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124 Life Sciences Building, Department of Biological Sciences, University of South Alabama, Mobile, Alabama 36688-0002
Charles E. Beard
114 Long Hall, Box 340361, Department of Entomology, Soils and Plant Sciences, Clemson University, Clemson, South Carolina 29634-0315
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Smittium, the most speciose genus of the "gut fungi" (Zygomycota: Trichomycetes), is found attached to the hindgut cuticle of larval aquatic Diptera. Smittium spp. colonize several host families (e.g., Smittium culisetae in Chironomidae, Culicidae and Simuliidae), but some species appear to be specific to a single host family (e.g., Smittium morbosum Sweeney in Culicidae). The specificity of Smittium spp. within a host family has been difficult to resolve. This research presents evidence that certain Smittium spp. differentially colonize particular species of black fly (Diptera: Simuliidae) hosts as measured by differences in prevalence, abundance and fecundity. Reasons for this differential occurrence and fecundity in hosts are unclear but might include fungal responses to variations in host morphology, physiology, distribution or behavior. Variable fitness of Smittium spp., within a suite of available hosts, could be a factor in the diversity of this fungal group.
Key words: axenic cultures, colonization, Harpellales, Simuliidae, symbiosis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Misra 1998, Moss 1998). The largest order, Harpellales, comprise 34 genera, the most species-rich genus being Smittium (Legeriomycetaceae) (Lichtwardt et al 2003). Fifty-five species of Smittium have been found attached to the hindgut cuticles of several families of aquatic dipteran larvae, as well as larval may-flies (Ephemeroptera) and stoneflies (Plecoptera) (Lichtwardt and Williams 1990, Williams and Lichtwardt 1990, Lichtwardt et al 2001, Alencar et al 2003). Host specificity of Smittium usually has been discernible at the family taxon, although some species colonize several host families within an order. For example, Smittium culicis Manier has been found in variety of families of the lower flies (Diptera: Nematocera) including Chironomidae, Culicidae, Psychodidae, Simuliidae, Thaumaleidae and Tipulidae (Lichtwardt et al 1999). Advances in axenic culturing techniques (Lichtwardt 1964) have made Smittium spp. available for molecular (Sanger et al 1972, Gottlieb and Lichtwardt 2001, White 2002), nutritional (Horn and Lichtwardt 1981), physiological (Williams and Lichtwardt 1972a; El-Buni and Lichtwardt 1976a, 1976b; Horn 1989) and ultrastructural studies (Sato et al 1989).
Black fly (Diptera: Simuliidae) larvae are often one of the most dominant insects in the stream benthos (Cummins 1987, Adler et al 2004). Most larval black flies are filter feeders, using a pair of modified labral fans to remove particles from the water column (Currie and Craig 1987, Crosskey 1990). Larvae passively capture a wide array of food particles of varying sizes, ranging from 0.091 to 350 µm diam (Crosskey 1990), which regularly consists of animal matter, bacteria, detritus, diatoms, filamentous algae and fungal spores (Kurtak 1978, 1979). Larval simuliids worldwide are known to harbor 12 species of Smittium (Nelder, McCreadie and Beard unpubl data). Five of these species have been reported exclusively from larval simuliids, while the other seven species are known to colonize additional families of aquatic Diptera.
A Smittium-simuliid model was adopted in these experiments because both symbiont moieties can be maintained and manipulated under laboratory conditions. In addition, black flies were selected as the model host because they are a ubiquitous part of the macro-invertebrate stream fauna (Adler and McCreadie 1997).
In this paper we examine the occurrence and fecundity of five species of trichomycetes in various hosts. Occurrence, as used here, has two components. The first is prevalence (i.e., the number of host larvae with gut fungi divided by the number of hosts examined); it is expressed quantitatively as a percent. The second is relative abundance (i.e., the amount of hyphal growth in gut of the host [McCreadie and Beard 2004]). Accordingly we examined whether occurrence of several species of Smittium varies within a single species of black fly and whether occurrence of a single species of Smittium varies among several host species of black flies.
