Warwick

January 2003

Workpackage 1 and 2: 16S rDNA Synechococcus sequencing:

16S rDNA sequences are currently being amplified from marine Synechococcus laboratory cultures of PICODIV (mostly Blanes) and PROSOPE environmental samples. Most cultures have arisen from successive plating on solid agar and so are potentially clonal. Growth of environmental cyanobacteria in solid agar was conducted after initial periods of growth in non-selective liquid culture. In addition we are reviewing the potential of PICODIV clones libraries Bl000921 and Bl001221 to yield additional novel cyanobacterial 16S rDNA sequences.

Workpackage 2: Synechococcus ntcA DNA analysis:

Polyacrylamide gel electrophoresis of BstUI/ HaeIII digested ntcA DNA amplified from laboratory-cultured Synechococcus (see above) is allowing assessment of whether any potentially novel strains exist in our collection.

Workpackage 5: Dot blot analysis of environmental PCR products

All environmental samples received from the three PICODIV sampling sites have been DNA extracted and/or PCR-amplified to yield cyanobacterial-specific 16S rDNA PCR products (primers used are oxygenic phototroph-specific 107f/1313r). Products have been purified and quantified in preparation for dot-blot analysis with cyanobacterial group-specific oligonucleotide probes. Each PCR product is blotted onto a nylon membrane in triplicate alongside appropriate control strains for producing calibration curves after probe hybridisation. Application of each cyanobacterial group-specific oligonucleotide probe, along with the eubacterial-specific probe EUB338 will allow analysis of relative temporal and spatial distribution of recognised cyanobacterial groups.

We are also currently performing PCR amplification of environmental DNAs, using oxygenic phototroph-specific primers, provided from two other environments
i) seawater samples obtained by F. Partensky during the Prosope cruise in the Mediterranean Sea
ii) samples obtained off the coast of Chile (provided by Osvaldo Ulloa) which encompass oxygen minimum zone waters.
These will also be hybridised with the several marine Synechococcus clade specific oligonucleotides designed during the project.

October 2002

Sequencing of clone libraries:

We are currently sequencing 16S rDNAs retrieved from the following PICODIV clones libraries: He008003, He001005, RA001006, RA000609, Bl000921 and Bl001221. These libraries were generated from environmental DNA PCR amplified using the oxygenic phototroph-specific primers 107f/1038r. Library plasmids containing marine 16S rRNA genes have been screened by PCR using two primers pair combinations (see note 1) to assess the basic identity of the insert DNA and therefore its suitability for subsequent sequence analysis. Whilst most 107f/1038r generated 16S rDNA PCR products screened in this way prove to be cyanobacterial-like upon sequencing, a small minority of clones have been still shown to contain non-cyanobacterial DNA inserts upon sequencing even though these are supposedly cyanobacterial-specific PCR primers. Fig.1 shows phylogenetic analysis of those cyanobacterial 16S rDNA sequences retrieved from the PICODIV clone libraries listed above which we have obtained so far. [For some clades e.g. V-IX only one sequence has been included for each clade]. In the very near future 30-40 more sequences will be returned and we will then incorporate all these sequences into ARB and redraw phylogenetic trees. For any novel clades identified specific oligos will be designed once full-length sequences of these clones has been obtained. Thus far, all environmental sequences tree within clades already known and for which oligos have already been designed.

ntcA DNA analysis:

We have generated PCR products from a diverse collection of laboratory cultured cyanobacteria (see note 2) using primers specific for the enzyme-encoding gene ntcA for the evaluation of genotypic diversity of this important functional gene by RFLP analysis. To this end, PCR products have been produced from available strains and purified awaiting digestion by the restriction enzymes HaeIII and BstUI which have previously been shown to have appropriate restriction sites within the ntcA gene. It is necessary for digests to be visualised on native polyacylamide gels, as small differences of length in digested DNA require a high-resolution separation electrophoresis technique to be employed to make band patterns discernible.

Extraction and amplification of environmental DNA from filters:

We currently have in –80 oC storage a collection of filters used to obtain marine biomass from PICODIV sampling sites for the annual cycle. Extraction of whole cell DNA contained on these filters has begun with its subsequent PCR amplification by oxygenic phototroph-biased primer pair 107f/1313r planned. These PCR products are to be immobilised on nylon membrane as dot blots and subjected to probe hybridisation assays using a range of 16S rDNA-directed oligonucleotides that have been designed against the currently recognised groups of marine cyanobacteria. This work is on-going.

Notes:

1. PCR primers used for screening clone libraries are 172f/359r and 172f/689r (359r and 689r Nubel et al., 1997 AEM 63: 3327-3332 .

2. Cultures strains of cyanobacteria from which ntcA DNA has been PCR amplified include:

• Strains collected during PICODIV sampling: Bl107, Bl17, Bl36/2, Bl119, Bl19, Bl152, Bl8.
  
• Strains collected during PROSOPE sampling: 157/13, 117, 534, 157/21.
• Strains collected during Red Sea sampling: RS9911, RS9920.
• Strains from WH and Scripps: CC9311, WH8018, WH8103.

Future publications

i) Focusing on marine cyanobacterial environmental sequences from clone libraries [but we may need to include dot blot hybridisation data together with this especially if no new novel clades are identified (from clades already known from cultures)]

ii) TEM of marine Synechococcus strains ultrastructure with Wenche & Jahn as co-authors

May 2002

• Download Year 2 report

February 2002

Clone libraries (107f - 1038r) BL 001221 and RA 000609 have been screened for cyanobacterial sequences. Clone library BL 000921 is nearly completely screened (60 out of 82 clones have been screened and all were cyanobacterial sequences). In contrast to BL 000921, clone libraries BL 001221 and RA 000609 contained only 23% and 20% cyanobacterial sequences respectively. BL 0001221 has been sequenced and RA 000609 has been sent out for sequencing. The remaining 23 clones from BL 000921 will be screened. All positive cyanobacterial clones from BL 000921will be sent for sequencing. We are assuming that the BL000921 library coincides with a Syn bloom (or at least higher numbers) compared to the other samples which contain a lot fewer cyano sequences, and that the specificity of the 1038 primer is such that when Syn numbers are low it will amplify heterotrophs.

Below is a phylogenetic tree (download as pdf file) describing the distribution of the sequenced clones from BL 001221 (KO#), BL000921 (BL#) and one clone from RA 000609 (KOra#). This analysis shows 4 representatives from the Prochlorococcus genus and many marine Synechococcus sequences with representatives falling into 6 of the seven clades. To determine whether RFLP analysis of the the clones prior to sequencing was useful for separating into different clades we performed RFLP anlaysis and sequencing of several clones. We found that RFLP pattern no. 1 is distributed throughout the tree, so clones are scattered in the tree & in different clades, although they share the same RLFP pattern. Such clones included KO11, KO15, KO33, KO40, KO43, KO51, KO64 and KO74. Clones KO26 and KO30 were from RFLP pattern no. 4 and were found clustered together in the same clade.

