Andrew Dopheide

Molecular Characterization of Ciliate Diversity in Stream Biofilms

ABSTRACT Free-living protozoa are thought to be of fundamental importance in aquatic ecosystems, but there is limited understanding of their diversity and ecological role, particularly in surface-associated communities such as biofilms. Existing eukaryote-specific PCR primers were used to survey 18S rRNA gene sequence diversity in stream biofilms but poorly revealed protozoan diversity, demonstrating a need for protozoan-targeted primers. Group-specific PCR primers targeting 18S rRNA genes of the protozoan phylum Ciliophora were therefore designed and tested using DNA extracted from cultured protozoan isolates. The two most reliable primer combinations were applied to stream biofilm DNA, followed by cloning and sequencing analysis. Of 44 clones derived from primer set 384F/1147R, 86% were of probable ciliate origin, as were 25% of 44 clones detected by primer set 121F/1147R. A further 29% of 121F/1147R-detected clones matched sequences from the closely related phylum Apicomplexa. The highly ciliate-specific primer set 384F/1147R was subsequently used in PCRs on biofilm DNA from four streams exhibiting different levels of human impact, revealing differences in ciliate sequence diversity in samples from each site. Of a total of 240 clones, 73% were of probable ciliate origin; 54 different putative ciliate sequences were detected from throughout seven taxonomic ciliate classes. Sequences from Oligohymenophorea were most commonly detected in all samples, followed by either Spirotrichea or Phyllopharyngea. Restriction fragment length polymorphism profile-based analysis of clones suggested a potentially higher level of diversity than did sequencing. Nevertheless, newly designed PCR primers 384F/1147R were considered to provide an effective molecular basis for characterization of ciliate diversity in stream biofilms.

Evaluating a multigene environmental DNA approach for biodiversity assessment

BackgroundThere is an increasing demand for rapid biodiversity assessment tools that have a broad taxonomic coverage. Here we evaluate a suite of environmental DNA (eDNA) markers coupled with next generation sequencing (NGS) that span the tree of life, comparing them with traditional biodiversity monitoring tools within ten 20×20 meter plots along a 700 meter elevational gradient.ResultsFrom six eDNA datasets (one from each of 16S, 18S, ITS, trnL and two from COI) we identified sequences from 109 NCBI taxonomy-defined phyla or equivalent, ranging from 31 to 60 for a given eDNA marker. Estimates of alpha and gamma diversity were sensitive to the number of sequence reads, whereas beta diversity estimates were less sensitive. The average within-plot beta diversity was lower than between plots for all markers. The soil beta diversity of COI and 18S markers showed the strongest response to the elevational variation of the eDNA markers (COI: r=0.49, p<0.001; 18S: r=0.48, p<0.001). Furthermore pairwise beta diversities for these two markers were strongly correlated with those calculated from traditional vegetation and invertebrate biodiversity measures.ConclusionsUsing a soil-based eDNA approach, we demonstrate that standard phylogenetic markers are capable of recovering sequences from a broad diversity of eukaryotes, in addition to prokaryotes by 16S. The COI and 18S eDNA markers are the best proxies for aboveground biodiversity based on the high correlation between the pairwise beta diversities of these markers and those obtained using traditional methods.

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

ABSTRACT 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.

Metal concentrations in stream biofilm and sediments and their potential to explain biofilm microbial community structure

Concentrations of metals associated with sediments have traditionally been analysed to assess the extent of heavy metal contamination in freshwater environments. Stream biofilms present an alternative medium for this assessment which may be more relevant to the risk incurred by stream ecosystems as they are intensively grazed by aquatic organisms at a higher trophic level. Therefore, we investigated zinc, copper and lead concentrations in biofilms and sediments of 23 stream sites variously impacted by urbanisation. Simultaneously, biofilm bacterial and ciliate protozoan community structure was analysed by Automated Ribosomal Intergenic Spacer Analysis and Terminal Restriction Fragment Length Polymorphism, respectively. Statistical analysis revealed that biofilm associated metals explained a greater proportion of the variations observed in bacterial and ciliate communities than did sediment associated-metals. This study suggests that the analysis of metal concentrations in biofilms provide a good assessment of detrimental effects of metal contaminants on aquatic biota.

