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Seasonality of trichomycetes in larval black flies from South Carolina, USA

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

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

Trichomycete fungi are common endobionts of aquatic insect larvae, but little is known of their ecology. In this study, the seasonality of trichomycete colonization of larval black flies (Diptera: Simuliidae) was investigated in three streams in northwestern South Carolina. At least eight species of trichomycetes were found in two species of black flies, and 93.8% of 1819 larval black flies examined contained trichomycetes. Significant differences were found in the seasonal prevalence of Harpella melusinae, Simuliomyces microsporus, and Paramoebidium spp. at one of three sites. At this site, the lowest mean prevalence for H. melusinae occurred in winter (67%) versus the other seasons (96–100%), whereas mean prevalence was lowest in summer for Simuliomyces microsporus (1%) versus the other seasons (2–21%) and lowest in summer for P. spp. (9%) versus the other seasons (45–67%). Significant differences in levels of colonization among seasons were not detected. Conjugations of H. melusinae (representing early stages of sexual reproduction) occurred most frequently in the spring and winter (up to 14% of larvae). Sexual reproduction (represented by zygospores) of Legeriomycetaceae occurred most frequently in the spring and fall (up to 17% of larvae).

Key words: aquatic insects, black flies, Simulium, symbiosis, Zygomycota

	INTRODUCTION

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Trichomycete fungi are obligate endobionts of aquatic and terrestrial arthropods and were discovered by Leidy in 1849. More than 200 species of trichomycetes are known from 15 orders of arthropods (Lichtwardt 1986 , Misra 1998 ). Larval black flies (Diptera: Simuliidae) are hosts for at least 20 species of trichomycetes (Crosskey 1990 ). Most species of trichomycetes colonizing black flies inhabit the hindgut. Harpella melusinae, however, is known only from the midgut peritrophic matrix of black flies (Lichtwardt 1986 ). This was the only trichomycete known to colonize black fly midguts until Labeyrie et al (1996) recorded Stachylina spp. in the midguts of Simulium vittatum. The trichomycetes found in black flies are represented by two orders, Amoebidiales and Harpellales. In black flies, the non-fungal (Misra 1998 ) order Amoebidiales is represented by unbranched species of Paramoebidium in the hindgut. Harpellales is represented by two families, the Harpellaceae and the Legeriomycetaceae. Harpellaceae are found in the black fly midgut and are characterized by an unbranched vegetative thallus and determinate growth. Legeriomycetaceae are found in the black fly hindgut and are characterized by a branching thallus and indeterminate growth.

Most trichomycete investigations have dealt with taxonomy. Ecology of trichomycetes has received less attention, with only five relevant publications (García et al 1995 , Grigg and Williams 1990 , LaBeyrie et al 1996 , Lichtwardt and Williams 1988 , Taylor et al 1996 ) and two theses (Slaymaker 1998 , Taylor 1992 ). To redress the paucity of ecological information, we examined the composition and seasonality of the trichomycete biota in larval black flies at three sites in northwestern South Carolina, USA, for two years. Specifically, we tested the following null hypotheses: (i) The prevalence of each trichomycete taxon is independent of season, and (ii) the level of colonization is independent of season.

	MATERIALS AND METHODS

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Study sites and host collections – Three sites with historically predictable black fly populations were chosen for the trichomycete study (Table I ). The streams were sampled from Aug 1995 through Nov 1997. Two of the sites (Brasstown Creek and Six Mile Creek) supported populations predominantly of Simulium pictipes (Diptera: Simuliidae) and one site (Willard's Pond outflow) had a population predominantly of S. vittatum cytospecies IIIL-1. Each site was sampled on a three-week cycle and water temperature was recorded for each sample (larvae collected on one date, at one site). Black fly larvae were collected randomly with forceps from the inhabited substrate. They were transported to the laboratory on moist filter paper in a Petri dish and kept at 5 C until processed. Voucher specimens of larval S. pictipes and S. vittatum cytospecies IIIL-1 and slides of each trichomycete genus have been deposited in the Clemson University Arthropod Collection. A culture (ARSEF 6810) of Smittium culisetae from Willard's Pond (June 17, 1996) has been deposited at the USDA-ARS Collection of Entomopathogenic Fungal Cultures in Ithaca, New York, USA.