Also we have sequenced 16S from several non clonal Syn isolates from Blanes (provided by Laure) and these are also included in the tree. These include Blanes cultures 103,107, 108 and 59/8 (designated KO103 etc). BL107 is interesting because it represents a sequence for a clade which up to now does not contain cultured counterparts. This culture is being sent by Laure for isolation of a clonal strain in the near future.

Nick Fuller is currently repeating use of the Syn clade specific probes on Red Sea annual cycle environmental samples that have been amplified with a new primer set 172f-1313r. The 172 primer hits all Syns and Pros except the HLII Pro calde (which we know is dominant in the Red Sea). The data is currently being analysed and will be compared with previous data using 107f-1313r target amplified env. DNA.

Richard Hastings the new postdoc has just started and will finish screening the 107-1038 libraries. Also, we intend to use the 172f primer to make one or two (final!) clone libraries to see if we are 'missing' Syns at the three coastal sites.

December 2001

Karen is proceeding to screen the clone libraries we have constructed using primer set 107f - 1038r. Clone libraries BL 001221 and RA 000609 have already been screened using primers 358f - 781r A & B. The library BL001221 had 28% cyanobacterial clones (23/total 82). RA000609 contained 15% cyanobacterial clones (11/total 73). The cyanobacterial clones are being purified and will be sent out early next week. The remainder of four clone libraries will be screened and sequenced in the next few weeks. Thirty-five random clones were also sequenced from the above two libraries and 6 of these clones were found to be cyanobacterial. The BLAST results showed that of the 6 cyanobacterial clones 1 clone was 99% similar to Prochlorococcus marinus, 3 clones were 98% similar to Synechococcus WH7803, 1 clone was 99% similar to an uncultured Synechococcus from environmental samples and 1 clone was 99% similar to an unidentified cyanobacteria. These sequences are being checked and will be included in new trees and alignments shortly.

We have also received from Roscoff 4 environmental samples (010412, 010613, 001219, 010516) which will be used for DNA extraction for additional library construction. Due to the large number of heterotrophs found in the previous libraries constructed with primers 107 and 1038, the new libraries will be constructed using primers 172 and 1313 to increase the number of cyanobacterial clones. Primer 172 is another new primer that should be cyanobacterial specific - as defined by in silico alignments and database searching in ARB.

Several new Synechocooccus strains were obtained from Laure from the Blanes site and were screened using ntcA primers. These appeared to contain novel RFLP patterns so 16S sequences are currently being obtained from these non-clonal cultures [strains BL103, BL107, BL108 (from a deep sample 1800m!! in Blanes Bay ) and BL59/8] using the 172f and 1313r primer set (which should give us specifically cyanobacterial sequence - though note the 172 primer does not recognise HLII Prochlorococcus).

Nick Fuller has recently completed an annual cycle data set of Prochlorococcus and Synechococcus population dynamics for the Red Sea system. Selected data from this cycle is included as two Powerpoint attachments (download file1 and file2). Seemingly Prochlorococcus HLII genotypes dominate the water column in the Gulf of Aqaba and can be found down to depth even in stratified water columns. The LL genotypes appear to be present only from late July - October and rapidly disappear thereafter but are never found in surface waters. The late appearance of LL genotypes may be a result of the seeding population of LL genotypes which only enter the Gulf slowly in water moving north out of the Red Sea proper.

The Synechococcus population (file2) is relatively minor compared to Prochlorococcus in the Gulf of Aqaba, but we see a dominance of an ecotype, and hence genotypes, representative of the RS9910 cluster (see attached tree) and that other clades (e.g. SynWH8103, SynWH7803, SynMinos11, SynRS9901, SynRS9918) are minor components of the Synechococcus population throughout the sampling period. This is noteworthy since strains WH8103 and WH7803 represent widely used laboratory Synechococcus isolates, and at least at Station A in the Red Sea they do not appear to be abundant genotypes. Moreover, phycocyanin (PC)-rich strains represented by the SynRS9918 clade were relatively easily isolated into culture but again represented a minor component of the Synechococcus population in situ. In contrast, we have isolated several representatives of a Red Sea clade that indeed appears to be a dominant component of the Synechococcus population at this site (i.e. the RS9910 cluster). Thus far this clade has not been previously reported from any oceanic region and these strains will be an extremely valuable resource for future physiological studies, particularly since they are relatively abundant both in stratified oligotrophic waters and well-mixed water columns. Also noteworthy is the transient bloom of the RS9901/16 ecotype in April 1999, another clade not previously described. These dot blots will be repeated shortly using the 172f- 1313r primer set which should amplify the Synechococcus signal we obtained here.

We have also obtained pigment data for selected month depth profiles and are currently correlating this with TTGE profiles of 'total' photosynthetics which have been performed on monthly samples and a couple of depth profiles from the monthly sampling regime. The TTGE gels were run using primer set 359f- 781r which is specific for cyanos/chloroplasts. TTGE gels are attached (file1) . We essentially see v. good correlation of the TTGE profiles with the Syn and Pro signals - Syns are present in higher numbers in the winter months than Pros and this is backed up by TTGE data. Conversely Pros are more dominant during the rest of the year and this again is confirmed by TTGE. Interestingly there is a major dominant TTGE band which lies between Syns/Pros and Trichodesmium and well away from eukaryotes we know, so we are currently trying to identify this sequence (by construction of a clone library and running clones on TTGE gels).

Question from Daniel: The labels on the right side of the TGGE slides are for the standards or do they correspond to band you have sequenced?

Dave's answer: Sorry I should have explained this. Yes, these are standards from the respective culture or PCR product. What we intend to do is make a clone library probably of just one of the monthly Red Sea samples - run clones on TTGE gels and then sequence bands that migrate the same distance. This is to avoid cutting out bands directly which seems problematic. The only thing to bare in mind is that the sequences we will get from these clones will be relatively short and so not so good for phylogenetic analysis. We are particularly interested though to find the identity of the 'band' that migrates close to cyanos on these gels & that is relatively abundant.

So in summary we now have a v. good suite of Syn probes for use with the EU coastal natural samples. One final point is if the Roscoff and Helgoland groups have isolated any Synechococcus strains (especially PC rich ones) (frozen ideally since this is just for PCR) then we will screen these with our ntcA primer set to see if there are any sequences we are still missing.

September 2001

Dr Karen Orcutt the new postdoc started work in mid-May. Since then Karen has been screening the 107f and 1038r clone libraries already made from several sites. Single read sequencing of several clones of the BL001221 library suggest that again the primers can amplify non-cyanobacterial sequences! Thus we are currently screening these libraries with 359f & 781r primers which are specific for oxygenic phototrophs. Further sequencing will proceed of positive clones. Of the two Blanes libraries screened so far this seems more of a problem with the winter sample when there are likely less Synechococcus present.