Preferential feeding by the ciliates Chilodonella and Tetrahymena spp. and effects of these protozoa on bacterial biofilm structure and composition.

ABSTRACT Protozoa are important components of microbial food webs, but protozoan feeding preferences and their effects in the context of bacterial biofilms are not well understood. The feeding interactions of two contrasting ciliates, the free-swimming filter feeder Tetrahymena sp. and the surface-associated predator Chilodonella sp., were investigated using biofilm-forming bacteria genetically modified to express fluorescent proteins. According to microscopy, both ciliates readily consumed cells from both Pseudomonas costantinii and Serratia plymuthica biofilms. When offered a choice between spatially separated biofilms, each ciliate showed a preference for P. costantinii biofilms. Experiments with bacterial cell extracts indicated that both ciliates used dissolved chemical cues to locate biofilms. Chilodonella sp. evidently used bacterial chemical cues as a basis for preferential feeding decisions, but it was unclear whether Tetrahymena sp. did also. Confocal microscopy of live biofilms revealed that Tetrahymena sp. had a major impact on biofilm morphology, forming holes and channels throughout S. plymuthica biofilms and reducing P. costantinii biofilms to isolated, grazing-resistant microcolonies. Grazing by Chilodonella sp. resulted in the development of less-defined trails through S. plymuthica biofilms and caused P. costantinii biofilms to become homogeneous scatterings of cells. It was not clear whether the observed feeding preferences for spatially separated P. costantinii biofilms over S. plymuthica biofilms resulted in selective targeting of P. costantinii cells in mixed biofilms. Grazing of mixed biofilms resulted in the depletion of both types of bacteria, with Tetrahymena sp. having a larger impact than Chilodonella sp., and effects similar to those seen in grazed single-species biofilms.

A comparison of bacterial, ciliate and macroinvertebrate indicators of stream ecological health

We evaluate the reliability of communities of bacteria and ciliated protozoa as indicators of freshwater ecological health. Samples of epilithic biofilm were taken from 18 freshwater streams, impacted by varying types and degrees of catchment modification. Communities of bacteria and ciliates were characterised using DNA fingerprinting techniques (automated ribosomal intergenic spacer analysis and terminal restriction fragment length polymorphism, respectively) and macroinvertebrate data also obtained, for comparison. Similar to the macroinvertebrates, the taxa richness of ciliate communities was reduced in more developed stream catchments; significant differences in the evenness of ciliate communities were also detected. We could identify no significant relationship between the richness of bacterial taxa and the percentage catchment development. However, a significant trend was detected between bacterial community structure and the predominant catchment land use (rural vs. urban) using a Bray–Curtis measure of similarity, a relationship not detected for the ciliate and macroinvertebrate communities. These findings indicate that stream bacterial, ciliate and macroinvertebrate communities each respond differently to various catchment conditions and highlight the potential of microbial communities to provide novel, alternative indicators of stream ecosystem health.

Functional Gene Composition, Diversity and Redundancy in Microbial Stream Biofilm Communities

We surveyed the functional gene composition and diversity of microbial biofilm communities in 18 New Zealand streams affected by different types of catchment land use, using a comprehensive functional gene array, GeoChip 3.0. A total of 5,371 nutrient cycling and energy metabolism genes within 65 gene families were detected among all samples (342 to 2,666 genes per stream). Carbon cycling genes were most common, followed by nitrogen cycling genes, with smaller proportions of sulphur, phosphorus cycling and energy metabolism genes. Samples from urban and native forest streams had the most similar functional gene composition, while samples from exotic forest and rural streams exhibited the most variation. There were significant differences between nitrogen and sulphur cycling genes detected in native forest and urban samples compared to exotic forest and rural samples, attributed to contrasting proportions of nitrogen fixation, denitrification, and sulphur reduction genes. Most genes were detected only in one or a few samples, with only a small minority occurring in all samples. Nonetheless, 42 of 65 gene families occurred in every sample and overall proportions of gene families were similar among samples from contrasting streams. This suggests the existence of functional gene redundancy among different stream biofilm communities despite contrasting taxonomic composition.