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The microdistribution of the trichomycete Smittium culisetae in the hindgut of the black fly host Simulium vittatum

     Department of Biological Sciences, Life Sciences Building, Room 124, University of South Alabama, 307 University Blvd., Mobile, Alabama 36688-0002

Charles E. Beard ¹

     Department of Entomology, Box 340365, Clemson University, Clemson, South Carolina 29634-0365

	ABSTRACT

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We examined the distribution of hyphae of the trichomycete fungus Smittium culisetae (Harpellales: Legeriomycetaceae) in the hindgut of a larval black fly (Simulium vittatum, cytospecies IS-7) by analyzing its prevalence and relative abundance. Hyphal prevalence was highest in the posterior colon (93.1%) and rectum (86.3%), with low prevalence (12.0%) in the anterior colon. Relative abundance of hyphae was highest in the posterior colon, followed by the rectum; relative abundance of hyphae in the anterior colon was lower. Hyphae of S. culisetae were not observed in the pylorus. We used a novel method of quantifying the relative abundance of S. culisetae in the host hindgut. The hindgut was observed with an ocular grid, and abundance was expressed as the ratio of grids occupied by hyphae to the number of grids occupied by hindgut.

Key words: aquatic insects, habitat selection, Simuliidae, symbiosis, Zygomycota

	INTRODUCTION

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Trichomycetes (Zygomycota) are a cosmopolitan class of filamentous fungi that are obligate commensals in the guts of various arthropods (Lichtwardt 1986, 1996). Members of the order Harpellales are found in the aquatic (immature) stages of mayflies (Ephemeroptera), stoneflies (Plecoptera) and nematoceran flies (Diptera) (Lichtwardt 1996). Seven genera and about 35 species of Harpellales are known from black fly larvae (Diptera: Simuliidae) (unpubl., Lichtwardt 1986, Lichtwardt and Williams 1990, Williams and Lichtwardt 1990, Labeyrie et al 1996, Lichtwardt and Arenas 1996). Black fly larvae become infected with Harpellales upon ingestion of trichospores (Lichtwardt 1996). After sporangiospore extrusion (trichospore germination), young thalli attach to the peritrophic matrix in the larval midgut (Harpellaceae) or to the hindgut cuticle (Legeriomycetaceae). Extruded sporangiospores of Smittium culisetae attach to the hindgut cuticle within 30 min of trichospore ingestion in mosquitoes at 24 ± 2 C, and developing thalli produce new trichospores within 22 h (Williams and Lichtwardt 1972), which are released into the gut lumen and exit with fecal material. When hosts molt, fungal thalli are shed with the hindgut lining (Lichtwardt 1986). This fungus is one of the relatively few species of trichomycetes that has been successfully cultured (Lichtwardt 1996) and readily produces large numbers of trichospores (e.g., Horn 1989).

Larval black flies can be found in streams ranging from temporary trickles to large rivers and are often a dominant part of the stream-macroinvertebrate community (Adler and McCreadie 1997). Larvae adhere to solid substrates in the stream and obtain food by filter feeding, scraping and collecting-gathering (Currie and Craig 1988). Because larval black flies require running water and adults generally refuse to mate in captivity, black flies historically have been difficult to manipulate under laboratory conditions (Crosskey 1990). As a result, little experimental work (e.g., Labeyrie et al 1996, Beard and Adler 2000) has been conducted examining the relationships between black flies and hindgut trichomycetes. The larval hindgut consists of three regions (Fig. 1); (i) the short, funnel-like anterior pylorus, (ii) the long simple, tubular colon and (iii) the terminal rectum (Crosskey 1990). Using host larvae of Simulium vittatum cytospecies IS-7, we examined the distribution of Smittium culisetae (Legeriomycetaceae) in the hindgut by analyzing the prevalence (presence/absence of hyphae) and relative abundance (quantity of hyphae) in each hindgut section. We tested the hypotheses that colonization prevalence and relative abundance vary in the sections of the black fly hindgut. We also independently tested the effects of larval host age, trichospore dosage and time after inoculation on variation in colonization prevalence and abundance (Table I).