On the Synechococcus strain side the Prosope cultures are still being made clonal by repeated plating (though for some isolates this appears difficult).

>From 16S rDNA sequences of ca 19 Red Sea Synechococcus isolates as well as other sequences obtained directly from clone libraries and cultures elsewhere several clade specific oligonucleotides have been designed. Wash curves have been performed for optimising hybridisation conditions, and will be used on environmental samples from the Red Sea in dot-blot hybridisation shortly.

Optimisation of T/DGGE conditions for analysis of photosynthetic picoplankton is also being performed. This will likely utilise primers 359f and 781r.

Karen Orcutt and Nick Fuller are currently (28th August - 1st October) in the Indian Ocean where i) seawater samples for DNA and RNA extraction are being taken along a transect as well as in depth profiles. ii) attempts to isolate novel picoeukaryotes are being performed. and iii) on-board incubations are being undertaken using 13C-NaHCO3 to assess the active photosynthetic organisms. This will be done by separation of 13C and 12C DNA by CsCl centrifugation.

June 2001

Maintenance & storage of the 107f and 1038r clone libraries already made is currently underway. Of these we are starting single read sequencing of more of these clones & including currently starting to sequence selected clones of the BL001221 library. Further sequencing will proceed.

On the Synechococcus strain side 'new' cultures (mostly from the Prosope cruise) are currently being made clonal by repeated plating, & sequencing of rDNA from these strains will complement those sequences for which we already have full length sequences. Full length sequences have now been obtained for virtually all of the Red Sea Synechococcus isolates we have obtained. These 'full length' sequences + 9 single reads from clones of the BL000921 library have been deposited in Roscoff. A phylogenetic tree showing the 7 major Syn clades that can now be distinguished from cultured strains is attached.

We have also started to ascertain if a new 23S reverse primer will be useful to obtain cyanobacterial & chloroplast specific 16S sequences in combination with the 107f primer from environmental samples. Two clone libraries have just been constructed using these primers with environmental DNA from the Red Sea (19.4.99) and off the coast of Chile. The primers themselves seem to work reasonably well with cultured Syn isolates although we have noticed there are 1 or 2 strains that do not appear to amplify with this primer set. This may suggest bias with the 23S reverse primer. This will be further investigated however.

We have recently obtained DNA samples from Klaus from the Southern Ocean Iron Ex experiment. Future work will construct 16S clone libraries from these.

A seawater sample from Blanes has been received for FISH analysis. Since Nyree left, this expertise & methodology will have to be re- optimised before we analyse this sample. (I may think about sending Nick & Karen over to Roscoff to optimise FISH). On this subject the Microbiology FISH paper is out next month - it can be cited as West et al., (2001) Microbiology 147: 1731-1744.

December 2000

Syn cultures. Thus far, Richard has been proceeding through the task of making several of the Syn Prosope cultures clonal. So he has plated single colonies twice now and will soon freeze down liquid cultures he is currently growing and they will be screened through petB-D or ntcA RFLP analysis and different RFLP types then selected for 16S sequencing. Nick Fuller in my lab has also now several potentially clonal Syn cultures obtained from the Red Sea. We are currently screening these for RFLP types and have already started 16S sequencing of these strains. Initial RFLP screening using petB-D intergenic region, ntcA and rpoC suggests these strains are 'different' to any we have from other locations. This RFLP data also provides some good preliminary evidence for horizontal gene transfer in these strains. As soon as we have sequences from these clonal cultures I will get Richard to send you an update. Unfortunately this will probably be into the New Year.

Of the non-clonal Roscoff cultures only RCC61 and RCC45 have grown on plates. These are also now being grown to increase culture volume and will be screened as above.
We have amplified 16S genes from the clonal Syn cultures received from Roscoff (RCC307, 310-313) and cloned inserts are currently awaiting sequencing by QIAGEN.

Clone libraries. The initial clone libraries (Helgoland [08/2000], DYF and MIO [PROSOPE Cruise]) developed with universal 16S rRNA PCR primers have been fully screened as follows: insert present (plasmid specific primers), insert amplifiable with CYA359F/1313R (oxygenic phototroph specific; followed by RFLP to minimise sequence types generated), and insert amplifiable with CYA359F/1038R (Synechococcus/Prochlorococcus specific). The results are presented below:

October 2000

Wkpage 1 Cultures and sample collection

• 12 Synechococcus cultures isolated during the Prosope cruise have been plated and clonal cultures are being isolated.
• Synechococcus cultures RCC30, 37, 43, 44, 47 45, 61* and 307, 310-313** isolated by F. Partensky and D. Vaulot: these cultures are also currently being made clonal * and/or clonal cultures have already been isolated**. 16S, rpoC1, petB-D intergenic region, pstS and rnpB sequences have been obtained from each strain in order to undertake a multi-locus sequencing approach to these isolates.
• Over fifty Synechococcus 'strains' have been isolated from the Red Sea. From these we have around 300 sub-cultures which are currently being made clonal and a RFLP restriction polymorphism analysis begun using ntcA and petB-D primers to determine unique RFLP patterns. These genes are being used in preference to 16S since they have a higher variability.

Wkpage 1 Sample collection

Two depth profiles for biodiversity studies were collected during the Promolec field workshop at Station A in the Red Sea during September 2000. Seawater samples were pre-filtered through 3um, collected onto 0.45 um supor filters and frozen in lysis buffer. DNA extraction and clone libraries are yet to be undertaken.

Wkpage 2 PCR methodology/ Clone libraries

Primers being used for construction of clone libraries are as follows:
i) 16S general eubacterial primers
ii) oxygenic phototroph-specific 359f and 1313r
We will use HaeIII + EcoRI as a RFLP screen for the clones

Libraries constructed thus far:
a) Helgoland August 2000 (no April sample was available to us)
b) Prosope cruise : sample sites MIO and DYF
both using general eubacterial primers.

We have picked 70 clones from each so far. Of these 27/27, 27/27 and 25/28 clones screened so far contain PCR inserts from each of the libraries mentioned above respectively. For the Helgoland library (the only one screened thus far) surprisingly 26/27 clones are amplified using the 359f and 1313r suggesting a very high proportion of chloroplast/cyano sequences in this library. These clones will then be screened with the cyanobacterial specific primer 1038, and RFLP screening undertaken. Unique RFLP patterns will be sequenced.

Wkpage 3 Probes

A list of marine Synechococcus and Prochlorococcus-specific probes have been defined (see Probe database). The Synechococcus-specific probes are currently being optimised. The Prochlorococcus-specific primers will be part of a FISH paper to be submitted to Microbiology shortly (as part of Promolec)

Workpackage 4 FISH

FISH work has been performed on Prochlorococcus populations in the Mediterranean (Prosope) and Red Sea. This work has demonstrated a dominance of HLII in the Red Sea and HLI in the former. Interestingly the distribution of HLI genotypes is confined to surface waters in the Prosope sample but HLII genotypes dominate throughout the water column in the Red Sea (in a stratified water column). LL genotypes are present in both water columns only at depth. The FISH Red Sea data will be part of the Microbiology paper mentioned above (part of Promolec).