In addition to occurrence we also examine trichomycete fecundity, defined here as the number of trichospores produced by thalli attached to the hindgut of the host. Trichospores are the asexual reproductive propagules, and the number of mature trichospores produced by a fungus can be used as a measure of fitness. Here fitness is used in the broad sense (i.e., the probability of contributing to the next generation). The four hypotheses to be tested in this study are: (i) occurrence of different Smittium spp. does not vary in a single host species; (ii) fecundity of different Smittium spp. does not vary in a single host species; (iii) Occurrence of Smittium spp. does not vary among species of hosts; (iv) fecundity of different Smittium spp. does not vary among species of hosts. This is the first study to report differential occurrence and fecundity of Smittium species among black fly hosts.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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. Hence, only the most pertinent details will be given here. Stock cultures of Smittium brasiliense Alencar, Lichtwardt, Ríos-Velásquez & Hamada, Smittium culisetae Lichtwardt, Smittium megazygosporum Manier & Coste, Smittium morbosum Sweeney and Smittium near typhellum (TABLE I) were maintained on plates of 3.7 g/L Brain Heart Infusion agar (Difco®) at 23–25 C, with monthly transfers to fresh plates; a sterile water overlay was not used on our stock cultures. Hyphal subcultures were transferred (see Lichtwardt 1986 for methods) to new plates 10 d before the start of an experiment. Plates were covered with a sterile water overlay to induce trichospore production, and trichospores were harvested by filtering the overlay through a glass-wool/aquarium-floss plug and centrifuged at 900 g for 10 min (Horn 1989). Trichospore concentration in the resulting suspension was determined with a counting slide (Haemacytometer, improved Neubauer scale). Inocula were aliquots of 4000 trichospores/mL of host-rearing water.
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). Eggs of Simulium vittatum were obtained from a colony at the University of Georgia (Athens, Georgia). Remaining hosts species were collected as eggs in the field at these locations: Simulium innoxium, Six Mile Creek, Pickens County, South Carolina, USA: 34°48.1'N, 82°50.7'W; Simulium verecundum s.s. and Simulium tribulatum, Three-mile Creek, Mobile County, Alabama: 30°37.4'N, 88°10.6'W and 30°37.4'N, 88°0.6'W, respectively. Field-collected eggs were refrigerated (4 C) until needed. Hatching occurred when eggs were placed in aerated water at 22 C. Larval age hereafter refers to the interval after eggs were placed in 22 C water. Larvae were fed fish-food slurry of 4 g of Tetra® fish food suspended in 1 L of water and 1 min of agitation in a blender. All experiments were conducted in Percival® incubators at 22 C with a light/dark regime of 16/8 h. Each treatment container consisted of a 12 cm diam x 11 cm tall round polypropylene plastic container fitted with a lid. Air supplied to container water by air stones created currents simulating the lotic conditions required by larval black flies.
Assessment of relative abundance, fecundity and prevalence.— –
The abundance of hyphae in a larval host was assessed following a modification of McCreadie and Beard (2003). From each experimental container 10 larvae were examined for trichomycetes. Larvae were placed in a drop of tap water under a dissecting microscope. The hindgut was removed, cleared of food, and the area with the densest amount of hypha(e) was viewed at 400x through a 10 x 10 mm ocular grid under phase-contrast microscopy. The number of grid squares that contained one or more hyphal brachlets were counted; relative abundance was expressed as the percentage of grid squares containing hypha(e) to the total number of grid squares covering the hindgut. When fecundity (i.e., mean trichospore production) was the dependent variable of interest (TABLE II), the numbers of grid squares that contained one or more trichospore(s) were counted and the results again expressed as a percent. The term prevalence (percent colonized) refers to the number of Smittium-colonized hosts divided by the number of hosts examined.
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). Ten larvae from each container were selected randomly and, depending on the experiment trichomycete relative abundance, prevalence or fecundity was assessed. Experiments 1–3: Smittium spp. occurrence within a single host. Simulium vittatum larvae were exposed to colonization by trichospores of Smittium brasiliense, Smittium megazygosporum, Smittium morbosum and Smittium nr. typhellum. Twelve containers were inoculated for each experiment (i.e., 3 containers per treatment level x 4 [levels] species). Response variables in these experiments were hyphal abundance and prevalence. This experiment was repeated three times.