The level of colonization by Harpellales was assigned a value according to increasing amounts of mass (thalli and trichospores). Because of the three-dimensional nature of the gut, the large numbers of thalli encountered, and the branched nature of the thalli or sporangia, counting the number of individual trichomycete thalli was not practical. To record relative amounts of trichomycetes, a scale was used with 0 indicating no thalli, 1 indicating fewer than 10 overlapping thalli, 2 indicating more thalli such that there were at least 10 overlapping thalli, and 3 indicating sufficient thalli or trichospores to span the width of the lumen at least at one point. The presence of sexual structures (conjugations and zygospores) of Harpellales was noted for each larva. Levels of Paramoebidium spp. were quantified by counting the number of thalli.

Data analysis – The proportion (or percentage) of larvae colonized was calculated for each sample. To determine whether prevalence of trichomycetes (number of larvae colonized) was independent of season (spring, summer, fall, winter), analysis of variance (ANOVA) on the proportion of larvae colonized per sample date was performed with season, year and the interaction of year by season as the independent variables. Seasons were defined as follows: spring, Mar 21–Jun 20; summer, Jun 21–Sep 20; fall, Sep 21–Dec 20; winter, Dec 21–Mar 20. Proportions of colonized larvae were subjected to an arcsin transformation prior to analysis (Zar 1999 ). To assess differences in the seasonal levels of colonization by Harpellales, ANOVA was performed on mean levels (0–3) of colonization in larvae. Seasonal levels of Paramoebidium spp. were compared using mean numbers of thalli per sample. Multiple comparisons among seasons were performed using Fisher's LSD test. Significance of the results of ANOVA was supported by parallel results with the nonparametric Kruskal-Wallis test (Conover 1980 ).

	RESULTS

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Trichomycete taxa – At least eight species of trichomycetes were found in the larval black flies. The only trichomycete colonizing the midguts of the black flies was Harpella melusinae (Harpellaceae). Species of the family Legeriomycetaceae included Genistellospora homothallica, Simuliomyces microsporus, and Smittium culisetae. One larva at Willard's Pond outflow (Oct 1996) was colonized by Smittium megazygosporum (and S. culisetae) (Beard and Adler 2000 ). A species of Pennella near hovassi had trichospores that averaged 28.1 x 6.5 µm (range 25.0–35.0 x 5.0–7.5 µm, n = 12 spores from five samples) and zygospores that averaged 77.9 x 14.2 µm (range 75.0–82.5 x 12.5–15.0 µm, n = 6 spores from five samples). Members of the Legeriomycetaceae that were too young to be identified to genus were recorded as undetermined Legeriomycetaceae. The species composition of the unidentified Legeriomycetaceae was assumed to be similar to the identified species composition. Zygospores were found on S. microsporus, P. nr. hovassi, and possibly G. homothallica. Zygospores of S. microsporus are characterized by "Type I" zygospores (Moss et al 1975 , Lichtwardt 1986 ) and they occurred at Six Mile Creek. Zygospores of P. nr. hovassi are characterized by "Type III" zygospores (Moss et al 1975 , Lichtwardt 1986 ) and they occurred at Willard's Pond outflow. Zygospores of G. homothallica might have been present at Willard's Pond outflow, co-occurring with the zygospores of P. nr. hovassi, but they could not be separated in mass.

Paramoebidium consisted of three morphotypes. P. chattoni (sensu Moss 1970 ) was large (up to 2 mm long) and attached to the anterior two thirds of the hindgut. Paramoebidium "B", an unidentified, intermediate-size form with a characteristic bulbous base, was attached to the same area of the gut as P. chattoni and was possibly the juvenile form of this species. P. curvum was found in the posterior third of the hindgut or on the bases of the rectal papillae.

Prevalence – Nearly 94% (CI = 92.6–94.9%) of the larvae (n = 1819) contained trichomycetes. The permanent streams (Brasstown Creek and Six Mile Creek) had mean trichomycete prevalences of more than 95% for two years. The temporary outflow of Willard's Pond had 70.2% mean prevalence in larvae (Table II ).