April 2000

The post-doc for PICODIV Richard Howarth will commence work in Warwick early June.

Other work pertaining to PICODIV in the coming month:

• Dave will be actively requesting information & sorting out organisation of the PICODIV workshop.

• Several marine Synechococcus strains have been isolated form the Red Sea & are currently been made clonal by plating.

• Flow cytometry data for a yearly picophytoplankton analysis in the Red Sea will be completed towards the end of May. This data set will complement those samples taken specifically for PICODIV in Sept. 2000.

Bremerhaven

January 2003

Download full report in Word format

Workpackage 1 Cultures

From samples taken in August and September 2002, two 24 well plates were established and most showed sufficient growth to be photographed and re-inoculated. Out of 45 plates from previous sampling times, we now have 543 cultures that have shown sufficient growth to be transferred to 50-ml flasks for further growth. Of these 10 cultures have been sent to the Frauenhofer Institute in Stuttgart for investigation for novel bioactive compounds. Ten of these cultures have been prepared for pigment analysis and this will be sent to Partner x. We are starting to screen about 20 cultures a week for SSCP analysis. This involves taking a 1.5ml sample from the culture, boiling it and using 5 l of the supernatant in a PCR reaction. Those cultures producing a PCR product will be analysed by SSCP. Out aim is to have at least 30-40 new sequences from SSCP bands to identify the cultures taken at the monthly sampling sites.

Workpackage 2 Environmental Sequences

Full length sequences (publication grade) were established for sequences from the novel alveolate clade and for the novel stramenopiles. Those for the first were sent to Agnes, the latter to Ramon. Sequence analysis of clones from the novel red algal clade was completed. We received an additional sequence from Ramon. These are currently analysed phylogenetically.

Workpackage 4 Probe measurement DNA-Chips (see complete report with Figures in Word file)

The results presented in the previous report indicated that the location of a probe plays an important role if the complete 18S-DNA is hybridised to a DNA-chip. Only those probes resulted in a significant signal-intensities that were located up to a maximum distance of ~ 900 bp from the 5'-end of the 18S-gene. Therefore probes should either be located in this area of the gene or if this is not always possible, DNA should be fragmented prior to a hybridisation. In the last report it was shown that a fragmentation of the 18S-DNA results in signals that could not be observed if the complete 18S-DNA was used. One major task of the past three months was to develop a protocol that allows a cheap a reproducible fragmentation of the 18S-DNA. In addition to that new probes were added to the previously used set of probes on the chip and tested in hybridisation-experiments. The hybridisations of the past three months have been carried out on DNA-chips that had been spotted by companies.

Worpackage 5 Sampling

We continued with monthly sampling until December 2003, meaning that we now have two full years of monthly samples. Preliminary experiments (e.g. see our presentation this summer in Roscoff) indicated that although diversity seen in SSCP analyses from month to month may change significantly, less differences can be observed when comparing the same month from two different years. We will now analyse SSCP profiles for the two year sampling period.
From the monthly sampling period flow samples and TSA FISH filters were sent to Roscoff, Pigment filters and DNA filters were sent to Barcelona.

October 2002

Workpackage 1 Cultures

Rough cultures were established every two months from April 2000 to May 2002. A total of 20 cultures have been deposited with Roscoff. There are now 424 cultures with visible growth and all of these can be processed now for SSCP. A total of 106 cultures have already been screened with SSCP and identified where unialgal. 104 Cultures have sufficient growth that they can be screened with flow cytometry, of these 20 have been screened. Ten cultures have had their pigments extracted for HPLC characterisation.

For future culture analysis an agreement is about to be signed with the Fraunhofer Institute in Stuttgart (FIS). The FIS will support us with the establishment of cultures and will screen cultures for economically interesting substances.

Workpackage 2 Environmental clones

We have begun to establish full-length sequences for selected clones:

• 5 sequences for the “novel red algal clade”
  
• 2 sequences for novel stramenopiles (sent to Ramon)
  
• 2 sequences for alveolates (sent to Agnes)
  

Workpackage 3 Probe development

We have finished all of our probe development but still outstanding are more FISH tests with the Hetero01 probe. This will take place in November when additional staff will be trained with this probe.

Workpackage 4 Probe measurements (DNA chips)

Most of our efforts in this reporting period were concentrated on this workpackage. Download report in pdf format.

May 2002

• Download Year 2 report

Culture from AWI screened by SSCP and partial sequencing

We have begun an extensive screening of picoplankton cultures using SSCP fingerprinting and subsequent partial sequencing (ca. 500 bp starting with the 528F primer) of the 18S rDNA. This method allows to identifiesy cultures lacking obvious morphological characters. It also works with very little material so that cultures can be checked that grow poorly. About 30 cultures can be checked simultaneously on a regular gel. Yet approx. 10-20% of cultures tested produced in the end “good” sequences and were identifiable:

Table . Best taxonomic affinity of cultures based on % similarity of the obtained sequence to known sequences

 Probes

Prymnesiophyte new class level probe Prym03. This probe has been tested through FISH showing good signals with all target cells but a few target species not showing a reaction. We are unsure of the reason for this lack of signal. Signals with picoplankton cells, e.g. E. huxleyi are very faint and will likely require TSA enhancement.

Prymnesiophyte new order level probe Cocco01. These probes, which require a competitor have been tested through FISH without a competitor. Some minor cross reactivity with non-target species has been noted and thus further tests will include a competitor. Signals with picoplankton cells, e.g. E. huxleyi are very faint and will likely require TSA enhancement.

OLI probes (AWI) for clades OLIGr01 and OLIGr04 have been tested through dot blots and are specific. The other three probes for clades (OLIGr01, OLIGr02F, and OLIGr03) can only be tested with negative controls because Roscoff discarded the clones from their clone library for these unknown novel clades of Haptophyte algae. Our strategy is to use these probes blindly on filters to see if we can recover the morphology of the cells of these novel clades detected through clone libraries.

Cryptophyte probe Crypto01-07. Testing of these probes will begin in June 2002 with a a new student and these probes are also the primary focus of a new EU project Micropad QLRT 2001-0193, which will begin in October 2002 for continuous testing of the Helgoland time series using these probes adapted to DNA microchips

February 2002

Please dowbload Linda's report as a pdf file.  You will find update on:

• SSCP for cultures and natural samples
• DNA chips. Data can be downloaded as Excel file.
• The novel red algal group investigated by Klaus

Decembre 2001

CLONE LIBRARIES AND CULTURES

Sampling -Two samples were taken at Helgoland and processed as usual.