View larger version (57K): [in this window] [in a new window]   FIG. 1. Hindgut of the larval black fly Simulium vittatum. py = pylorus, ac = anterior colon, pc = posterior colon, rc = rectum, ap = anal papillae. Scale bar: 250 µm

	MATERIALS AND METHODS

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Maintenance of fungal cultures and inoculum preparation – Smittium culisetae used in our experiments (U.S.D.A.-A.R.S Collection of Entomopathogenic Fungal Cultures in Ithaca, New York, ARSEF 6810) was isolated from a wild specimen of Simulium vittatum cytospecies IIIL-1 (Diptera: Simuliidae) collected in a temporary pond outflow (USA. SOUTH CAROLINA. Pickens County, Clemson University, 34°39.4'N 82°49.2'W). Stock cultures were maintained on plates of 3.7 g/L Brain Heart Infusion agar (Difco 0037-15-0) at 23–25 C, with monthly transfers to fresh plates; a sterile water overlay was not used on 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 after 6 d, and trichospores were harvested by filtering the overlay through a glass-wool/aquarium-floss plug 4 d later. The resulting trichospore suspension was centrifuged at 900 g for 10 min and the trichospore plug rinsed once in water (Horn 1989). Trichospore concentration in the resulting suspension was determined with a counting slide (Hemacytometer, improved Neubauer scale). Depending on the particular experiment, spore suspension sufficient to achieve 4000 or 8000 trichospores/mL of host treatment water then was added to the treatment containers (see below). The 4000 spore/mL dosage is based on our preliminary laboratory trials to achieve high, though not atypical, levels of trichomycete infestations, similar to those we have seen in the field.

Hosts maintenance – Eggs of Simulium vittatum cytospecies IS-7 were obtained from a colony at the University of Georgia (Athens, Georgia). This colony is free of trichomycetes, nematodes and microsporidians (Adler unpublished data, Beard and Adler 2000). For each experiment, eggs were transferred from storage (refrigerator, 4 C) to 1-L rearing containers (described below) with 500 mL of aged tap water. Eggs did not hatch while stored at 4 C, but hatching occurred when eggs were placed in 22 C water. Hatching and molting are asynchronous, therefore the exact age of individual larvae cannot be tracked. Larval age hereafter refers to the interval after eggs were placed in 22 C water, to provide a uniform point of reference. Aeration was supplied by aquarium air pumps fitted with AS2 Sweetwater® air-stones. Host larval food was based on fish-food slurry described by McCreadie and Colbo (1991), consisting of Tetra® fish food suspended in water (4 g/L) by blending. Larvae were fed daily by adding 10 mL of the slurry to each container.

All experiments were conducted in five-shelf Precival® incubators at 22 C with a light/dark regime of 16/8 h. Each incubator was supplied with a five-level pump-driven manifold system. Air to each incubator was supplied by an external Sweetwater® 5.5 CFM pump. The main airline (1.25 cm vinyl tubing) was attached to a five-valve main manifold inside the incubator. Each valve of this main manifold was attached to a 12-valve secondary manifold with 0.4 cm vinyl tubing, and each of these secondary manifolds supplied air to a single incubator shelf via 0.4 cm tubing. Thus, 60 airlines (five shelves with 12 lines/shelf) were fitted to each incubator, which in turn supplied air to 60 treatment containers. Air supplied to container water created currents, simulating the running water required by larval black flies.

Each treatment container consisted of a 12 cm (diam) x 11-cm (height) polypropylene plastic container fitted with a screw-top lid. A length (15 cm) of stiff plastic tubing (to prevent flotation of the airstone) was inserted into a 1 cm diameter hole cut into the screw-top near the lip and was pushed to the bottom of the container. Air tubing (0.4 cm) was inserted into the top of the stiff tubing, pulled through the opposite end of the stiff tubing and fitted with an AS1 Sweetwater® air-stone. An additional hole cut in the container lid provided access for feeding and dosing larvae.

Experimental protocol – One d (24 h) before the start of each experiment, 40 larvae were removed from rearing containers and placed in treatment containers with 500 mL of aged tap water. Although larval age varied among experiments (see below), age within each experiment was constant. After this 24 h acclimation period, larvae were dosed daily with spores and fed fish-food slurry. In all experiments, treatment water was changed and containers redosed with trichospores every 2 d until the end of the experiment (Horn 1989 and others typically have dosed the larvae once during experiments). Undosed containers were used as controls. Once an experiment was terminated, larvae were removed, placed on moist filter paper in Petri dishes, and held at 4 C 1–2 d. At this temperature, larvae voided the gut contents without molting. There were three replicates in each experiment.

Larval ages for experiments 1 and 2 were 22 d and 19 d, respectively. Larvae were dosed with trichospores every 2 d at a rate of 4000 spores/mL of treatment water. Both experiments were terminated at 6 d. Younger larvae, age 14 d, were used in experiments 3 and 4. Because these larvae were smaller, 8 d were allowed before the termination of the experiment; otherwise larvae would have been too small to manipulate for analysis of the hindgut fungi. Experiments 5 and 6 (larval age 20 d) were the same as experiments 1 and 2, with the exception that containers were dosed at the higher rate of 8000 spores/mL. Experiment 7 was designed to determine if the distribution of hyphae in the hindgut remained constant over time (starting with 20 d old mid-late instar larvae). Hence, larvae were removed at four different times (treatments) (i.e., 2, 6, 10 and 14 d after the initial inoculation). Three replicates, each dosed at 4000/mL, were used for each treatment (time). Larval age was 20 d at the beginning of the experiment.

