÞned, is the study of factors and processes that determine the dis- Although ecologists appreciate the role of symbiosis tribution and abundance of organisms across a heter- in ecological communities, deÞning symbiosis has ogeneous landscape (Krebs 2008). The mechanisms been problematic. In its simplest form, symbiosis is an that control the distribution and diversity of species interspeciÞc interaction deÞned by Þtness effects on are scale dependent (Adler and McCreadie 1997, Mc- each participant and expressed simplistically as Ϫ,0, Creadie and Adler 1998, McGill 2010). If distribution or ϩ, with no indication of degree (Table 1). Mutu- alism, for example, is a ϩϩinteraction, with no im- 1 Corresponding author: Department of Biological Sciences, Uni- plied symmetry, that is, no assumption of whether versity of South Alabama, Mobile, AL 36688 (e-mail: jmccread@ both species beneÞt equally (symmetrical relation- jaguar1.usouthal.edu). 2 Department of Entomology, Soils & Plant Sciences, Clemson ship) or one participant receives greater beneÞt University, Clemson, SC 29634Ð0315. (asymmetrical relationship).

0046-225X/11/0289Ð0302$04.00/0 ᭧ 2011 Entomological Society of America 290 ENVIRONMENTAL ENTOMOLOGY Vol. 40, no. 2

Table 1. Simple two species interactions

Interactiona Species 1 Species 2 Competition Ñ Ñ Predation ϩ (predator) Ϫ (prey) Parasitism ϩ (parasite) Ϫ (host) Grazing ϩϪ/ϩ Fig. 1. Cost of symbosis in a simple three-species inter- Mutualism ϩϩaction. The symbiote (S) imparts a cost (s) to the host (H) Commensalism ϩ 0 but returns nothing directly. If the host can transmit the Neutralism 0 0 symbiote to a competitor (C), then there are two possible costs of competition, that from an uninfected competitor a Ϫ,0,ϩ are expressed in terms of the directional effects on growth (CU) and that from an infected (CI) competitor. rate, survival, reproductive ability, and fecundity; based on Odum and Barrett (2005).

tinuum of symbiotic associations (Hochberg et al. Downloaded from https://academic.oup.com/ee/article/40/2/289/409195 by guest on 24 September 2021 2000, Thompson and Cunningham 2002, Neuhauser We follow the original concept of symbiosis deÞned and Fargione 2004, McCreadie et al. 2005b). From an by de Bary (1879) and subsequently embraced by evolutionary perspective, natural selection should fa- Boucher (1985), Sapp (1994, 2004), Agnew et al. vor a shift from parasitism (ϩϪ) to commensalism (ϩ (2003), and Tamas and Andersson (2003): a relation- 0) and eventually to mutualism (ϩϩ) (Price 1997). ship in which two species live in close physical asso- Phylogenetic evidence suggests that certain fungal ciation, with a degree of dependence on the associa- pathogens have shifted from pathogenicity to a non- tion. To this deÞnition, we add a time element, with at lethal form of parasitism (Humber 2008). As a corol- least one species existing in the association for a sig- lary, prevalence of commensals (e.g., trichomycetes) niÞcant part of its life. Hence, relationships between in larval black ßies is typically greater, reaching 100% ßowering plants and pollinators, though mu- (Beard and Adler 2002), than prevalence of parasites tualistic, would not be symbiotic. Because a size dif- (e.g., microsporidia), which typically is below 5% ference typically exists between symbiotic partici- (McCreadie and Adler 1999). pants, we follow the usual convention of referring to Symbioses are set in communities and their inter- the smaller species as the symbiote and the larger actions and consequences can manifest across multi- species as the host. Some authors use the term “sym- ple taxa and different trophic levels (Klepzig et al. biosis” in a narrower sense, referring only to mutual- 2001, Omacini et al. 2001). The community in which ism (e.g., Hentschel et al. 2000, Gross et al. 2003). We symbioses operate also must be viewed in the context Þnd such a deÞnition too restrictive for three reasons. of the abitoic environment. An early example dem- First, the nature of the interactions between species onstrating the dynamic nature of symbiotic interac- living in close association is frequently unknown. Sec- tions in response to environmental conditions was ond, such a narrow deÞnition does not emphasize the documented for mycorrhizal associations with scotch ßuid nature among commensalism, mutualism, and heather (Calluna vulgaris [L.] Hull) (Wells et al. parasitism (e.g., McCreadie et al. 2005b). Third, hav- 1930). The relationship is mutualistic when soil nitro- ing symbiosis and “mutualism” as terms for the same gen is low, but turns pathogenic toward the heather as relationship is redundant. nitrogen increases. Understanding host-symbiote dy- A few comments about parasitism are in order. Par- namics thus requires a shift of perspective from a asitism occurs when one species beneÞts, usually the pairwise view of associations to a multispecies net- symbiote, and the other is harmed, typically the host. work of interactions (e.g., Stanton 2003) set within the Demonstrating a harmful effect by the parasite can be abiotic environment. difÞcult, although in some cases, the parasite causes A simple graphical model demonstrates how the patently deleterious effects to the host, including nature of symbiotic interactions can be viewed in a death. A parasite is often viewed as causing no great community network (Fig. 1). Symbiote S takes re- harm because eliminating the host would destroy the sources from host H but supplies nothing directly to parasiteÕs habitat. Begon et al. (2006), however, argue the host. The cost to the host is s, and if only this this view is incorrect and that selection should favor pairwise interaction is considered, the interaction rep- parasites that maximize their Þtness, which at times resents parasitism. When the host has a competitor might be achieved through decreased virulence to the (C), which by deÞnition is a ϪϪinteraction (Morin host but at other times by increased virulence. Many 1999), a cost is imparted to the host. If the host trans- pathogens of insects, for example, rely on killing their mits the symbiote to the competitor, two new costs host to increase transmissibility and Þtness. Hence, a arise from the perspective of the host: CU, the cost to pathogen can be considered an extreme parasite hav- the host when competing with an uninfected com- ing effects on a population similar to those of preda- petitor, and CI, the cost to the host when competing tors. with an infected competitor. Interesting dynamics ap- Ͻ Ͼ pear if CI CU.Ifs CI, the symbiote is still a parasite from the host perspective. If s ϭ C , the symbiote is a Dynamics of the Symbiotic Relationship I commensal. Although a cost to the host results from Both theory and empirical evidence suggest that the symbiote, the cost is offset by the reduced cost of parasitism and commensalism are points along a con- competition due to the symbioteÕs infection of the April 2011 MCCREADIE ET AL.: SYMBIOTE ECOLOGY OF BLACK FLIES 291 competitor; the symbiote gains, the net cost to the host microsporidia, and mermithid nematodes). The vast Ͻ is 0, and by deÞnition, commensalism exists. If s CI, majority of the roughly 190 known species of symbi- the relationship is mutualistic from the host perspec- otes in larval black ßies are considered parasitic (Table tive; the cost of the symbiote is outweighed by the 2). However, this perspective belies the dynamic na- reduced cost of competition and the host gains a net ture of symbioses under different environmental con- beneÞt. ditions and a dearth of information about bacterial The primary implication of this model is that the symbiotes, many of which are probably mutualistic. dynamic nature of symbiosis is expected to change Coelomycidium simulii. This is found nearly spatially (e.g., different competitors in different loca- worldwide in a remarkably broad range of simuliid tions) and temporally (e.g., population dynamics of a host taxa, suggesting that it is a complex of species. competitor in a single location). A challenge for ecol- Although it is one of the most widespread parasites of ogists is to link local ecological and evolutionary pro- black ßies, its prevalence is typically Ͻ4% of a larval