Sequencing. Three (out of 6) of the putative red algal clones were sequenced in house, the others are still under work. I attempted to clone 18S PCR products from 9 unicellular red algae, including Cyanidium caldarium and Galdieria sulfuraria. Due to a bad cloning kit this was successful only for 5 species, NOT including the latter ones. The cloning experiments will be repeated this or next week. The clones already obtained will be sent to Quiagen for sequencing.

Cultures. More efforts were made to obtain cultures. From precultures started until April 2001 a total of about 550 are now established in 50 ml cultures. Of these 225 have grown visibly and will be analysed by flow during the next weeks. We continue with new 50 ml cultures from precultures started between May and August 2001, i.e. a total of 744 and we awaiting signs of visible growth in the cultures before proceeding to the next step.

 PROBE DEVELOPMENT

Hetero01 tests positive with all heterokonts so far tested in dot blot tests. Negative with haptophytes and dinoflagellates so far tested. Our DNA supply for various heterokonts is rather limited and we are awaiting a broader spectrum of heterokont DNA to be sent to us from Bob Andersen. The probe requires the use of a competitor but so far the tests at the dot blot level have shown that the competitor is not necessary. In-situ tests will begin shortly.

Prym03, the new probe only for the class Prymnesiophyceae to the exclusion of the Pavlovaceae, has also been tested in dot blots and shows positive within the class and negative with other classes so far tested. We need to test it against the Pavlovaceae, which hasn't been done to date.

Cocco 01-, the probe for all of the coccolithophorid algae also shows strong positive tests with all coccolithophorids tested so far in the dot blot analyses. This probe has been tested against a very broad spectrum of haptophytes and shows a very high specificity.

Testing for the OLI probes will begin shortly as we have now received clone material from the Roscoff lab.

Two genus level probes for Prymensium, PryMGl01A and PrymGlo2B, have been tested at both dot blot and in situ levels, with one probe giving much brighter signals than the other in-situ (Fig. 1).

 DNA chips

The aim of an application of microchip-technology in the PICODIV-project is to develop a tool to facilitate the monitoring of picophytoplankton in water samples. Currently we are in the process to optimize hybridization conditions of class level probes. In a first experiment there have been tested probes for the detection of diatoms, bolidophyceae, dinophyceae, prymnesiophyceae, pelagophyceae and chlorophycea. Therefore chips have been spotted with the probes CHLO 01, CHLO 02, PRYM 01, PRYM 02, PRYM 03, DINO B, DINO E-12, BOLI 01, BOLI 02 and PELA 01. Additionally the probe EUK 1209 has been spotted to detect all eukaryotes. The chips containing the probes mentioned have been hybridized in a first experiment to 18S PCR-products of a diatom, two dinophyceaes and a bolidophyceae, which were isolated so far in the PICODIV-project by Dr. Klaus Valentin. Under the chosen hybridization conditions it appeared that there were specific signals for all tested PCR-products. Unfortunately there have also been some unspecific signals, which have to be eliminated by changing the hybridization conditions. To eliminate nonspecific binding more stringent have been chosen for the next attempt. Under those conditions probe DINO E-12 bound to all the 18S PCR-products amplified from the PICODIV-clones, whereas none of the other probes gave a hybidization-signal. As the clones are not completely sequenced yet it is difficult to interpret these results correctly. To make the interpretation of the hybridization-results easier, it was decided to use fully sequenced 18S-PCR-products for the optimization of the hybridization-conditions. Therefore in further experiments first 18S-PCR products from the prymnesiophyceae Prymnesium patelliferum and from the dinophyceae Alexandrium tamarense have been used for the hybridization of the DNA-Chips. In these experiments Alexandrium tamarense got bound specifically by DINOE-12, whereas probe DINO B did not give a signal. Prymnesium patelliferum did not bind to the prymnesiophyceae-specific probes PRYM 01, PRYM 02 and PRYM 03 (Fig. 2). In the first panel of Figure 2 all of the positive controls have worked but the specific probe has not. In the second panel of Figure 2, the specific probe has worked and the positive controls were absent. The eukaryotic probe EUK 1209 did not bind to one of the tested PCR-products. A possible explanation for this result is, that due to target secondary-structures the probes do not have access to the binding side. In order to facilitate binding of the probes EUK 1209, PRYM 01, PRYM 02, PRYM 03 and DINO B helper-oligonucleotides have been designed. The helper-oligonucleotide bind close to the binding-sides of the probes and by doing this they are supposed to decrease inhibitory effects of secondary-structures of the target DNA in the hybridization process. Currently the effect of the helper oligonucleotides is under testing.

The first hybridization experiments indicate that there is quite a good chance for the development of a DNA-microchip for the detection of pico-phytoplankton. It could be shown that it is possible to hybridize 18S-PCR-products to microchips, even though details of the hybridization conditions remain to be optimized.

October 2001

Clone libraries

As discussed in Barcelone no new clone libraries for the helgoland site were established because one full year is allready covered. Attempts were made to establish clone libraries for the baltic sea and the "Bodden" but DNA was not amplifiable, probably due to heavy phenol contaminantions. Three clone libraries were established for the IronEx cruise conducted by the AWI last winter. The samples were from the iron patch before iron fertilization (1.11.00), after two weeks of fertilization (St. 107), and a paralell sample from outside the patch (St. 108). For each library 32 clones were sequenced with 528F. Sample 1.11.00 contains essentially only ciliate sequences, St. 107 and St .108 show a very high degree of diversity. We will sequence at least additional 50 clones from the latter libraries. Full length sequencing will begin for the new isolates near the red algal class.

Sampling

Routine sampling for the Helgoland site was carried out as planned. Samples were send to Wenche for EM. DNA was isolated an analysed by SSCP (see below). For each sample 96 precultures were started.

 Cultures

We continued with the routine analysis of precultures started throughout the months before. All precultures are screened with LM and those that show growth are transferred to 50 ml flasks. Many of these continue to grow and are further checked by flow cytometry. Some look uniform in that they produce a distinct cloud in this analysis. We are now in the process of checking these clones by SSCP (see below).

 SSCP analysis

We have now established the SSCP method for fingerprinting and culture testing. DNA samples from 2001 from the helgoland site were analysed with this method and it turned out that there is a high degree of diversity present in the eucaryotic picoplankton. Only few band are detectable in more than one sample. For the ironex samples the bias of the entire cloning procedure was tested by running SSCP profiles from (i) the total DNAs, (ii) from the full-length 18S rRNA products, and (iii) from the pooled clone libraries. The profiles look more or less the same indicating that there is no obvious bias in any of the steps. We have established a simple procedure to do SSCP profiles from crude cultures. From several cultures such profiles were done. It turned out that those cultures that look uniformly in the flow analysis usually produce only one band in the SSCP analysis whereas those that contain more than one population in flow analyses produce multiple band in SSCP. In the future we will routinely analyse our cultures by SSCP. Since we use the 528F primer for SSCP we will be able to directly sequence the SSCP product(s) and compare them to our clone libraries.