For each experiment, 10 larvae (five in Experiment 7) were selected randomly from each replicate and the relative abundance of hyphae assessed as follows. Larvae were placed in a drop of tap water under a dissecting microscope. The hindgut then was removed and cleared of food. Under phase-contrast microscopy, the anterior colon, posterior colon and rectum were viewed at 400x through an ocular grid (10 mm x 10 mm reticle). The number of grids that contained one or more hyphae were counted and relative abundance expressed by the ratio (percentage) of grids containing hyphae to the number grids occupied by gut. The sections of the hindgut often were larger than the ocular grid area; therefore, landmarks were used to provide consistency in analyzing each section of the hindgut. Hence, the anterior part of the colon just posterior to the cuticular teeth, the posterior part of the colon just posterior to the colonic bend and the area of the rectum just anterior to the rectal papillae were measured for each larva.

Statistical analysis – All statistical tests were considered significant at P < 0.05 and followed the methodology of Zar (1996). The experimental protocol produced two response variables: prevalence, which is defined as the presence or absence of hyphae among each section of the hindgut, and relative abundance (i.e., the number of hyphae per given area of hindgut in a specific section). To determine if there was a significant difference in the prevalence of hyphae among gut sections (i.e., anterior colon, posterior colon, rectum), data from experiments were pooled and subjected to Chi-square analysis. Multiple comparisons were used after a significant Chi-square analysis to determine where differences in prevalence occurred. Multiple comparisons were performed on the arcsine-transformed percentages, using a "Tukey-type" test based on the q_4k statistic, where = 0.05, 4 = df, and k = number of groups. In the case of abundance, each experiment was analyzed separately. These data were analyzed with a one-way anova on acrsine-transformed percentages, with section of the hindgut as the main effect (treatment). For significant anovas, Tukey multiple comparisons were used to determine where significant differences in relative abundance occurred.

	RESULTS

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Of the 235 larvae examined in this study, only 10 (4.3%, CI = 2.1–7.7%) lacked hyphae in any part of the hindgut. Eight of these larvae had light head capsules (unsclerotized) suggesting the lack of hyphae was the result of a recent larval molt (and consequently expulsion of the hindgut fungi with the cuticle). Furthermore, uninfected larvae were spread evenly among experiments (i.e., one or two uninfected hosts detected per experiment). Spore production also was noted in colonized larvae. Hence, the rearing system developed for this study not only maintained larvae during the course of all experiments but readily allowed for infection of hosts as well as growth and reproduction of S. culisetae.

Prevalence by hindgut section – Pooling the data from experiments 1–6 (i.e., larvae examined 6 or 8 d after inoculation) showed that distribution of hyphae (based on prevalence) varied significantly among each section of the hindgut examined (Table II). Most larvae had hyphae in the posterior colon (93.1%) and rectum (86.3%), but fewer larvae had hyphae attached to the anterior colon (12.0%). This trend was consistent when each experiment was examined individually and when data from Experiment 7 (i.e., larvae examined 2, 6, 10 and 14 d post-inoculation) also were considered. Hyphae were never observed in the pylorus (experiments 1–7, n = 235).

View larger version (26K): [in this window] [in a new window]   FIG. 2. Relative abundance of hyphae in the black fly hindgut. Each experiment was analyzed with arcsine-transformed percents; however, raw data and confidence intervals are presented graphically for comparative purposes. All anovas were significant at P < 0.001. For each graph, bars with different letters are significantly different at a family error rate of P < 0.05. Experiments 1–4 were conducted with a 4000 spores/mL of host treatment water; experiments 5 and 6 used 8000 spores/mL. Experiments 1, 2, 5, 6 used larval ages of 19–21 d; experiments 3 and 4 used larval ages of 14 d. ac = anterior colon, pc = posterior colon, rc = rectum

View larger version (16K): [in this window] [in a new window]   FIG. 3. Relative abundance of hyphae in the black fly hindgut over time (Experiment 7). Data were analyzed using arcsine-transformed percents; however, raw data and confidence intervals are presented graphically for comparative purposes. All anovas were significant at P < 0.001. For each graph, bars with different letters are significantly different at a family error rate of P < 0.05. This experiment was conducted with 4000 spores/mL of host treatment water; all larval ages were 20 d at the start of the experiment. Days PI (post inoculation) indicate the number of days after the initial inoculation that larval hindguts were assessed for hyphae abundance. ac = anterior colon, pc = posterior colon, rc = rectum