cesses with higher-order ecogeographic features host population, though rates 10 times higher have Downloaded from https://academic.oup.com/ee/article/40/2/289/409195 by guest on 24 September 2021 (Thompson 1999). Metapopulation studies show that been recorded (Crosskey 1990, McCreadie and Adler the outcome of a species interaction or the trajectory 1999). Patently infected larvae are packed with spher- of a particular population is not homogenous over a ical sporangia throughout their hemocoel. Infected large spatial scale; rather, outcomes vary among local larvae eventually rupture and zoospores are released populations (Hughes et al. 1997, Thompson 1999). (Tarrant 1984). Transmission from host to host occurs Most studies examining species interactions such as vertically (Tarrant 1984) and possibly horizontally, competition have inadequately small time scales (Cal- perhaps through an intermediate host (Lacey and laway and Walker 1997, Thompson 1999). To evaluate Undeen 1988). the importance of symbiotic interactions in the ecol- Microsporidia. Microsporidians are pathogenic, ob- ogy and evolution of organisms, the geographic struc- ligate, intracellular symbiotes that originally were con- ture of these interactions, coupled with appropriate sidered basal because they lack mitochon- time scales, must be considered. dria (Agnew et al. 2003). The trait now is considered a secondary loss resulting from life as obligate intra- cellular parasites, and recent evidence indicates that The Hosts microsporidia are fungi (Lee et al. 2008). Larval black ßies occupy aquatic habitats ranging Once ingested, microsporidia usually infect gut ep- from temporary trickles to large rivers and are often a ithelial cells (Agnew et al. 2003) and then the fat body, dominant part of the lotic macroinvertebrate commu- the principal larval tissue supporting microsporidian nity (Adler and McCreadie 1997). Larvae adhere to development in black ßies (Adler et al. 2004). solid substrates in ßowing water and obtain food as production in fat body cells leads to patent infections Þlter feeders, scrapers, collector-gatherers, and pred- visible as white or reddish, lobate cysts (Adler et al. ators (Currie and Craig 1988). The winged adults are 2004). Like other pathogens of black ßies, microspo- terrestrial and capable of considerable dispersal. Most ridia increase the duration of the larval stage (Mau- adult females require a blood meal to develop eggs, rand et al. 1975). Microsporidian prevalence in larval although some species can develop the Þrst clutch black ßies is usually Ͻ3% (Table 3). are re- without a blood meal and other species never take leased as the larva decomposes, but whether interme- blood (Adler et al. 2004). Reviews of black ßy biology diate hosts are needed for horizontal transmission of have been presented by Laird (1981), Kim and Merritt microsporidia of black ßies is unknown. Vertical trans- (1988), Crosskey (1990), Adler et al. (2004), mission from adult females to larvae, via infected eggs, Malmqvist et al. (2004), and Adler and McCreadie occurs in at least one species (Tarrant 1984). (2009). Thirteen described species attack larvae in North America (Adler et al. 2004). Detailed information for microsporidians of black ßies is given by Jamnback The Symbiotes (1970), Va´vra and Undeen (1981), Weiser and Un- Black ßies, both as adults and immatures, are hosts deen (1981), Crosskey (1990), and Adler et al. (2000, for an entourage of symbiotes living in and on their 2004). bodies. Little is known about most symbiotes living on Trichomycetes. Members of the order the host; we, therefore, focus on those organisms living are zygosporic trichomycete fungi that live in the guts in the larval host (Table 2). Many undetected symbi- of aquatic . Aquatic insects are the most otes, such as , undoubtedly remain to be dis- common hosts of trichomycetes and Diptera is the covered in black ßies. In addition, the vast majority of most commonly colonized order. Trichomycetes gen- microbial organisms currently cannot be cultured erally are considered commensals, but also can be (Moran 2006). Most symbiotes of black ßies are de- pathogens or mutualists, depending on environmental tected visually only when the hosts are patently in- circumstances (McCreadie et al. 2005b, Lichtwardt fected. We, therefore, restrict our review to symbiotes 2008). There are Ϸ9 genera and 15 species of tricho- or their manifestations that can be detected readily mycetes known from black ßies in North America under the microscope, with host dissection (i.e., (Nelder et al. 2006, White et al. 2006b). The preva- trichomycete fungi and ) or without lence of many species of trichomycetes varies over (i.e., the fungus Coelomycidium simulii Debaisieux, space and time from 0 to 100% (Taylor et al. 1996; 292 ENVIRONMENTAL ENTOMOLOGY Vol. 40, no. 2

Table 2. Symbiotes associated with larval black flies (Diptera: Simuliidae)

