Relative Diversity and Community Structure of Ciliates in Stream Biofilms According to Molecular and Microscopy Methods

Ciliates are an important component of aquatic ecosystems, acting as predators of bacteria and protozoa and providing nutrition for organisms at higher trophic levels. Understanding of the diversity and ecological role of ciliates in stream biofilms is limited, however. Ciliate diversity in biofilm samples from four streams subject to different impacts by human activity was assessed using microscopy and terminal restriction fragment length polymorphism (T-RFLP) analysis of 18S rRNA sequences. Analysis of 3′ and 5′ terminal fragments yielded very similar estimates of ciliate diversity. The diversity detected using microscopy was consistently lower than that suggested by T-RFLP analysis, indicating the existence of genetic diversity not apparent by morphological examination. Microscopy and T-RFLP analyses revealed similar relative trends in diversity between different streams, with the lowest level of biofilm-associated ciliate diversity found in samples from the least-impacted stream and the highest diversity in samples from moderately to highly impacted streams. Multivariate analysis provided evidence of significantly different ciliate communities in biofilm samples from different streams and seasons, particularly between a highly degraded urban stream and less impacted streams. Microscopy and T-RFLP data both suggested the existence of widely distributed, resilient biofilm-associated ciliates as well as ciliate taxa restricted to sites with particular environmental conditions, with cosmopolitan taxa being more abundant than those with restricted distributions. Differences between ciliate assemblages were associated with water quality characteristics typical of urban stream degradation and may be related to factors such as nutrient availability and macroinvertebrate communities. Microscopic and molecular techniques were considered to be useful complementary approaches for investigation of biofilm ciliate communities.Heterotrophic microeukaryotes such as ciliates are thought to be of considerable importance in aquatic ecosystems, as they are major predators of bacteria and constitute a nutritional resource for other protozoa, invertebrates, and probably fish larvae (9, 22, 36, 52, 62, 63, 71). In addition, protozoan bacterivory contributes to enhanced decomposition of leaf detritus—a vital nutrient resource in streams—by increasing turnover of bacterial populations through predation (57). It is not well understood, however, how ciliate diversity and community structure in streams are affected by changing environmental conditions, or how ciliate communities affect other stream biota and processes. The effects of various physical, chemical, and biological factors on freshwater protozoan communities have been considered by a number of studies, but most of these have focused upon planktonic organisms in lentic habitats (for example, see references 2, 11, and 44). However, the complex microbial communities in biofilms have been recognized as important contributors to critical ecological processes, such as auxotrophic primary production, nitrogen fixation, and nutrient cycling, and may underpin the function of stream food webs (31, 45, 61). The few studies which have investigated benthic habitats in lotic systems have found evidence of the existence of diverse communities of abundant ciliates (3, 20, 56) and shifts in community structure in response to ecophysiological parameters (30, 42, 43). With one exception, however, these investigations were based on aquatic sediments, and the organisms within epilithic biofilms have continued to receive little attention.Most studies of ciliate diversity and ecology have utilized microscopy-based methods of identification (for example, see references 3 and 56), as ciliate cells are relatively large and morphologically diverse. Such methods demand a high level of taxonomic expertise, however, and are difficult and time-consuming—for example, many ciliates are fragile and fast moving, and they often require difficult fixing and staining protocols for reliable identification. Molecular biological tools offer the possibility of more accurate and efficient methods for protozoan study and may provide a useful complement to traditional approaches (12, 18, 28, 65), yet we know of only a few molecular studies of environmental ciliate diversity (18, 20, 37). A series of recent investigations used culture-independent analysis of 18S rRNA gene sequences to reveal the existence of diverse microeukaryote communities in assorted marine, anoxic, and extreme environments (40, 48, 66, 69, 70, 72). Furthermore, a growing body of evidence suggests the existence of significant genetic diversity among various ciliate taxa which has escaped detection by microscopy (14, 18, 23, 34, 60, 64, 78), pointing to the potential for molecular techniques to generate new insights into ciliate diversity and ecology, and suggesting a need for comparison of the effectiveness of these different techniques in environmental samples.Terminal restriction fragment length polymorphism (T-RFLP) analysis provides an efficient, inexpensive, and semiquantitative means for comparing microbial molecular diversity between different samples and has been widely used to investigate bacterial communities, although only a few studies have applied T-RFLP methods to the analysis of microeukaryote diversity (6, 16, 17). In this study, ciliate diversity and community structure were investigated in biofilm samples from streams representing a range of levels of anthropogenic degradation, with the objective of testing the null hypothesis that human impacts have no effect upon this important heterotrophic component of stream ecosystems. To achieve this, ciliate-targeted PCR primers were used in conjunction with T-RFLP and multivariate statistical analyses. Additionally, ciliate diversity measures obtained using molecular techniques were compared with those derived from microscopy-based methods in order to assess the relative effectiveness of these approaches.