View larger version (33K): [in this window] [in a new window]    FIG. 1. Percentage of larval black flies (Simulium pictipes) with trichomycetes at Brasstown Creek, Oconee County, South Carolina. 1995–1997. Conjugation detected in Harpella melusinae

View larger version (40K): [in this window] [in a new window]    FIG. 2. Percentage of larval black flies (Simulium pictipes) with trichomycetes at Six Mile Creek, Pickens County, South Carolina. 1995–1997. Conjugation detected in Harpella melusinae, Zygospores of Legeriomycetaceae present

View larger version (33K): [in this window] [in a new window]    FIG. 3. Percentage of larval black flies (Simulium vittatum cytospecies IIIL-1) with trichomycetes at Willard's Pond outflow, Pickens County, South Carolina. 1995–1997. * No stream flow, Conjugation detected in Harpella melusinae, Zygospores of Legeriomycetaceae present

Pennella nr. hovassi followed a trend similar to that for S. microsporus (highest in fall) at Six Mile Creek and Brasstown Creek except that the highest mean proportions were low (4% and 3%, respectively) and probably were influenced by single samples (Nov 1995). At Willard's Pond outflow, prevalences of P. nr. hovassi and Genistellospora homothallica were not significantly different by season (P > 0.05), but tended to be higher in spring (50% and 25%, respectively) and fall (30% and 21%). P. nr. hovassi and G. homothallica coexisted in approximately 50% of the larvae at Willard's Pond outflow. G. homothallica was not prevalent (<1%) at Six Mile Creek or Brasstown Creek.

At Willard's Pond outflow, Smittium culisetae reached its highest prevalence in summer although the number of colonized larvae (n = 4) was low. All larvae with S. culisetae (except one larva in Feb 1997) were found from Jun through Oct 1996 at Willard's Pond outflow (Fig. 3 ). S. culisetae was not found at Six Mile Creek or Brasstown Creek.

The prevalence of Paramoebidium spp. varied significantly by season and year at Six Mile Creek (Table III ), but a year by season interaction was not significant (P > 0.05). Prevalence in summer (9%) was significantly lower than in spring (45%), fall (67%), or winter (55%). At Brasstown Creek, prevalences in winter and summer were almost equal (13% and 14%) and prevalence in spring (38%) was higher than in winter. The prevalence in fall (43%) was highest (as at Six Mile Creek). Paramoebidium spp. occurred only in spring at Willard's Pond outflow.

Prevalence of sexual stages. Zygospores of the Legeriomycetaceae occurred in fall and spring of both years and in one winter (Dec 1995) at Willard's Pond outflow (Table IV ). The highest prevalence of zygospores was 50% in Apr 1997 at Willard's Pond outflow. Zygospores of the Legeriomycetaceae were detected only during the fall of both years at Six Mile Creek (Fig. 3 ). Conjugations of H. melusinae occurred during the spring peaks of prevalence for this fungus at Willard's Pond outflow, but not during the fall (1996). Conjugations of H. melusinae were found during late spring and early fall of 1997 at Six Mile Creek, but in only one sample at Brasstown Creek (25 Jan 1996).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The prevalence of trichomycete taxa in our study was not independent of season at one of three sites. The prevalence of H. melusinae in the fall and spring was high, which agrees with the findings of Slaymaker (1998) in Kansas and Taylor et al (1996) in England. The decrease in prevalence observed by these authors in Feb and Mar corresponds to our low levels in winter at Six Mile Creek. The number of larvae in summer in Slaymaker's (1998) study was low and H. melusinae was not found; however, in our study prevalence in summer was still high at Six Mile Creek, and in the study by Taylor et al (1996) . In contrast, El-Sherif (1975, cited by Taylor et al 1996 ) found little temporal change in the prevalence of H. melusinae, P. chattoni, or Stipella vigilans in North Wales. The level of colonization in our study was independent of season, although a relationship might have been detected if larger sample sizes had been available. Taylor et al (1996) observed peaks in colonization by H. melusinae from Apr to Jul and from Nov to Mar. Temporal differences in colonization necessitate sampling a site throughout the year to obtain a representative picture of the trichomycete mycota.

The rapid increases in the prevalence of H. melusinae at Willard's Pond outflow (spring, 1996 and 1997) indicate that black flies were quickly colonized by this trichomycete. Lichtwardt and Williams (1988) also found that H. melusinae can quickly colonize a population and "maintain very high levels of infestation." The source of the inoculum to establish H. melusinae is unknown. Conjugation was observed in H. melusinae from Willard's Pond outflow, and zygospores might have been produced from the conjugating thalli. The fate of the zygospores is unknown, but they might be a resistant stage that provides inocula when conditions are more favorable. The rapid appearance of H. melusinae also might indicate that the inocula are coming from ovipositing females and not from the local stream. Adult black flies in northeastern North America and Great Britain have been found with cysts in their ovaries that replace some or all of the ova. After oviposition, the cysts germinate and produce Harpellales spores that are presumed to inoculate larval black flies in the habitat. In this way, infected females could inoculate uncolonized streams or upstream habitats (review by Lichtwardt 1996 ).