Number of Symbiote Nature of relationship References nominal species Bacteria Bacillalesa 2 Parasitic Weiser and Undeen 1981, Reeves and Nayduch 2002 Rickettsiallesb 1 Commensalistic? Crainey et al. 2010 Chlorophyta Helicosporidia 1 Parasitic Boucias et al. 2001, Tartar et al. 2002 Fungi Blastocladiomycetes 1 Parasitic Tarrant 1984 Hyphomycetesc 1 Parasitic? Adler et al. 2004 Microsporidia 34 Parasitic Crosskey 1990, Adler et al. 2004 “Trichomycetes”d 36 Commensalistic Beard et al. 2003, McCreadie et al. 2005b, Nelder et al. 2006 Mutualistic, Parasitic Zygomycetese 7 Parasitic Nadeau et al. 1994, 1995; Adler et al. 2004 Downloaded from https://academic.oup.com/ee/article/40/2/289/409195 by guest on 24 September 2021 Nematoda Mermithidae 88 Parasitic Molloy 1981, Gordon 1984, Crosskey and Poinar 2002 Nematomorpha Gordioidea 1 Parasitic Adler et al. 2004 Protozoa Ciliophora 2 Parasitic Lynn et al. 1981, Batson 1983 Haplosporidia 1 Parasitic Beaudoin and Wills 1968 Mesomycetozoea 5 Parasitic White et al. 2006; Lichtwardt 2008 Trypanosomatidaf 1 Parasitic? Crosskey 1990 Stramenopila Oomycetes 2 Parasitic Nolan and Lewis 1974, Adler et al. 2004 Platyhelminthes Trematodag 2 Parasitic Busˇta and Nasˇincova´ 1986, Jacobs et al. 1993 Viruses Cypovirus 1 Parasitic Green et al. 2007 Iridescent virus 1 Parasitic Crosskey 1990 Virus-like particles 2 Parasitic Charpentier et al. 1986, Federici and Lacey 1987

a Many bacteria are found in larval guts (Snoddy and Chipley 1971), and the distinction between symbiotes and food or transients can be blurred, although some bacteria evidently can become pathogenic at times via septicemia (Weiser and Undeen 1981); those found in the gut are not included here as symbiotes unless classic symbiosis has been documented. b One report of Wolbachia in larvae has been conÞrmed molecularly (Crainey et al. 2010). c Hyphomycetes previously included asexual fungi that form conidia on separate, not organized, hyphae. Such a classiÞcation is no longer necessary, because molecular methods allow both sexual and asexual fungi to be grouped together among their relatives (Hibbett et al. 2007). d Trichomycetes traditionally have been recognized as fungi, but molecular evidence suggests the group is polyphyletic and has only ecological, rather than phylogenetic, relevance (Hibbett et al. 2007). All species that colonize black ßies are members of the order Harpellales and, therefore, are true fungi. e Zygosporic fungi previously classiÞed as are not well resolved as a monophyletic phylum (Hibbett et al. 2007). Entomoph- thorales, traditionally classiÞed as a group of zygosporic fungi, typically infect adults, but some evidence suggests that at least some species also infect larvae (Crosskey 1990, Adler et al. 2004). The no. of species is based largely on records from adult black ßies. f The single record from black ßies, known only from the original description, is based on material from the body cavity of a larva (Crosskey 1990). g Infections have been established only in the laboratory, but suggest that wild infections are likely (Busˇta and Nasˇincova´ 1986, Jacobs et al. 1993).

Beard and Adler 2002; Hapsari et al. 2009a, 2009b; spore extrusion from the trichospore, the thallus at- Nelder et al. 2010). taches to the midgut peritrophic matrix (Harpel- Larval black ßies are colonized by ingesting asexual laceae) or the hindgut cuticle () trichospores (Lichtwardt 2008). Following sporangio- and can produce new trichospores within 24 h (Vo-

Table 3. Prevalence of symbiotes of larval black flies from nine areas of North America (P.H. Adler and J.W. McCreadie, unpublished data)

Number % of infected larval hosts Area Latitude (Њ) Longitude (Њ) Collections Larvae C. simulii Mermithidae Microsporidia Alaska 60.81Ð60.33 161.82Ð159.41 18 798 1.253 1.253 3.383 Colorado 40.68Ð36.00 108.55Ð104.17 52 1,129 8.503 2.391 2.923 Florida 30.97Ð27.21 86.76Ð80.97 30 2,490 0.361 0.562 1.165 New Jersey 41.34Ð39.24 75.15Ð74.22 110 2,660 0.113 0.376 1.090 Northwest Territories 69.77Ð67.54 126.82Ð122.28 46 5,303 1.282 6.939 1.509 Ohio 40.76Ð38.43 84.77Ð80.63 41 1,260 0.159 1.825 0.238 Oregon 46.57Ð42.29 123.98Ð118.59 56 1,979 0.455 0.707 0.505 South Carolina 35.21Ð33.25 83.30Ð80.68 209 26,639 0.725 6.318 1.385 Wyoming 44.96Ð44.27 110.93Ð109.60 49 5,155 0.873 0.563 1.145 Totals 611 47,413 Mean (CI) 0.920 4.924 1.344 (0.835Ð1.010) (4.308Ð4.683) (1.242Ð1.451) April 2011 MCCREADIE ET AL.: SYMBIOTE ECOLOGY OF BLACK FLIES 293 jvodic and McCreadie 2007). Trichospores exit the host in fecal pellets. When a host molts, thalli are shed with the gut lining; thus, the mycota is regularly lost and renewed during host development (Lichtwardt 1986, 2008). Trichospores remain viable under moist conditions for several months (Williams 1983, J. W. McCreadie, unpublished data). Sexual zygospores are produced in some species. Trichomycetes can be pathogenic to female black ßies, replacing host eggs with fungal cysts. Thus, the symbiote gains dispersal service and the host loses its egg clutch (Moss and Descals 1986, Rizzo and Pang Fig. 2. Probability of not Þnding at least one host black