View larger version (131K): [in this window] [in a new window]   FIG. 4. Hyphae abundance of Smittium culisetae in the hindgut, anterior colon, and posterior colon from the black fly Simulium vittatum. The posterior colon is heavily infested with hyphae, whereas few hyphae are present in the anterior colon. The junction between the anterior and posterior colon is indicated by the arrow. ac = anterior colon, pc = posterior colon. Scale bar: 200 µm

	DISCUSSION

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Most species of trichomycetes are restricted to specific regions of the arthropod alimentary canal (i.e., foregut, midgut and hindgut). For example, members of the families Harpellaceae and Legeriomycetaceae attach to the lining of the midgut or hindgut, respectively (Lichtwardt 1986, 1996). Various physiological adaptations ensuring spore extrusion and holdfast formation in the appropriate location within the gut have been investigated with Smittium spp. in the mosquito host Aedes aegypti (Horn 1989). In S. culisetae, spore extrusion consists of a two-phase response. In the first phase, which occurs in the larval midgut, trichospores are exposed to a pH of 10 in the presence of potassium. In Phase II, the trichospores pass into the hindgut where pH drops to 6–8. The sporangiospore is extruded and attaches to the hindgut cuticle with holdfast material.

Although the restriction of trichomycetes to the foregut, midgut and hindgut of arthropods is well documented (Lichtwardt 1986), little has been reported on the distribution and abundance of thalli within each of these regions (e.g., hindgut). For example, Smittium morbosum occupies the mosquito pylorus (anterior part of the hindgut) and thalli penetrate the midgut epithelium and Malpighian tubules (Horn 2000). Simuliomyces microsporus attaches to Paramoebidum chattoni (sensu Moss 1970), which in turn attaches to anterior sections of the hindgut in black flies (Beard and Adler 2002). Smittium culisetae in mosquitoes is restricted to the rectum (Horn 1989). In contrast, this study showed colonization of S. culisetae within the black fly hindgut was most abundant in the posterior colon, followed by the rectum, with little colonization in the anterior colon. Thus, distribution of S. culisetae thalli within the gut might be dependent on the host species or family. However, we showed that distribution was independent of larval age, trichospore dosage and time after inoculation. Results of the relative abundance measurements might have been affected by molting and the amount of time thalli had to grow relative to molting.

Black flies show many physiological similarities to mosquito larvae; therefore we can speculate that sporangiospore extrusion of S. culisetae probably follows the same sequence of events within the black fly gut as it does in the mosquito gut. High abundance in the posterior colon and rectum in our study might reflect the point where the transit time through the gut and peak spore extrusion coincide. Structural differences in the hindgut cuticle also might contribute to the observed pattern of relative abundance. Most colonization occurred in the posterior colon where the narrow lumen of the anterior colon expands into the distinctly curved portion of the posterior colon (Fig. 1). Thus, this loop might act as a trap for spores. Peristalsis also is active in the hindgut of mosquitoes (Jones 1960) and might play a role in orienting the extruded spores to a proper attachment attitude in relation to the cuticle in specific sections. Attachment in the rectum was independent of the amount of inoculum; therefore, hyphal crowding probably has little influence on the site of attachment. Physiological differences in the sections of the hindgut also might be expected to govern the areas of attachment. For example, the pylorus could be high in amino acids, ions (sodium, potassium) and other hemolymph components found in the Malpighian filtrate (Bradley 1985) that empties into this area. Many of these chemicals are reabsorbed or modified as food moves through the hindgut.

Now that we know that the distribution of Smittium culisetae varies within the hindgut, we can begin to examine the underlying reasons for the distribution. Other species of trichomycetes should be examined to compare distribution patterns. We need to investigate the environment of the microhabitats occupied by the fungi, and other host types should be examined to see if the patterns vary by host. These investigations will add to our limited knowledge of the physiology of this group of fungi.

	ACKNOWLEDGMENTS

 
We wish to thank S. A. Axsmith, D. T. Ihle, A. F. McCreadie, M. P. Nelder and B. Reynolds for their assistance in the laboratory. We thank P. H. Adler for reviewing the manuscript and E. W. Gray for providing black fly eggs. Financial support for this study was obtained from NSF Grant DEB 0075269 awarded to the authors.

	FOOTNOTES

 
¹ Corresponding author. E-mail: cbrd{at}clemson.edu

Accepted for publication April 8, 2003.

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