Miceallenous

We have established cultures from several unicellulare red algae received from the culture collection in Göttingen, Germany, and for some species I brought from Gießen. From half of the cultures we successfully isolated total DNA and from a total of 5 cultures we yet were able to amplify the 18S. These will now be sequenced to more firmly establish the "new red algal class" seen in the phylogentic analyses.

Progress with the Heterokonta probe Heter01

The probe for Heterkonta has been tested in dot blot hybridizations without the addition of the competitor. The dot blot resulted in predicted signals at 55°C hybridization temperature and a salt concentration of 2xSSC in the washing steps. The probe was tested in the dot blots with the following organisms:

As targets: Amphora proteus, Navicula diplomeis, Thalassiosira fluviatilus, Amphora capitella, and Fragellariopsis sublineata.

As non target organisms: Alexandrium tamarense 31/4 (Western Europe), A. tamarense GTP01 (America), A. ostenfeldii PS16, A. taylori (P901), A. minutum L20/2, A. fundyense GT-7, A. catenella (P807), Prymnesium parvum, P. nemamethecum, P. patelliferum.

We will raise the stringency to 1x SSC in the wash, which removes non-target signals that appear after 2 hours exposure. We need more target material within the other groups besides the diatoms and more non/target groups to test. We will ask our partners to provide the needed DNA. The probe will be tested with amplified rDNA in FISH with and without the use of the competitor, which at present is not needed for the dot blots as far as we have tested.

Current status of the PicoDiv-Chip

The goal of using DNA-microarray-technology in the PicoDiv-Project is to develop a technique which offers the possibility to identify the organisms in a taken sample by just one experiment.

Such a DNA-microchip would contain probes which are specific for organisms in the pico-phytoplankton. The microchip would be hybridized to a labled population of PCR-products from the 18S-RNA amplified from a sample. The hybridizationsignals would give informations about the organisms present in the sample.

In the course of the development of such a DNA-microchip it has to be investigated in a first experiment whether the probes which are present in the lab and tested by insitu-hybrididaztion, are suited to be used in a microarray-experiment. A set of 12 probes has been selected to be tested. Those probes are „Higher Group Probes" and „Class Level Probes". First it is planed, to hybridize the probes on a DNA-micro-chip to PCR-products from different clones isolated in the PicoDiv-project. This experiment is supposed to answer the question wether the probes bind specifically to the PCR-product from a single clone. If the experiment shows that the probes are suited for a hybridization on a microchip, the next step would be to reproduce the results concerning organisms found in the PicoDiv-samples by the cloning-stratagie.

Currently the project is in the phase that the DNA-micro-chips are to be produced very soon. Therefore results from the first set of hybridization-experiments are expected to be available in the next few months. The first spotting of the probes began on 1 Oct 2001 and the first hybridisations will be performed using fluroescently labelled PCR products from known cultures bodn to class level probes.

A similar set of tests will be made with these probes using a dot blot format on sampels already taken in 2000.

We have contacted several companies and have made a short list of the instruments we wish to use. We are engaging in a rental with option to have a lease/purchase contract with these companies for the DNA chip-reader. We have been given permission to use the spotter at the University of Bremen to spot the probes. 

June 2001

Samples: Full samples were taken and processed in May (He010516) and June (He010619) at the Helgoland site. Wenche received samples which this time were filtered at 1 x g, seemingly resulting in good quality of the cells. Also we received filters from the Bodden and Baltic sea sampling conducted by Ulf Carten’s group in Rostock.

Precultures/cultures: For the May and June samples, respectively, 96 precultures were started. Since the Barcelona Meeting we dedicated two days per week to culturing and monitoring of cultures. Several hundred precultures were checked and from about half of them 50 ml cultures were started. Many of these started growing to visible densities.

Clone libraries: Clone libraries were established and screened by minipreps for the three IronEx samples. 32 clones from each library were sent for sequencing. It seems as if the sample taken before the release of the iron looks pretty uniform whereas the others are highly diverse. For these, additional clones will have to be sequenced. IronEx DNA was also sent to Dave for prokaryote analyses.

Sequences: We received and analyzed the sequence data for the final Helgoland sample, i.e. He010218. The sample was highly redundant, i.e. only about 30 different sequences were found among 170 analyzed clones. Most of the sequences group with the prasinophytes. Most interesting is a cluster showing only 96% identity to Micromonas species. (I will sent unique sequences to Daniel shortly). Today we received 90 sequences for the IronEx samples.

Plans: Work will slow down a bit in July because of the holiday season. We will make sure, however, that sampling continues on a monthly basis as planned. In August the Bodden/Baltic sea samples will be processed. We will continue with the analysis of cultures and will put some efforts in paper writing (one year succession in Helgoland, IronEx data, “strangophiles” among our sequences.) I suggest that we discuss the issue of papers describing the new classes we found on the occasion of my excursion to Menfig from Sept. 14-22.

January 2001

We have now sequences form the October library and the December
library is coming well. For fingerprints and the PICODIV monitoring
throughtout the second year (which for this purpose I will start now) I
took a sample at Helgoland on Jan 17 and will continue on a monthly
schedule. I tried to do SSCP with DNA samples and cloned 18S fragments
and it looks promising. Some bands in the DNA samples and single bands
from the clones. I now have to establish the method here.
I am still in the process of analysing and combining all the sequence
data collected so far: I have now a full set of 528f sequences for 3
libararies (he000427, he000803, Orkney 0004) and partial data for 2 more
(He0003 and He 001005). In order to eliminate redundant clones and
obvious contaminations (i.e. sea urchins, molluscs, crustacea...) I will

December 2000

See Klaus analysis of the clone library in the new page devoted to this topic.

I am still in the process of analysing and combining all the sequence
data collected so far: I have now a full set of 528f sequences for 3
libararies (he000427, he000803, Orkney 0004) and partial data for 2 more
(He0003 and He 001005). In order to eliminate redundant clones and
obvious contaminations (i.e. sea urchins, molluscs, crustacea...) I will
align all the datasets and sent you the output.

It looks as if we will get samples from the last Polarstern cruise, i.e.
the iron experiment. A student took for me samples from inside the iron
patch before and after adding the iron, and from a control patch. I am
looking foreward very much for the analysis of these.