2005, Lichtwardt 2008). The occurrence of these ovar- Downloaded from https://academic.oup.com/ee/article/40/2/289/409195 by guest on 24 September 2021 ßy infected with C. simulii, as a function of number of hosts ian fungal cysts is not universal (Yeboah et al. 1984, examined; prevalence taken from Table 3. Labeyrie et al. 1996, White et al. 2006a), and we do not understand what prompts the symbiote to become parasitic in the adult. The cells settle in the stream substrate, encyst, and Mermithid Nematodes. The nematode family produce cystospores that presumably are ingested by Mermithidae includes the most commonly encoun- the larval host (Lichtwardt 1976, 2008). No evidence tered parasites of black ßies (Poinar 1981), but their of a negative relationship has been documented, sug- taxonomy is poorly resolved (Crosskey 1990, Adler et gesting that these symbiotes are typically commensals, al. 2004). The free-living stages of simuliid mermithids although the possibility of mutualism cannot be ex- (eggs, postparasitic juveniles, and adults) live in the cluded. streambed. After hatching, juveniles enter the larval host by piercing the cuticle with a protrusible stylet Symbiote Distributions and make their way to the host hemocoel (Molloy 1981). Multiple infections in a single host are common When quantifying spatial distributions of symbiotes, (Ezenwa and Carter 1975, Mondet et al. 1976). De- a useful parameter is prevalence, which is the percent velopment in the host varies with temperature, from of host individuals colonized by a symbiote compared a week in warm areas to several months during the with the number of host individuals examined. Esti- winter in northern areas. At the end of the parasitic mates must be made cautiously because bias can be stage, the worms exit the larva, causing host death, or introduced if prevalence is calculated after a signiÞ- they are retained in the larva and emerge from the cant proportion of uninfected larval hosts have pu- pupa or adult (Poinar 1981). The postparasitic juve- pated. With this caveat, prevalence can be used at niles move to the streambed where they molt to adults, scales from the stream reach (i.e., a stream section on mate, and lay eggs; the free-living phase lasts up to two the scale of meters) to biomes and continents. For years (Poinar 1981). Prevalence of 1Ð10% is typical for example, the prevalence of the fungal symbiote C. most locations (Crosskey 1990) (Table 3). simulii was 1.11% (n ϭ 90 host larvae) in Gold Run Nematode infections that persist into the adult usu- Creek, AK (24 June 2004), 1.25% in the state of Alaska ally cause host sterility, although females of at least (n ϭ 4,238) and 0.92% in North America (n ϭ 47,413) some host species can blood-feed, mate, and search for (authors, unpublished data). oviposition sites (Anderson and Shemanchuk 1987). Sample size is critical when examining distributions Infected females and males feminized by the infection of black ßy symbiotes, particularly microsporidians, exhibit oviposition behavior after an upstream ßight nematodes, and C. simulii, for which prevalence is low (Crosskey 1990). During oviposition, the juvenile and many hosts must be examined to discover them. worms exit the host through the abdominal wall and We are conÞdent that, on average, Ϸ0.92% of larvae in enter the stream. Upstream ßights of adult hosts coun- North America show patent infections of C. simulii ter downstream drift of infected larvae. Accounts of (Table 3), based on the large sample size (n ϭ 47,413) mermithid parasites of black ßies are available (Phelps and narrow conÞdence interval of our estimate and DeFoliart 1964, Finney 1981, Molloy 1981, Poinar (0.835Ð1.010%). The importance of sample size is dem- 1981, Gordon 1984, Crosskey 1990). onstrated by using this prevalence as typical of a site Mesomycetozoea. The genera Amoebidium and Par- and applying the binomial distribution. Based on our amoebidium, once constituting the order Amoebidi- estimate of prevalence as the probability of a success- ales in the fungal class Trichomycetes, are now con- ful trial (infected host), and the number of hosts sidered protists in the Mesomycetozoea (Lichtwardt examined as the number of trials, a total of 325 hosts, 2008). Amoebidium attaches to the external cuticle, on average, must be examined to be 95% sure of not usually near the anus, and is infrequent on black ßies. missing the patent infection (Fig. 2). Researchers At least two species of Paramoebidium are found in should consider correlation analyses between the North American black ßies (Adler et al. 2004). They number of sites or hosts examined and estimates of attach to the hindgut cuticle as unbranched thalli. prevalence to identify cases where sample size can Reproduction of Paramoebidium is achieved by the bias results. McCreadie and Adler (1999), for example, release of amoeboid cells, often in the host exuviae. found that sample size had a signiÞcant effect on the 294 ENVIRONMENTAL ENTOMOLOGY Vol. 40, no. 2

Table 4. Correlation coefficients (with P values) for prevalence ing the distributions would be niche partitioning, of symbiotes of larval black flies and geographic coordinates from whereby each symbiote species occupies the same nine areas of North America; data from Table 3 location in the hindgut in the absence of other com- Mean degrees petitors but extends into different, presumably less Symbiote Latitude Longitude favorable, areas of the gut as competitors colonize the host. Although interspeciÞc interactions among C. simulii Ϫ0.029 0.140 trichomycetes appear to inßuence species distribu- (0.941) (0.720) Mermithidae 0.357 0.022 tions in a host, colonization of black ßies by tricho- (0.346) (0.955) mycetes is not signiÞcantly inßuenced by the presence Microsporidia 0.362 0.594 of certain nontrichomycete symbiotes (Kim and Adler (0.339) (0.092) 2005). Habitat Selection among Hosts. Patterns of species

