Ecology and genetic analysis of Katablepharis CRE, a heterotrophic flagellate that ‘blooms’ in the Columbia River estuary during the spring Peter Kahn, Tawnya Peterson, Peter Zuber Center for Coastal Margin Observation and Prediction & Institute of Environmental Health, Oregon Health & Science University, Portland, OR
[email protected]
4. Spatial and temporal distribution of Katablepharis CRE in 2013 assessed through qPCR
1. Background • Heterotrophic protists play important roles in the complex estuarine food web by shaping bacterial populations through predation, transferring prey carbon to higher trophic levels, and remineralization of nutrients such as nitrogen and phosphorus. • While difficulty in their identification using light microscopy has often led to heterotrophic protists being lumped into broad classifications, there is a strong need to develop methods that increase the spatial and temporal resolution of observations of particular organisms in order to discover the drivers of population structure and ecological function. • The Columbia River estuary contains large amounts of allochthonous detritus which is the primary source of organic matter driving ecosystem processes. This organic matter can fuel a high level of bacterial growth and productivity, which in turn can fuel propagation of grazing heterotrophic protists via the microbial loop. However, major heterotrophic protists (primary consumers and bacterivores) in the estuary are not well characterized.
Cercozoa
Chrysomonadida
Ciliophora
Dinophyceae (H)
Dinophyceae (M)
Katablepharidaceae
Estuary Mouth SATURN-03
North Channel
South Channel
Columbia River SATURN-04
Oregon
Pacific Ocean
8.0E+03 6.0E+03 4.0E+03 2.0E+03 0.0E+00 1-Jan B.
2 miles
A.
Other
1.0E+04
1-Mar
1-May
1-Nov 35.0
Katablepharis CRE USE gene copy numbers mL -1
1.6E+04
% of heterotrophic SSU Sequences
1-Sep
1.8E+04
100%
75%
50%
25%
Apr-07
Aug-07
Apr-08
Jul-08
Plume
Mid-Salinity
Freshwater
Mid-Salinity
Freshwater
Plume
Mid-Salinity
Freshwater
Plume
Mid-Salinity
Freshwater
Plume
Mid-Salinity
Freshwater
0%
Sep-08
Fig. 1. SSU sequence libraries of heterotrophic protists from April and August 2007, and in April, July and September 2008 . Freshwater = 0; Mid-Salinity = 15; Plume = 28-31. “H” refers to putative heterotrophic dinoflagellates, while “M” refers to putative mixotrophic dinoflagellates.
• SSU sequence libraries from 2007 and 2008 revealed an abundance of sequences related to Katablepharis in spring mid-salinity waters, a non-pigmented heterotrophic flagellate that has been found to be an important primary consumer and bacterivore associated with particles in other aquatic systems. Katablepharis sequences comprised over 40% of all protist sequences in spring mid-salinity samples. • Events occurring in the spring, such as upriver diatom blooms and spring runoff, deliver organic matter to the estuary that could fuel katablepharid proliferation.
3. Unique sequence element within the D2 region of Katablepharis CRE 28S rRNA gene
Gene copies mL -1
1.4E+04
25.0 1.2E+04 1.0E+04
20.0
8.0E+03
15.0
6.0E+03 10.0 4.0E+03 5.0
2.0E+03 0.0E+00
0.0
S B S B* S B S* B S* B S B* S* B* S B S B S B S* B S B* S B S* B S B S S* S NC
EM
1
3
1, 2, 3
2 1
ITS 1 18S
44
% similarity to 99 Katablepharis japonica:
ITS 2 28S
5.8S
49
43
46
Unknown
43 99
USE 59 No significant similarity
• 1. What is the temporal and spatial extent of this unique element amongst Katablepharis CRE and other katablepharids in the Columbia River system? -All Katablepharis CRE sequences recovered from the estuary contain this region. The only other katablepharid detected in the system, Leucocryptos does not appear to have this element. • 2. Is the unique element found in any other organisms in the Columbia River system or elsewhere? - No, so far Katablepharis CRE is the only organism linked to positive amplification from the unique element. This region has also failed to align with the NCBI sequence database and metagenomic databases. However, several other unique sequence elements have been detected from other protist taxa, including the parasitic dinoflagellate Eudubosquella and deepwater diplonemids. • 3. Can the unique element be used as a taxonomic marker to study the ecology of Katablepharis CRE ? - USE-specific primers for quantitative PCR (qPCR) and probes for fluorescence in-situ hybridization (FISH) were developed based on this element and used on estuarine samples collected across a five site river-to-ocean transect from 2013.
