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Department of Entomology, Box 340365, Clemson University, Clemson, South Carolina 29634-0365
John W. McCreadie
Department of Biological Sciences, University of South Alabama, Mobile, Alabama 36688
Peter H. Adler
Department of Entomology, Box 340365, Clemson University, Clemson, South Carolina 29634-0365
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ABSTRACT |
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A total of 2063 mid- to late-instar larval black flies were collected from 64 stream sites in South Carolina and screened for the presence of the trichomycete fungus Harpella melusinae. Sixteen of 18 host species were colonized by H. melusinae on at least one occasion. Prevalence of H. melusinae in larvae of Simulium tuberosum cytospecies "A" was highest in acidic streams with low conductivity, whereas H. melusinae colonized larvae of Simulium verecundum most frequently in slower-moving streams. Ecological conditions, therefore, can serve as predictors of the prevalence of H. melusinae. Prevalence in host larvae was significantly lower in the Piedmont ecoregion than in the Mountain ecoregion. Prevalence did not differ in the host species S. verecundum across ecoregions, suggesting that different prevalences among host species might indicate some host preference. The prevalence of H. melusinae differed significantly between two univoltine host species (Simulium venustum and Prosimulium magnum) at the same site but not between two multivoltine host species (S. tuberosum cytospecies "FG" and S. tuberosum cytospecies "CDE"), suggesting that host life history could be important in determining fungal prevalence.
Key words: aquatic insects, fungi, Simulium, symbiosis, Zygomycota
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Adhering to in-stream substrates, they obtain food largely by filter feeding (Currie and Craig 1987). Larvae are hosts to a variety of symbionts, including fungi in the class Trichomycetes (Zygomycota), a cosmopolitan group of filamentous fungi that live as obligate commensals in the guts of various arthropods (Lichtwardt 1986, 1996). These fungi are particularly common in the aquatic stages of insects such as black flies, but little is known about their ecology.
Black flies are one of the taxonomically best-known groups of aquatic insects in North America, in part because their polytene chromosomes facilitate discovery and identification of species (Adler and McCreadie 1997). Most other groups of stream insects (e.g., stoneflies, midges) that support a trichomycete mycota are inadequately known as immatures at the species level (McCafferty et al 1990, Coffman and Ferrington 1996), precluding the detection of host associations and limiting ecological studies.
In this study, we surveyed larval black flies in South Carolina, U.S.A., for the trichomycete Harpella melusinae (Harpellales: Harpellaceae). This trichomycete thrives only in the midgut of larval black flies and can be detected easily in preserved specimens. It is an unbranched fungus that attaches to the peritrophic matrix of the host (Fig. 1), whereas branched trichomycetes (Harpellales: Legeriomycetaceae) attach to the hindgut cuticle and are not readily detected in preserved larvae (Adler et al 1996).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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. The foothills ecoregion was combined with the Piedmont ecoregion based on no significant differences in aquatic parameters. Collection sites, preselected from 1:125 000 maps, were chosen, based on accessibility and stream flow. Each stream site was sampled by walking swaths from bank to bank while hand-collecting mid- to late-instar larvae from all available substrates. Rationale for this sampling protocol is given by McCreadie and Colbo (1991). As in previous studies of this type (e.g., Quinn and Hickey 1990, McCreadie and Colbo 1991, McCreadie and Adler 1998), black fly species in the samples were assumed to represent the local occurrences. Samples were collected from May to Jul 1992 and 1993, and from Feb to Apr 1993.
We followed the protocol of McCreadie and Adler (1998) for measuring stream variables at each site: canopy cover, conductivity, depth, discharge, dissolved oxygen, dominant streambed-particle size, pH, riparian vegetation, seston, velocity, water temperature and width. These variables are useful predictors of the distribution of larval black flies among stream reaches (McCreadie and Colbo 1991, McCreadie and Adler 1998), as well as of some of the parasites that infect these larvae (McCreadie and Adler 1999). Latitude and longitude were taken from 1:125 000 maps.