distributions and the processes driving these patterns Downloaded from https://academic.oup.com/ee/article/40/2/289/409195 by guest on 24 September 2021 detection of mermithid prevalence in larvae, but ac- are scale dependent (Schneider 1994). The impor- counted for this effect by incorporating the variable tance of scale-related processes for the distributions of into their prediction equations. The lack of signiÞcant larval black ßies, and by extension the distributions r values (P Ͼ 0.05) from a correlation analysis (Table of their symbiotes, has been viewed at two scales 4) provides further conÞdence in our estimates of of study, microdistribution and macrodistribution prevalence. Because trichomycetes can be relatively (Colbo and Wotton 1981, Adler and McCreadie 1997). frequent in black ßies (Beard 2002, Beard et al. 2003, Microdistribution of larvae in a stream is usually on a Nelder et al. 2009), large samples are not as critical for scale of 100 m or less, typically referred to as the conÞdently estimating their prevalence. stream reach (Minshall 1988). Macrodistribution in- Habitat Selection in Hosts. The distribution of sym- volves the distribution of larvae among streams and biotes in larval hosts has been examined most exten- encompasses a scale of kilometers; thus, it is the dis- sively for trichomycetes, with the Þrst level of habitat tribution of larvae in and among entire streams, wa- selection occurring shortly after trichospore inges- tersheds, ecoregions, biomes, and continents. tion. Sporangiospore extrusion (i.e., trichospore ger- Host-Symbiote Microdistributions. Although verti- mination) and thalli attachment are believed to be cal transmission is known or suspected for a variety of triggered by host-gut conditions, particularly changes black ßy symbiotes, horizontal transmission appears to in pH and potassium concentrations (Misra 1998, be the dominant form of dissemination to hosts. Hor- Horn 2001, Lichtwardt 2008). izontal transmission requires the symbiote to enter the Within the hindgut, species of the stream and Þnd a new host. Each host, therefore, can colonize speciÞc habitats. For example, Smittium cu- be viewed as a potential habitat patch to be colonized, lisetae Lichtwardt occupies the rectal area of the hind- with each patch situated among areas of habitat (i.e., gut in mosquitoes (Horn 2001), whereas it is most the stream) unsuitable for colonization. When we frequent in the posterior colon in black ßies (Mc- refer to the stream as unsuitable for colonization, we Creadie and Beard 2003), possibly because of mor- mean unsuitable only for the infective stage of the phological differences in the host hindguts (Vojvodic symbiote, because some symbiotes, such as nema- and McCreadie 2009). This example raises the possi- todes, colonize the streambed after exodus from the bility that the symbiote is responding to differences in host. the habitat landscape in a manner akin to free-living Little is known about the dynamics of host (patch) organisms. We suggest that investigations of symbiote colonization by symbiotes at the scale of the stream distributions in hosts would beneÞt from application reach. However, trichomycete Þtness varies among of landscape ecology. An intriguing example of habitat host species (Nelder et al. 2005). Selection, therefore, selection in black ßies is exhibited by Simuliomyces should favor increased host speciÞcity, and coloniza- microsporus Lichtwardt, which typically attaches to tion should entail a suite of host-recognition cues. thalli of Paramoebidium, which in turn attach to the Evidence suggests that abiotic stream conditions in- anterior host hindgut (Beard and Adler 2002, Siri and ßuence both trichomycete Þtness (Vojvodic and Mc- Lo´pez Lastra 2010). Creadie 2007) and colonization dynamics (Beard et al. When colonization space is limited, sessile organ- 2003). Therefore, the gut trichomycete mycota should isms inßuence both intraspeciÞc and interspeciÞc dis- be the result of the interplay between the host and its tributions (Connell 1961, McCook et al. 2001, Fujii and habitat. Hiradate 2007). Because multispecies occurrences of Although our understanding of colonization of the trichomycetes in a single host individual are common host is poor, simple simulations provide a starting point (Lichtwardt and Williams 1988, Kim and Adler 2005) for hypothesis testing. Given the unidirectional ßow of and the gut presents a Þnite amount of habitat, com- streams, the probability that an uninfected host con- petitive interactions among trichomycetes are ex- tacts a symbiote propagule from an infected host is pected. Three Smittium species in larvae partially a positional relationship between an infected demonstrate growth patterns consistent with inter- host I and an uninfected host S. That is, an individual speciÞc competition; the location of each species of class S downstream of an individual of class I has the changes in the presence of another species (Vojvodic potential to become infected by I, but an individual I and McCreadie 2009). A possible mechanism explain- downstream of an individual S does not have the April 2011 MCCREADIE ET AL.: SYMBIOTE ECOLOGY OF BLACK FLIES 295