He000427: Clone library established and sequenced, 96 precultures started, some precultures transferred to fresh media and sent to Bente/Wenche
He000803: Clone library established and sequenced, 96 precultures started, some precultures transferred to fresh media, nothing sent yet to Bente/Wenche because cultures are not grown densely enough; DNA sample sent to Richard Howarth for 16S analyses.
He001005: Clone library established and partly sequenced, 96 precultures started, DNA sample sent to Richard Howard.
He001206: DNA isolated, clone library to be established, 144 precultures started
Orkney0004: Clone library established and sequenced, no cultures

October 2000

Clone libraries. I (Klaus) now have sequence data for two clone libraries, i.e. Helgoland March 2000 and April 2000. These have not been completely analysed, i.e. not all redundant clones have been removed from the tree. As soon as this is done all the sequences will be sent to Roscoff database.
The DNA filter from the Celtic sea somehow did not make it from the ship to my desk.
Other clone libraries are well under way:
- A clone library was established for the Orkney cruise
- another from a Sample taken at Helgoland on August 3.
- For both a substantial number of clones was yet analysed by minipreps and both will soon (this month or in november) be sent for sequencing.
- A clone library for a sample taken at Helgoland on Oktober 10 will be established this month.
- The next samples will be taken in december at minimum light conditions and in Jan/Feb for minimum
temperature conditions.
- I also sent a DNA sample from the Helgoland sample ("He 000803") to Daves Lab.
- Hopefully I will receive a few samples from the next antarctic cruise by our vessel (Polarstern) this
winter.

Cultures. I routinely prepare 8 rows of 6 serial 1:10 dilutions for two media (IMR and Drebes) = 96 precultures per sample = 192 for two samples (He 000427 and He000803). In my experience 10-20 % show significant growth after 3-4 weeks. These are then transferred to 50 ml flasks. Klaus sent Bente several cultures from the He 000427 sample that are now being analysed. From the He000803 sample Klaus prepared several cultures that are now grown enough to be sent to Bente.

Sequencing. I contacted Qiagen and was told that nobody except me yet sent any clones for sequencing!!! Did the others not prepare any clone libraries?
I also contacted another company (AGOWA) for sequencing services and got a better offer, i.e. 16.- DM for a 500 bp run (excluding miniprep) versus 25.- DM per 500 bp run (including miniprep). I will send them my next clone library.
Seqlab (Bio-Service@SEQLAB.de; Firma: Sequence Laboratories Göttingen GmbH http://www.seqlab.de) contacted me and I told them that we are still interested in their services for full length sequening of the inserts. They feel confident to do a publication-ready analysis of a full 18S for ca. 138.- DM. As soon as I have decided which clones are to be fully sequenced I'll let them do a set of 20 - 50 sequences. (Qiagen asked for 350.- DM, Agowa for 280.- DM for this service)

July 2000

We got something growing in the samples taken in April. A crude mixture of course. I will prepare dilutions this week.
Of the DNA sample taken in april I now did 170 minipreps resulting in about 80 possibly unique clones. These will be sequenced next week.
I will receive a sample from the celtic sea that i can process in august
The next sample in helgoland will be taken at the end of july.
I will get a few samples from a antarctica trip conducted by Uli Bathmann from the AWI.

June 2000

From the sample taken in march at Helgoland I prepared approx. 500 - 1000 18S clones. Of these I minipreped 47, resulting in 45 inserts of the expected size. The minipreps were then digested with a cocktail of 7 different enzymes (each working in the same buffer, and cutting the vector only in the polylinker). From the resulting pattern I estimated that at least 22 clones contained different inserts. Then 48 clones each were sent to two different companies for sequencing. 83 sequences were obtained. (In comparison Quiagen provided better results.) All were BLAST searched at Genebank (RDP did not work properly then). All were aligned and a tree was built. The following results were obtained:
- of the 83 sequences 41 were distinguishable
- 42 sequences, among these 5 distiguishable clusters, were most similar to Dinoflagellates
- 18 sequences (12 clusters) represented different algal groups: bolido-, Bacillario-, Chryso-, Crypto-, and mainly Prasinophytes/phyceae (8 sequences)
- The remaining sequences were grouped in 5 clusters, all showing highest similarities to ciliates.
That is, 50 out of 83 sequences potentially belong to photosynthetic organisms.

Because of the observed diversity I decided to have at least 48 additional clones sequenced. Perhaps we should dicuss in Roscoff which ones should be sequenced completely for tree building.
The sample taken in April at Helgoland (spring bloom) was also further processed:
DNA was isolated, PCR reactions carried out, checked for quality and PCR products were cloned. I obtained >500 clones that are currently being analysed.
I also received two samples from the may cruise to the Orkneys. In both samples DNA was heavily degraded. Therefore I got PCR products only from one sample. Interestingly two major bands were present. I will clone these products this week.

April 2000

Sampling.  We took one sample at Helgoland at the end of March. This sample was used to establish a good DNA isolation procedure and will be used to generate a first set of sequences (see below). Another sample will be taken today (27/4/00) and/or next week. That is because spring bloom has just started and will probably go on for a couple of weeks. We will obtain additional samples from the Orkney Islands (next week) and from the Celtic Sea (end of May) on the occasion of cruises carried out by other projects.
In the future we will be informed by the Biologische Anstalt Helgoland about significant situations concerning sampling along with their routine daily monitoring program. We plan for at least 5 sampling times/year (spring bloom, early summer, summer, fall, winter LL [low light], and winter LT [low temperature]) or at times of biological relevance as judged by the Helgoland monitoring program. These times are known to represent distinct communities as judged by the time series data at Helgoland.

Cultures. The sample to be taken today and/or next week will be used to establish a first set of cultures using different media, temperatures, and light intensities with the help of Bente Edvardsen who happens to be in Bremerhaven for the next 3 weeks.

DNA isolation. The sample taken at the end of March was used to test different DNA isolation protocols for their yield and DNA quality. These included CTAB, Phenol, PAN kit III, PAN plant spin columms and two protocols of the Macherey Nagel Nucleo spin bond kit . Yield was judged by agarose gel electrophoreses, quality by Eco RI digestion and PCR efficiency. Of all the methods tested CTAB and PAN plant spin kit methods turned out to give best results. For the time being we decided to use the PAN kit in order to have a highly reproducable method that easily can be adopted by others. (For those who are interested: test kits and informations are available under pan@vilstal.net.pan-biotech.de or by phone +49 (0)8543-3636.
The sampling and DNA isolation protocol now established looks like the following:

• Seawater (at least 0.5 L, up to 5 L) is filtered through a 3 µm filter. The filtrate is either used for culturing (see above) or subsequently filtered on a 0.22 µm (diam. 45 mm) filter at low pressure (200 - 400 mbar). In our experience one such a filter has a capacity of approx. 0.5 L prefiltered seawater. Filters are air-dried on 3MM paper or paper towels, soaked with a small volume of 50 mM Tris; 100 mM EDTA,; pH 7.5, and shock-frozen in liquid nitrogen. They can then be stored at -20°C.
• For DNA isolation filters are transferred into 1.5 ml eppendorff tubes prefilled with lysis buffer from the PAN Plant Spin Kit. and then processed according to the manufacturors recommendations with the following modifications: the initial 65°C incubation is extended from 30 to 60 min and the DNA is eluted twice with 100 µl elution buffer instead of only once. One prep takes about 2 h.
• Typically we obtained 500 - 1000 ng good DNA from 500 ml of seawater. As low as 10 ng of this DNA are enough to produce a visible amount of 18s rRNA PCR product under stringent conditions.