Host identification and detection of Harpella melusinae –
All black fly larvae were fixed on site in three changes of acetic acid-ethanol (1:3) and stored in the laboratory at 4 C until processing. Larvae first were identified morphologically. To make cytospecific identifications of larvae that could not be identified morphologically, silk-gland polytene chromosomes were prepared using the procedure of Rothfels and Dunbar (1953). Chromosome-banding patterns were compared with standard maps available in the literature (e.g., Landau 1962, Rothfels et al 1978) and on file in our laboratories. Voucher larvae and microscope slides of H. melusinae are deposited in the Clemson University Arthropod Collection.
From each collection, 22–64 middle to late-instar larvae (mean = 28) of each available species of black fly were selected for midgut examination. Larvae patently infected with mermithid nematodes and other parasites were not selected. At 54 sites, only one host species was collected in sufficient numbers for statistical analysis and at 10 sites two host species were abundant enough for analysis, for a total of 64 sites examined. Each larva examined was placed in a drop of aged tap water and its midgut peritrophic matrix removed and teased apart with fine needles to clear the contents. The peritrophic matrix then was placed on a microscope slide, with a coverslip and scanned for H. melusinae under a compound microscope (450 x), using phase contrast. A larva was counted as colonized by H. melusinae only if distinctive features of this fungus were detected, including holdfasts or well-defined trichospores. Lack of these characters resulted in classifying the larva as not colonized.
Statistical analysis –
For each host species, the ratio of larvae colonized by H. melusinae to the number of larvae examined was considered the prevalence. The relationship between prevalence (dependent variable) and stream conditions at each stream site (independent variables) was examined by regression. Stream variables (conditions) often are highly intercorrelated (e.g., Ciborowski and Adler 1990, McCreadie and Adler 1998). When intercorrelated variables are used as independent variables in regression analyses (see below), a condition known as multicollinearity occurs (Neter et al 1990). Multicollinearity affects confidence intervals and significance tests of regression coefficients, resulting in models that are unreliable. Because of the multicollinearity problem, principal components analysis (PCA) commonly is used in ecological studies (e.g., McCreadie and Adler 1998, 1999). Determining which original stream variables have significant loadings in each of the significant PCs was based on rank correlation between the PC and the original stream variables (Ludwig and Reynolds 1988). The significance level for these correlations was set at P < 0.01 (McCreadie and Adler 1998), which provides a compromise between the chances of making a type I error (due to large number of tests) and a type II error (if multiple test adjustment is used).
In PCA, PCs replace the original stream variables as independent variables for regression analyses. Based on the recommendation of Noru
is (1985), only PCs with eigenvalues (amount of variation contained in the PC) 
1.0 were used as independent variables. Separate PCAs were performed for each set of stream conditions in which each host species was found. Stepwise multiple regression (
= P < 0.05) was used to determine which of the PCs representing significant variation in each analysis had relationships with prevalence (Neter et al 1990).
At three collection sites, two different host species were collected in sufficient numbers to determine if the prevalence of H. melusinae was independent of host species. Independence was tested using contingency table analysis with the Chi-square test statistic.
A one-way analysis of variance was used to examine prevalence of H. melusinae across ecoregions, regardless of host species. Simulium verecundum was the only host taken in sufficient numbers across (in two of the three) ecoregions for this test. Because two ecoregions were compared, a t-test was used.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The influence of host species on prevalence could be analyzed on two occasions for S. venustum and Prosimulium magnum. Chi-square analysis showed a significant difference in prevalence between these two hosts on both occasions (Table IV). In contrast, the one collection in which S. tuberosum "FG" could be compared to S. tuberosum "CDE" had no significant difference in the proportion of colonized larvae (Table IV).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Harpella melusinae was relatively common (34%) in the black flies of our study compared with the prevalence of other symbionts (maximum of 6.5% McCreadie and Adler 1999). In our study, 16 of 18 (89%) host species were colonized by H. melusinae. Although two host species (S. nyssa and S. podostemi) were not colonized, they have been found with H. melusinae in other collections (unpublished data), indicating they are also hosts. In a similar study of black flies infected with chytrids, mermithids and microsporidia, only 25 of 43 (58%) host species were patently infected (McCreadie and Adler 1999). Trichomycetes are advantageous for studies of host-symbiont relations because colonizations are detected readily, whereas many symbionts (e.g., microsporidia) produce subclinical infections that routinely go undetected.