controlled laboratory experiments. Black ßies are dif- Þcult to rear through multiple generations in the lab- oratory, and only a single colony of a single species exists (Gray and Noblet 1999). Additionally, many species of black ßy symbiotes currently cannot be cultured. If hosts are viewed as habitat patches, host speciÞcity becomes a study of habitat selection by symbiotes among different types of habitats. Mermith- ids, microsporidians, and C. simulii in black ßies are speciÞc at the host-family, or lower, taxonomic level (Crosskey 1990). In contrast, some of the trichomy- Fig. 3. Number of times an individual of class S (unin- cetes in black ßies also colonize hosts in related fam- fected) is directly downstream of an individual of class I ilies such as and Culicidae (Lichtwardt Downloaded from https://academic.oup.com/ee/article/40/2/289/409195 by guest on 24 September 2021 (infected) as a function of the percent of habitat patch 1986, 2008). In each group of symbiotes, some species occupied and density of I and S individuals. are catholic in the host taxa they colonize (habitat generalists), whereas other species are restricted to a potential to infect the upstream host S. We can ex- narrow group of host taxa (habitat specialists), imper- amine the interaction between host position and host fect taxonomy of the symbiotes notwithstanding. density by asking how the number of times a host I Many symbiote-host records have limited use be- directly upstream of a host S changes with population cause the hosts or symbiotes are not identiÞed to size N (S ϩ I). Consider a small area of host habitat species. Of further concern is that reports of host (e.g., a rock) of size a ϫ a. For simplicity, we assume speciÞcity could be more apparent than real. For ex- this area is not affected by other local population ample, a host species might be the most commonly patches, that each larva occupies the same amount of infected by a particular symbiote simply because it is space (1/a2), and that each larva in class I or S is the most abundant (Mokry and Finney 1977). When distributed randomly in this area. Regardless of the reports of host infections take relative density of host density of I to S, the probability of S being directly species into account, more conÞdence can be placed below I (i.e., in a position to intercept symbiote prop- in claims of host speciÞcity. For example, the nema- agules) increases exponentially (Fig. 3). Hence, in tode Mesomermis camdenensis Molloy in a stream in contrast to typical equations used to model disease New York state infected 10Ð25% of larvae of the Sim- transmission (e.g., Holt 1994, Turner et al. 2002, Begon ulium tuberosum (Lundstro¨m) complex (Molloy et al. 2006), the contact required for transmission 1979), but Ͻ1% of the S. venustum complex. Both host among I and S classes does not vary in a simple linear complexes were collected at the same place and time manner. Combining Þeld and laboratory data with and both were reported as abundant. However, each more realistic assumptions would provide insight into of these host complexes in this area contains at least symbiote-habitat patch dynamics of stream insects, Þve species (Adler et al. 2004). Though not reporting especially if grounded in metapopulation theory. speciÞc values of relative abundance, Colbo and Por- The life cycle and transmission dynamics of tricho- ter (1980) provided evidence that the nematode Me- mycete trichospores and preparasitic mermithids dif- somermis flumenalis Welch prefers Prosimulium mix- fer from those of C. simulii and microsporidians to the tum Syme and Davies over Stegopterna mutata extent that simple simulations would not apply di- (Malloch). rectly. For example, the hindgut trichomycete mycota Only a few studies have examined host speciÞcity is lost with each host molt (Lichtwardt 1986, 2008); under laboratory conditions. Four species of hindgut hence, individual hosts have the potential to oscillate trichomycetes (genus Smittium) in the larval host Sim- between colonized and uncolonized states. Nema- ulium vittatum Zetterstedt differ signiÞcantly in hy- todes, once leaving a patch, pass through free-living phal abundance, trichospore production, and preva- stages lasting weeks to months in the streambed lence, indicating various degrees of host speciÞcity (Crosskey 1990) and negating the positional effects in (Nelder et al. 2005). Laboratory studies of mermithids our simulation. also have shown various degrees of host preference Host attributes can inßuence the distribution of (Bailey and Gordon 1977, Colbo and Porter 1980). symbiotes. For example, age of the host can inßuence Host-Symbiote Macrodistributions. Characteristics spatial distributions. In some species of nematodes, of host populations (e.g., species abundance and com- individuals that infect early host instars complete de- position) and the streams in which the hosts are found velopment in the larva, whereas those infecting older change over spatial axes (McCreadie and Colbo 1991, larvae are carried through to the adult stage (Mondet 1992; Adler and McCreadie 1997; McCreadie and et al. 1976). Accordingly, nematodes infecting a new Adler 1998). The spatial ecology of symbiotes, there- habitat patch (young larva) would be most likely to fore, is coupled to the scale-dependent processes that remain in a single section of stream, whereas those inßuence both the hosts and the streams in which infecting an older habitat patch (mature larva) could these hosts are found. Two broad mechanisms inßu- be exported from the stream reach via the adult host. encing symbiote macrodistribution can be envisioned, Most of what can be gleaned about host speciÞcity those acting directly on the symbiotes and those op- in black ßies comes from Þeld collections rather than erating indirectly on the symbiotes through effects on 296 ENVIRONMENTAL ENTOMOLOGY Vol. 40, no. 2 the hosts. Most Þeld studies have focused on the pat- terns of symbiote distribution, with little consider- ation of the mechanisms responsible for the patterns. The recognition of spatial patterns of symbiote di- versity depends on taxonomic resolution of the host and symbiote. However, symbiote identiÞcations to species are not always possible. For example, only free-living adult nematodes, rather than the parasitic juveniles, currently can be identiÞed to species (Poi- nar 1981), and heavily parasitized larval hosts can be difÞcult to identify. Thus, no large-scale studies based on species-level identiÞcations of symbiotes and hosts Fig. 4. Test of host speciÞcity by mermithid nematodes have been conducted to decouple factors inßuencing in the larval host Simulium tuberosum in streams of South Downloaded from https://academic.oup.com/ee/article/40/2/289/409195 by guest on 24 September 2021 the distribution of each host species from those inßu- Carolina; data from McCreadie and Adler (1999). encing the distribution of each symbiote. However, a handful of studies have laid the foundation (e.g., Beard et al. 2003, Nelder et al. 2009), and molecular tech- and latter studies might have been examining dispersal niques promise to facilitate identiÞcation. versus population growth, respectively. Studies of macrodistributions of simuliid symbiotes Because the postparasitic stage of mermithids can typically are based on samples from fewer than 30 sites last several months or more (Gordon 1984), the oc- and usually from restricted geographic areas where currence of these symbiotes is often associated with biogeographic factors would not play a signiÞcant role, stream conditions. The occurrence of mermithids in S. as with mermithids (Ezenwa 1973, 1974a, b; Bruder tuberosum and S. ubiquitum Adler, Currie & Wood (as and Crans 1979; Colbo and Porter 1980; Colbo 1990), S. tuberosum F.) over a large area of South Carolina is correlated with stream oxygen levels (McCreadie and microsporidians (Ezenwa 1973, 1974a, 1974b; Va´vra and Undeen 1981; St-Onge and Charpentier 2008), Adler 1999), and the presence of mermithids in larval and trichomycetes (Nelder et al. 2009). A few studies hosts in Quebec is associated with stream depth (St- Onge and Charpentier 2008). have sampled a large number of sites or a broad geo- Symbiote distributions over many sites can provide graphic region. For example, Ebsary and Bennett insight into host speciÞcity by regressing symbiote (1975) sampled 198 streams for nematodes and mi- prevalence against the relative abundance of the tar- crosporidia on the island of Newfoundland; although get host. A simple linear function would indicate pro- the island is within a single ecoregion (Boreal Shield), portional allocation among individuals of a host (i.e., it is several hundred km across. Anderson and DeFoliart no host speciÞcity). Deviation from a simple linear (1962) sampled 215 streams in Wisconsin but provided function would indicate a preference or aversion for little information on site locations. In South Carolina, 115 a speciÞc host. Reworking the dataset of McCreadie sites over three distinct ecoregions were sampled for the and Adler (1999), we examined patent nematode in- trichomycete melusinae Le´ger and Duboscq fections in the host S. tuberosum for 115 stream reaches (Beard et al. 2003) and mermithids, microsporidans, and in South Carolina. A simple linear function indicates C. simulii (McCreadie and Adler 1999); however, only nematode infection no greater than expected from the former study considered the association between host frequency in the population (Fig. 4). An R2 of symbiote prevalence and ecoregion. 38.2% indicates that factors in addition to host fre- Few studies have examined the association between quency are responsible for variation in prevalence stream conditions and symbiotes. In South Carolina, among sites, likely including stream conditions. prevalence of the trichomycete H. melusinae in the The inßuence of ecoregion on the composition of host Simulium tuberosum (Lundstro¨m) is highest in the host assemblage (McCreadie and Adler 2008) sug- acidic streams with low conductivity, but is highest in gests that symbiote composition and diversity also larvae of S. verecundum Stone and Jamnback in slow- should change over ecoregions. Of particular interest moving streams (Beard et al. 2003). The prevalence of are the patterns and processes that inßuence symbiote H. melusinae in black ßies in Thailand is highest in assemblages within and among zoogeographic re- cooler water (Hapsari et al. 2009a). Hence, coloniza- gions. The only data on symbiote occurrence over a tion of host patches among streams is the result of the continental scale of which we are aware are our un- interplay between patch characteristics (host species) published data (Table 3). Correlation analysis be- and stream conditions. In contrast, the presence of the tween mean prevalence at each location in Table 3 and trichomycete Harpella among 19 stream sites in coastal mean latitude and longitude indicates lack of a signiÞcant Alabama and Mississippi is associated with dissolved coefÞcient, suggesting that symbiote prevalence does oxygen and water temperature (Nelder et al. 2009). not change along simple northÐsouth or eastÐwest gra- Although differences among studies could reßect bio- dients, notwithstanding the higher taxonomic levels used geographic factors, the factors determining the occur- for nematodes and microsporidians (Table 4). rence of symbiotes at a site (Nelder et al. 2009) might Based on large populations, small body size, and short differ from those inßuencing prevalence (Beard et al. generations, which lead to high dispersal rates, free- 2003, Hapsari et al. 2009a). In other words, the former living microbes are assumed to have few dispersal bar- April 2011 MCCREADIE ET AL.: SYMBIOTE ECOLOGY OF BLACK FLIES 297 riers; therefore, cosmopolitan distributions might be ex- pected (Finlay and Clarke 1999, Coleman 2002, Fenchel and Finlay 2004). However, emerging evidence chal- lenges the idea that microbes are all cosmopolitan (Whit- taker 2003), even though the extent to which microbial symbiotes of larval black ßies are cosmopolitan is cur- rently difÞcult to assess. Many symbiote species (e.g., C. simulii and various microsporidians and trichomycetes) are recorded from black ßies in multiple zoogeographic regions (Crosskey 1990), but at least some of these so- called species of symbiotes are probably species com- plexes (Adler et al. 2004, White et al. 2006b). At the other extreme are symbiotes that appear to have restricted Fig. 5. Rank-abundance curves for microsporidian sym- Downloaded from https://academic.oup.com/ee/article/40/2/289/409195 by guest on 24 September 2021 distributions; however, these distributions also must be biotes of larval black ßies collected from the Piedmont and treated cautiously because they might reßect a lack of Sandhills ecoregions of South Carolina, 1992Ð1993. discovery or reporting. richness relation (Cornell and Lawton 1992, Cornell 1993), has not been investigated for black ßy symbi- Symbiote Diversity otes, although the relationship has been examined for Patterns of diversity among free-living organisms the hosts (McCreadie et al. 2005a). are a central focus of ecological studies. The study of The most familiar and generally accepted patterns diversity, however, explicitly requires a consideration of diversity include species-area curves, rank-abun- of scale. Spatial patterns of microbial diversity have dance graphs, latitudinal gradients, and local-regional not received the same attention as those of plants and diversity relationships (Cornell 1993, Rosenzweig (Green and Bohannan 2006). Patterns and 1995, Hawkins and Diniz-Filho 2004). We focus on processes of symbiote diversity for black ßies rarely two of these patterns of symbiote diversity, rank-abun- have been considered. dance (Whittaker) plots and species-area curves. For At the local scale, diversity can be considered the microsporidians, the problem of low prevalence number of species in a deÞned area, and is referred to means that estimates of symbiote diversity must come as ␣-diversity; for simplicity, we can deÞne the area as from a large number of hosts, which in practical terms the stream reach. One could argue that if we consider means using data from many sites. Hence, our rank- the larval host as a patch, ␣-diversity could be the abundance curves are highly modiÞed plots in which number of symbiotes in a single individual. However, we use prevalence among hosts of different species for microsporidians (P.H. Adler and J.W. McCreadie, from a large number of collections as proxies of di- unpublished data) and probably mermithids (Cross- versity. key 1990), multispecies infections are infrequent and Given these cautionary notes, an example of a rank- ␣-diversity in these groups would default to 1, with abundance plot is provided for two adjacent ecore- little explanatory or predictive value. We, therefore, gions of South Carolina (Fig. 5). The Piedmont ecore- use the same levels of scale as for the host. Although gion has a gentle rolling topography, clay-textured the number of species of trichomycetes at a particular soils, and streams with circumneutral pH; the Sandhills location varies seasonally, ␣-diversity at a single loca- ecoregion has a rolling to rugged hilly landscape, sand- tion ranges from 5 to 6 species over longer periods of textured soils, and acidic streams (McCreadie and time (Beard 2003, Nelder et al. 2010). A similar num- Adler 1998). The datasets from these two ecoregions ber appears to be the case for microsporidians (Ledin are comparable in the number of sites (Piedmont ϭ 30, 1994). Many studies simply provide host-symbiote re- Sandhill ϭ 32) and host individuals (Piedmont ϭ cords from which estimates of ␣-diversity cannot be 4,235, Sandhill ϭ 4,295). The Shannon-Wiener diver- extracted (e.g., Ebsary and Bennett 1975, Va´vra and sity index is signiÞcantly higher (t ϭ 2.29, df ϭ 157, P Ͻ Undeen 1981). 0.05) for the Piedmont (H ϭ 0.475) than for the Habitat patches vary and, thus, species composition Sandhills (H ϭ 0.361), even though richness (n ϭ 5) can be expected to vary among these patches. This is the same. Evenness is higher for the Piedmont turnover of species composition among habitats (e.g., ecoregion, which is reßected in the gentler slope of stream reaches) is known as ␤-diversity. We are not the curve (Fig. 5). Although theses curves are mod- aware of any studies that consider or calculate ␤-di- iÞed versions of typical rank-abundance plots, they versity of black ßy symbiotes. Regional diversity, such conform to a broken-stick model, which is perhaps not as for an ecoregion, is known as ␥-diversity and can be surprising given that infection of a single host by two considered either a linear (␥ ϭ ␣ ϩ ␤) or a multipli- species of microsporidians is infrequent (J.W. Mc- cative (␥ ϭ ␣␤) relationship between ␣-diversity and Creadie and P.H. Adler, unpublished data). As far as ␤-diversity (Lande 1996). Regional diversity has been we are aware, Fig. 5 is the Þrst plot of its kind for any documented for trichomycetes (e.g., Nelder et al. black ßy symbiote and demonstrates how even basic 2005) and microsporidians (Va´vra and Undeen 1981). patterns of diversity have been ignored. However, the often-observed relation between ␥-di- Species-area curves represent a fundamental pat- versity and ␣-diversity, the so-called local-regional tern of species diversity (Cornell 1993, Rosenzweig 298 ENVIRONMENTAL ENTOMOLOGY Vol. 40, no. 2