Our study suggests that the biologies of trichomycete taxa differ. For instance, Willard's Pond outflow had more larvae with members of the Legeriomycetaceae (Smittium culisetae) than with H. melusinae. These Legeriomycetaceae might have more effective ways to colonize insects in ephemeral streams, and the higher organic content of the stream might have allowed higher productivity of these fungi. Smittium culisetae occurred in the summer when temperatures exceeded 25 C at Willard's Pond outflow, but most other trichomycetes were not detected, perhaps indicating greater tolerance of higher temperatures by S. culisetae. At Six Mile Creek, the site with the most continuous population of black flies, Paramoebidium spp. and H. melusinae were common. Legeriomycetaceae fungi were less common, possibly indicating that they are more limited, perhaps by nutrition (e.g., no organic input from cattle at Six Mile Creek), compared with H. melusinae or Paramoebidium spp.

We examined only multivoltine species of black flies. In contrast, Lichtwardt and Williams (1988) examined both multivoltine species (e.g., Simulium tuberosum) and univoltine species (e.g., Prosimulium onychodactylum) of black flies, but did not compare trichomycete colonization in larval black flies with these two life histories. A comparison of this type might give insight into the epidemiology and biology of colonization potential for the trichomycetes. For example, a minimal intermolt interval at a given temperature, might be necessary for trichomycete reproduction to occur. The study by Labeyrie et al (1996) had one sample with both a univoltine species (Cnephia dacotensis) and a multivoltine species (S. vittatum). The prevalences of two taxa of trichomycetes were "considerably" higher in the univoltine species.

	ACKNOWLEDGMENTS

 
This work was supported in part by Grant No. DEB-9629456 from the National Science Foundation to PHA. We thank G. R. Carner, R. W. Lichtwardt, T. M. McInnis, A. G. Wheeler, and M. M. White for reviewing an earlier version of the manuscript, and W. C. Bridges and J. W. McCreadie for statistical advice. This is Technical Contribution No. 4665 of the South Carolina Agricultural and Forestry Research System, Clemson University.

	FOOTNOTES

 
¹ Corresponding author, Email: cbrd{at}clemson.edu

Accepted for publication July 25, 2001.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
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Conover WJ., 1980 Practical nonparametric statistics 2nd ed. New York: John Wiley & Sons. 493 p

Crosskey RW., 1990 The natural history of blackflies New York: John Wiley and Sons. 711 p

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García JJ, Campos RE, Maciá A., 1995 Observaciones ecológicas sobre Mansonia indubitans y Ma. titillans (Diptera: Culicidae) y sus enemigos naturales en Punta Lara, Argentina Rev Soc Entomol Argent 54:43-50

Grigg RD, Williams MC., 1990 Cyclical presence of Amoebidium parasiticum on mosquito (Culicidae) hosts in central Nebraska Mycologia 82:132-134

Labeyrie ES, Molloy DP, Lichtwardt RW., 1996 An investigation of Harpellales (Trichomycetes) in New York State blackflies (Diptera: Simuliidae) J Invert Pathol 68:293-298[Medline]

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Lichtwardt RW., 1986 The Trichomycetes: Fungal Associates of Arthropods New York: Springer-Verlag. 343 p

———. 1996 Trichomycetes and the arthropod gut In: Howard D, Miller D, eds. The mycota, animal and human relations. New York: Springer-Verlag. p 315–330

———, Williams MC., 1988 Distribution and species diversity of trichomycete gut fungi in aquatic insect larvae in two Rocky Mountain streams Can J Bot 66:1259-1263

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Moss ST., 1970 Trichomycetes inhabiting the digestive tract of Simulium equinum larvae Trans Brit Mycol Soc 54:1-13

———. Lichtwardt RW, Manier J-F., 1975 Zygopolaris, a new genus of Trichomycetes producing zygospores with polar attachment Mycologia 67:120-127

Slaymaker A., 1998 Diversity of trichomycete species and prevalence in aquatic hosts in two north-eastern Kansas streams [Master of Arts Thesis]. University of Kansas. 93 p

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———, Moss ST, Ladle M., 1996 Temporal changes in the level of infestation of Simulium ornatum Meigen (complex) (Simuliidae: Diptera) larvae by endosymbiotic fungus Harpella melusinae Lichtwardt (Harpellales: Trichomycetes) Hydrobiologia 328:117-125

Zar JH., 1999 Biostatistical analysis 4th ed. Upper Saddle River, New Jersey: Prentice Hall. 929 p

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