Host life history is a parameter that should be considered when comparing symbiont loads (Beard and Adler 2002), although it often is neglected. Significant differences existed in the prevalence of H. melusinae between two univoltine host species (P. magnum and S. venustum) at the same site and time. However, no significant difference occurred between two closely related multivoltine species, S. tuberosum "FG" and S. tuberosum "CDE". In one sample from New York, a difference in prevalence of H. melusinae was noted between two host species, the multivoltine species Simulium vittatum "IIIL-1" and the univoltine species Cnephia dacotensis (Labeyrie et al 1996). These differences might be related to life history (e.g., voltinism), host-microhabitat selection and its effect on propagule ingestion and the intrinsic physiology of the hosts themselves.
Harpella melusinae occurs in all ecoregions (including the Coastal Plain, unpubl data) in South Carolina but has the highest prevalence in the mountains, possibly reflecting host-species changes across ecoregions. Black fly species are not independent of ecoregion and, in some cases (e.g., Simulium dixiense), are limited to particular ecoregions (McCreadie and Adler 1998). The mean prevalence of H. melusinae across ecoregions for the one analyzed host species (S. verecundum) did not differ significantly. If ecoregion is a determining factor, the prevalence in the same host species would be expected to differ across ecoregions. The difference in prevalence across ecoregions for all host species, therefore, might be explained partly by differences in host-species composition across ecoregions. Thus, although H. melusinae colonizes a wide range of hosts within the family Simuliidae, it might prefer certain host species. Black fly species assemblages change seasonally (Adler and Kim 1986), thus the preferred host might occur seasonally and affect prevalence of colonizing trichomycetes. The mechanism governing seasonal host cycles often is attributed to temperature, but Hynes (1970) proposed that the constancy of photoperiod plays a role in governing seasonal life history in univoltine aquatic insects. Trichomycetes might be affected by photoperiod, whether directly or indirectly. Tannins released by autumn leaf fall also might affect the hosts. Rey et al (1999) found that tannins produce lesions in the midgut epithelium of aquatic Diptera, which might make host flies more susceptible. Tannins also have been shown to induce more virulence in the protozoan parasite Lambornella clarki of the treehole mosquito Aedes sierrensis (Mercer and Anderson 1994).
Given the intimate association between H. melusinae and its hosts, as well as the production of trichospores that are host-free for part of their existence (Lichtwardt 1986), trichomycete ecology can be viewed as a triangular relationship involving the fungus, host (black fly) and environment (streams), in much the same way parasite ecology is viewed (e.g., Fuxa and Tanada 1987). Although the influence of environment on the occurrence of insect pathogens is well known (Benz 1987), our study is the first to demonstrate that environmental factors, other than host, can be predictors of trichomycete prevalence in black flies. Taylor et al (1996) examined the relationship between environmental factors and trichomycete infestations of black flies in a stream in Hampshire, England. No simple relationship was found, but they speculated that infestation is related to factors such as temperature, suspended solids and host density.
Though not directly investigated, the influence of environment on the occurrence of H. melusinae in hosts can be envisioned to operate through two processes. First, Harpellales trichomycetes have a free-living trichospore stage that might survive in the field for several months (Williams 2001). Trichospores of Smittium culisetae, for example, can survive in distilled water at 4 C for four months with no apparent decline in viability (unpublished data). Stream conditions, therefore, have the potential to influence the distribution and viability of this free-living stage in much the same way that stream conditions influence the distribution of hosts (Ross and Merritt 1987, Adler and McCreadie 1997, McCreadie and Adler 1998). For example, trichospores are distributed by flowing water. Therefore, stream velocity could influence the number of trichospores available to potential hosts, explaining, at least partly, why velocity was a significant predictor of prevalence in S. verecundum.
Second, environmental conditions such as food and temperature influence the susceptibility of hosts to infection (Watanabe 1987). Food supply, for example, influences the size of black flies (Colbo and Porter 1979, McCreadie and Robertson 1998), which might influence susceptibility to nematode parasitism (Colbo 1982). These factors also might influence the susceptibility of larvae to other symbionts, such as trichomycetes.
Our investigation focused on the predictability of trichomycete prevalence. Although we showed that the distribution of H. melusinae among streams was related to some stream-site conditions, our knowledge of trichomycete ecology is in its infancy. Interpretation of field data would benefit from additional knowledge of how environmental conditions influence host susceptibility to fungi.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Accepted for publication October 21, 2002.
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LITERATURE CITED |
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