Table 5. Species-area regressions of the no. of microsporidian ers is to determine the best measure of area when species, log10 (S), and three proxies of habitat area, based on data examining species-area relationships for microbial in Table 3 symbiotes, or even if such relationships have any 2 meaning. Predictor (Log10) Intercept Slope F (1,7) PR(%) Surface-area polygon 0.299 0.060 0.43 0.534 Ñ Number of sites Ϫ0.253 0.450 6.44 0.039 40.5% Future Directions Number of hosts Ϫ0.240 0.221 2.48 0.160 Ñ Ecological studies of symbiosis in ßowing waters have lagged behind those in terrestrial systems. Little 1995, Hawkins and Diniz-Filho 2004). The relation- is known, for example, of the ecological interactions ship between species and area is described mathemat- among co-occurring symbiotes, particularly for black ically by the power function S ϭ cAz, where S is the ßy symbiotes where much of the literature contains number of species, A is the area under consideration, little more than host-symbiote records, often based on Downloaded from https://academic.oup.com/ee/article/40/2/289/409195 by guest on 24 September 2021 and c and z are Þtted constants. The equation gener- misidentiÞcations. Advances in symbiote ecology of ally is expressed in logarithm form (base 10) as larval black ßies, nonetheless, have been made over log(S) ϭ log(c) ϩ zlog(A), although some debate the past decade. We now know, for example, that the suggests a semilog expression (i.e., S ϭ log(c) ϩ relationship between trichomycetes and their black zlog(A)) should be used (Krebs 1999). The difÞculty ßy hosts is dynamic. Current researchers also appre- of applying this relationship to symbiotes is in deter- ciate the importance of scale in understanding the mining the measurement of area to be used. If we processes and patterns of symbiote diversity and that consider each host a patch, the number of hosts ex- stream conditions can be useful predictors of symbiote amined could be considered a measure of area. Host occurrence. speciÞcity, however, could complicate simple species- We believe four areas of research are needed to area relationships. Surface land-area polygons would advance understanding of symbioses in ßowing water. impart some bias because the land area to stream area First is a rigorous taxonomic treatment of the symbi- would not be constant over large areas. We suggest otes. The molecular techniques being used for tricho- that the number of sites examined is the least-biased mycetes will be invaluable for deÞning species limits method because collecting at a site typically entails in other symbiote groups. Second is the incorporation sampling from a relatively small section of a stream. of symbiotes into food webs. SigniÞcant progress al- Accordingly, as the number of sites sampled increases, ready has been made to integrate parasites into these the area of stream sampled might be expected to webs (Lafferty et al. 2008). Third is a shift of perspec- increase in a monotonic fashion. tive from a Þxed pairwise view of symbiotic associa- The log-log regression between three proxies of tions to a multispecies network of dynamic interac- area and the number of microsporidian symbiotes in tions. A simple example of a host-symbiote network each of nine geographic regions is presented in Table details the associations between black ßy hosts and 5. We found a signiÞcant regression only for the num- their trichomycete symbiotes for streams (Fig. 6). A ber of sites examined, which could indicate a species- major challenge will be to determine how host-sym- area relationship, a sampling effort relationship, or a biote networks integrate into other stream networks. combination of both. A challenge for future research- Fourth is the elucidation of how multiple symbiote