Experiments 4, 5: Fecundity of fungal species in a single host. Simulium vittatum larvae were exposed to colonization by Smittium brasiliense, Smittium megazygosporum, Smittium morbosum and Smittium nr. typhellum. Twelve containers were inoculated for each experiment (i.e., 3 containers per treatment level x 4 [levels] species). The response variable in these experiments was fungal fecundity. This experiment was repeated three times.
Experiment 6–11: Occurrence of Smittium culisetae among hosts. For these experiments the prevalence and relative thallial abundance of Smittium culisetae were compared among hosts. Experiments were designed as these paired-host comparisons: Simulium innoxium versus Simulium vittatum (experiments 6, 7, 8), Simulium verecundum s.s. versus Simulium vittatum Experiment 9) and Simulium tribulatum versus Simulium vittatum (experiments 10, 11). The number of experiments for each host was determined by the availability of host eggs. For experiments 6–9 each host pair had four treatment levels; four containers with 40 larvae each of species x, four containers with 40 larvae each of species y and four containers with 20 larvae of species x, plus 20 larvae of species y. Mixed containers produced two treatment levels (i.e., species x in the presence of species y, and the inverse, species y in the presence of species x). The response variables were relative thallial abundance and prevalence. Mixed containers were used to determine whether thallial abundance and prevalence in a host was independent of the presence of the other host species. In experiments 10 and 11 Simulium tribulatum and Simulium vittatum are isomorphic and cannot be separated morphologically; thus, mixed-species containers were dropped from these experiments.
Statistical analyses.— –
All measures of fecundity and relative fungal abundance (raw percentage data) were transformed into arcsine (arcsin) percents to achieve normal distributions (Quinn and Keough 2002). Mean arcsin percents, for each trial in each experiment, were analyzed with a one-way analysis of variance (ANOVA). Significant differences among treatment means were determined using the Tukey’s method of multiple comparisons. For each treatment, an experimentwise adjustment of P-values was made to preserve a family error rate of P = 0.05 for all comparisons between treatments (Zar 1996). Chi-square analysis was used to determine whether prevalence was independent of host. Differences between levels within a treatment were analyzed using post hoc comparisons by chi-square analyses with an experimentwise adjustment of P-values (i.e., P-value = 0.05/6 5 0.00833 for six comparisons).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). No significant differences in mean abundance were detected among these latter three Smittium species. With regard to prevalence, the only definitive statements that can be made are that Smittium brasiliense exhibited a significantly higher prevalence than Smittium nr. typhellum in experiments 1, 2 and 3 and that the prevalence of Smittium brasiliense was significantly higher than Smittium megazygosporum in Experiment 3. As can be seen in TABLE III, there was considerable overlap in prevalence among the species of Smittium in the larval host Simulium vitattum. Hence, relative abundance and prevalence do not appear to be completely coupled measurements of occurrence.
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). In Experiment 6, fecundity of Smittium brasiliense was significantly higher than both Smittium morbosum and Smittium megazygosporum, but not Smittium nr. typhellum.
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abundance, TABLE VI prevalence). With regard to relative abundance consistent results were noted in experiments 6–9 with no significant differences found between the abundance of Smittium culisetae in a host species treated in isolation or with another host species present. In other words no evidence was found suggesting that the presence of one host species affected the abundance of Smittium culisetae in the other host (TABLE V). With regard to the comparisons of Simulium innoxium and Simulium vittatum, some evidence of differences in the abundance Smittium culisetae between these larval hosts was found. In Experiment 7 both Simulium innoxium and Simulium innoxium w/Simulium vittatum were significantly more abundant than Simulium vittatum or Simulium vittatum w/Simulium innoxium. In Experiment 8 both S. innoxium and Simulium innoxium w/Simulium vittatum were significantly more abundant than Simulium vittatum w/Simulium innoxium. Little evidence was found suggesting that the prevalence of Smittium culisetae varied among the two hosts Simulium innoxium and Simulium vittatum. In only one case (Experiment 7) was any significant difference found and this was only between Simulium innoxium and Simulium vittatum.