NC
EM
S03
SC
S04
23-May-13
NC
EM
S03
SC
20-Jun-13
S04 NCS03 SC 18-Jul-13
5. FISH with USE-specific probes for Katablepharis CRE
A.
49
Fig. 2. Sequencing of the rRNA gene of the Columbia River Estuary Katablepharis (CRE) revealed a 332 bp region within the D2 region of the 28S rRNA gene that is unique to the CRE strain. This unique sequence element (USE) is GC-rich compared to the rest of the gene and shows no significant similarity to other katablepharids in the NCBI database, while the rest of rRNA gene aligns well with Katablepharis japonica and other sequenced katablepharids. Colored arrows indicate PCR primers designed in the 28S unique sequence element (purple), to be general for all eukaryotes (teal), or katablepharid-specific (light brown). Numbers within the arrows refer to the primer sets that were used to answer the following questions:
S04
• This qPCR assay confirmed the wide distribution of Katablepharis CRE in the Columbia River estuary with respect to salinity (0-26PSU), but with overall at each site higher abundances measured in bottom waters. High abundance of Katablepharis CRE USE gene copy numbers persisted until river discharge returned to pre-freshet levels.
28S
96
SC
Fig. 3 A. Sampling locations for qPCR and FISH analyses conducted for March-July 2013. Surface and bottom samples were taken monthly for five sites B. Annual Columbia River discharge (m3/s) measured at the outflow of Bonneville Dam for 2013 (daily mean). Gray arrows indicate sampling dates for qPCR and FISH analyses. C. Distribution of Katablepharis CRE USE in the Columbia River estuary estimated by qPCR from March-July 2013. Asterisks denote samples which were also analyzed with FISH.
General eukaryotic primer Katablepharid-specific primer
S03 4-Apr-13
C.
B.
Katablepharis CRE USE gene abundance (copies ml -1)
2
30.0
Salinity
28S USE primer 2
% GC:
1-Jul
Salinity
Apicomplexa
Bonneville Dam Outflow (m3/s)
2. Katablepharis sequences dominate mid-salinity SSU clone libraries during the spring
1.2E+04 Washington
1.2E+04 1.0E+04
Fig. 4 A. Epifluorescence micrographs of Columbia River estuary water A. DAPI-stained cells and B. the corresponding microscopic field using the USE-specific Katablepharis CRE probe. C. Comparison of Katablepharis CRE abundance estimated by FISH and qPCR using probes specific for its Unique Sequence Element (USE) within the large subunit for both approaches.
y = 2.5642x R² = 0.58408
8.0E+03 6.0E+03 4.0E+03 2.0E+03 0.0E+00 0.0E+00 5.0E+02 1.0E+03 1.5E+03 2.0E+03 2.5E+03
C.
Katablepharis CRE cell abundance (cells ml -1)
• FISH analysis detected a ~7 µm organism, which is the approximate size of katablepharids in other systems. • The slope of the regression between qPCR and FISH estimates suggest a ratio of 2.5 gene copies per cell. The R2 value for this correlation is low, likely because biases in DNA extraction and qPCR optimization, as well as potential variability in growth rate, causes discrepancies between the two methods.
6. Conclusions • Katablepharis CRE dominated protist assemblages (including autotrophic and heterotrophic fractions) during the spring months of this study. qPCR suggests a wide distribution of Katablepharis CRE with regards to salinity, but a preference for bottom waters Given their high abundance and repeatable temporal patterns, we hypothesize that they play an important role in estuarine biogeochemical and ecosystem function. • The unique sequence element of Katablepharis CRE confers extreme variability compared to the rest of the D2 region, with no significant similarity to any sequences in the NCBI database. The discovery of the unique sequence element (USE) within the 28S rRNA gene of Katablepharis CRE provided an excellent template to track the distribution of Katablepharis CRE in the system through USE-specific probes combined with quantitative and qualitative methods. • Unique sequence elements can increase the taxonomic resolution in studies of 28S rRNA protist diversity and be utilized to develop strainspecific probes for quantitative or qualitative monitoring of ecologically relevant heterotrophic species.