Fig. 6. Host-symbiote species web based on collections in South Carolina. Each pale, upper sphere represents one trichomycete species and each dark, lower sphere a larval host species. Links represent conÞrmed associations of each trichomycete species with each larval host species. April 2011 MCCREADIE ET AL.: SYMBIOTE ECOLOGY OF BLACK FLIES 299 species interact with one another to determine the Simuliidae) across a heterogeneous environment. Myco- microcommunity within a host individual. Field-col- logia 95: 577Ð583. lected material reßects compatible symbiote species Beaudoin, R. L., and W. Wills. 1968. Haplosporidium simu- but not failed colonization trials that result from in- lii sp. n. (Haplosporida: Haplosporidiidae), parasitic in compatible species interactions. Laboratory experi- larvae of Simulium venustum Say. J. Invertebr. Pathol. 10: ments could provide a starting point for understanding 374Ð378. the dynamics of multiple symbiote interactions within Begon, M., J. L. Harper, and C. R. Townsend. 2006. Ecology: individuals, populations and communities, 4th ed. Black- a host. well, Cambridge, MA. Boucher, D. H. 1985. The biology of mutualism: ecology and evolution. Oxford University Press, New York. Acknowledgments Boucias, D. G., J. J. Becnel, S. E. White, and M. Bott. 2001. In vivo and in vitro development of the Helico- We thank M. Blackwell for helpful comments on fungal

sporidium sp. J. Eukary. Microbiol. 48: 460Ð470. Downloaded from https://academic.oup.com/ee/article/40/2/289/409195 by guest on 24 September 2021 classiÞcation. This work was funded by National Science Foundation grants DEB-0075269 and DEB-0841636, the latter Bruder, K. W., and W. J. Crans. 1979. The black ßies (Simu- under the American Recovery and Reinvestment Act of 2009. liidae: Diptera) of the Stony Brook watershed of New This is Technical Contribution No. 5866 of the Clemson Jersey, with emphasis on parasitism by mermithid nem- University Experiment Station, and is based, in part, on work atodes (Mermithidae: Nematoda). New Jersey (Rutgers) supported by NIFA/USDA, under project number SC- Agric. Exp. Stn. Bull. 851: 1Ð21. 1700276. Busˇta, J., and V. Nasˇincova´. 1986. Record of Plagiorchis neo- midis Brendow, 1970 (Trematoda: Plagiorchidae) in Czechoslovakia and studies on its life cycle. Folia Para- ϩ References Cited sitol. 33: 123Ð129 2 plates. Callaway, R. M., and L. R. 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