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In experiments 10 and 11, Simulium tribulatum had significantly higher thallial abundance of Smittium culisetae when compared to Simulium vittatum in both trials. Prevalence of Smittium culisetae also was significantly higher (chi-square analysis) in Simulium tribulatum than in Simulium vittatum.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) and its low level of sporulation in our simuliid-host experiments might indicate that this species of Smittium was originally from a substandard host (Beard and Adler 2000). As reported by Williams and Lichtwardt (1972b), trichomycete isolates more readily colonize those hosts that are taxonomically similar to the original isolate’s host.
In experiments 6–8, no significant difference was found in Smittium culisetae relative thallial abundance and prevalence between Simulium vittatum and Simulium innoxium. The physiology of the black fly gut and the morphology of the labral fans were not studied here, and the following conjecture is an attempt to explain the observed results. The number of primary fan rays or the surface area of the labral fans might be related to the ability of a black fly larva to trap certain particle sizes (Davies 1966, Chance 1970). For example, a lack of difference between these two host species, in terms of relative abundance and prevalence, might be related to their similar labral-fan morphologies. Morphology of labral fans often is related to the speed of the stream in which a particular black fly species is typically found (Malmqvist et al 1999) and by the types of food ingested by certain species in specific streams (Carlsson 1962). These two species occupy similar habitats in nature (i.e., fast flowing or headwater streams); therefore they might have similar labral-fan morphologies (Nelder unpubl data).
The significantly higher prevalence and relative thallial abundance of Smittium culisetae in Simulium tribulatum, compared to Simulium vittatum, is interesting because these two host-species are morphologically indistinguishable as larvae (cryptic species). However over most of their geographical ranges they are ecologically distinct (Adler and Kim 1986). In nature Simulium vittatum inhabits streams originating from springs or streams that are generally pristine and unpolluted, whereas Simulium tribulatum is found in a wider range of stream types (Adler and Kim 1984, 1986). Therefore these results could indicate an ecological or behavioral (preference for certain streams) relationship for differential abundance of Smittium spp. in the hosts.
Smittium spp. might be better adapted to Simulium tribulatum, Simulium verecundum s.s. and their corresponding natural habitats, compared to Simulium innoxium and Simulium vittatum. Simulium verecundum s.s. and Simulium tribulatum occupy the Mountain, Piedmont, Sandhills, and Coastal Plain ecoregions of the southeastern USA, while Simulium vittatum and Simulium innoxium occupy the Mountain ecoregion of the same geographical area (McCreadie and Adler 1998). In general Simulium verecundum s.s. and Simulium tribulatum had higher prevalences and relative thallial abundance of Smittium culisetae than Simulium vittatum and Simulium innoxium and might be more suitable hosts for Smittium species.
Smittium spp. isolated from, and traditionally found in, nonsimuliid hosts can colonize larval black flies. We propose that the Smittium spp. investigated here can colonize across families of larval Diptera and that their host specificity can be recognized only at the ecological, rather than a taxonomic, level of host recognition (i.e., "filter-feeding" insect larvae). Williams and Lichtwardt (1972a) performed host specificity studies using axenic cultures of Smittium spp. and a larval-mosquito host (Aedes aegypti Linnaeus). Their semiquantitative data were not subjected to statistical analysis; consequently, no definitive conclusions concerning differential occurrence could be made. Differences in occurrence of Smittium spp. likely are associated with the behavior, habitat, morphology and physiology of the host larval black fly (Williams and Grigg 1990).
Although speculative, we hypothesize that primordial Smittium spp. might have been faced with periods in which favorable hosts were not available for colonization. A Smittium species’ ability to switch hosts and habitats is an important aspect of their survival and diversity. The ability to colonize a variety of hosts, especially across families, could be a useful trait for long-term survival. With this in mind, colonization of multiple families of hosts might be a primitive trait, whereas a restricted range of hosts is a specialized or derived trait.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Corresponding author. 114 Long Hall, Box 340315, Department of Entomology, Soils and Plant Sciences, Clemson University, Clemson, South Carolina, 29634-0315. E-mail: mnelder{at}clemson.edu
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