PCR. Few 18s rRNA PCR reactions using 1F and 1528R primers were yet carried out. At the moment we are in the process of optimizing the conditions. This includes DNA amounts, annealing temperatures, cycle numbers etc. As a measure for the desired result, i.e. maximum variabilty in PCR products under as stringent as possible annealing conditions, we use as markers (1) size, (2) different patterns in ?HA Yellow? and ?HA Red? gels, and (3) digestion patterns of triple digests with Hae III, Hinf I and Rsa I. We got good results at stringent, i.e. 53°C, annealing temperature. Annealing at as low as 45°C resulted in multiple bands of the wrong size. HA Yellow proved useful in that it demonstrated at least 8 different bands within a single PCR band. The triple digest produces a large number of bands that can make different PCR bands of the same size distinguishable.
Probably by the end of May we will have established initial clone libraries of 18s rRNA PCR products. Minipreps of these clones will be checked for the presence of different inserts, by using triple digests and HA Yellow/Red gels.

Sequencing.  For May and June, we plan to sequence the first sets of cloned PCR products. We are currently negotiating with different companies the possibility of having these clones sequenced comercially. It is already obvious that this will be possible at costs comparable or even lower than in-house sequencing. The best offer to date is 9 Ecu per clone and 300 bases given that we provide the company with an agar plate with 96 colonies on it. Inserts are amplified from these colonies and sequenced directly by the company. A second company offers to do minipreps and sequencing in a 96 well scale for maybe 10-15 Ecu per clone and for up to 650 bp. A complete SSU sequence can be done for approx. 70 Ecu.
Until June, i.e. in time for the meeting in Roscoff, we hopefully will have both companies do at least one 96 well sequencing of the samples taken at Helgoland.

Barcelona

January 2003

Download full report from Mikel with all Figures
Download full report from Ramon with all Figures

Wpk 1: Pigment analysis of Cultures (Mikel)

• Florenciella ultra: Pigment identification has been provided for a description of strain RCC446, a possible Dictyophyceae with 19but-fucoxanthin and fucoxanthin and without 19hex-fucoxanthin, a pigment composition typical of Pelagophyceae. Additional Dictyophyceae cultures from Roscoff RCC 355, 389, 392 and 394 were similar in pigment composition to RCC446. However, RCC382, another Dictyophyceae, presented the pigment suite of a diatom, with fucoxanthin as the main and only marker carotenoid. (Manuscript led by Wenche Eikrem)
• Prasinophyceae: Thirty strains of Prasinophyceae have been analyzed for pigment determination. The results have been used to cluster the strains according to their pigment composition. The pigment cluster respected the taxonomic classification. (Manuscript led by Mikel Latasa)
  

Wkp 2: Stramenopile sequence analysis (Ramon)

We have obtained 10 complete sequences of novel stramenopiles clones from Blanes Bay. We have also received 8 complete sequences from the Roscoff Coast, 2 from Helgoland and 2 more from Orkney cruise. These sequences were chosen to be representative of the overall diversity of novel stramenopiles in the PICODIV coastal sites. A tentative phylogenetic tree (using Neighbor Joining) relates these sequences with the basal branches of novel stramenopiles and confirms and expands the results of a huge diversity within this group. Soon, we will perform a database search to include in the analysis all sequences of basal heterotrophic organisms to obtain a better phylogeny. We are exploring a new way to align sequences that is totally automatic. It is based in the ClustalW program, together with the software Gblocks (Castresana 2000). Besides of being fully automatic, this approach has the advantage of being very flexible in the incorporation of new sequences.

Wpk 5: Pigment analysis of time series (Mikel)

All the samples from the time series of Roscoff and Blanes have been analyzed. The Roscoff series comprised 68 data points from 01 March 2000 to 14 November 2002 in bi-weekly intervals. The Blanes series comprised 21 data points from 20 March 2001 to 27 November 2002 in monthly intervals. Three samples, > 3 um, < 3 um and total (GF/F filters), per data point were sampled for HPLC pigment analysis. Samples were analyzed additionally fluorometrically.

Roscoff time series

The size fractionation was carried out successfully in technical terms. The sum of the two fractions showed a very good agreement with the total collected independently with a relationship almost identical to 1:1.

The Chlorophyll a distribution indicated a clear increase in the abundance of all algal sizes during the summer period. This increase was more pronounced for the large fraction. The median chlorophyll concentration was very similar for the large and the small fractions (306 and 268 ng L-1, respectively) indicating that the large differences observed in the graph below are due to episodic events.

These episodic events, however, constituted an important feature in the dynamics of these populations. In this sense, the coefficient of variation was 128% for the large fraction and only 58% for the small one. The ranges of the chlorophyll concentration were 123 to 4543 ng L-1 and 65 to 906 ng L-1, which indicate 37-fold and 14-fold differences for the large and small fractions, respectively.

The pigment data set from Roscoff has been subject to further analysis with CHEMTAX, a program to estimate the contribution of major algal groups to chlorophyll a biomass. This analysis showed the presence of 8 major algal groups: Dinoflagellates, Pelagophytes, Diatoms, Prymnesiophytes, Cryptophytes, Cyanobacteria and two pigment types of Prasinophytes.

CHEMTAX output revealed the dominance of Diatoms followed by Prasinophytes and Cryptophytes. This distribution had a different image in the two size fractions. In the large fraction, diatoms made up more than 80% of the pigment biomass (average) with small contributions from the rest of the groups. For the small fraction, however, the dominant group in terms of pigment biomass was the Prasinophytes (almost 50%) followed by diatoms and cryptophytes. Haptophytes, Pelagophytes and Dinoflagellates had minor contributions in Roscoff phytoplankton populations. Cyanobacteria constituted an insignificant fraction of the phytoplankton.

October 2002

Arctic cruise (Aug-Sept 2002)

• Download report by Carlos

Analysis of novel stramenopiles lineages

We are investigating the extent of phylogenetic diversity (and biogeographical distribution) of novel stramenopiles. The idea is to add to the phylogenetic tree presented in Massana et al. (2002) the sequences obtained during the coastal sampling in PICODIV and recently published sequences. We have currently obtained a phylogenetic tree using around 250 partial sequences. This tree suggests the existence of at least 12 independent lineages. Two of these can be related to groups for which there are known organisms (labyrinthulids and oomycetes), whereas the rest are novel groups. In order to confirm the phylogenetic clades observed using partial sequences, we have decided to sequence completely representative clones: 11 from Blanes, 9 from Roscoff, 2 from Helgoland and 1 from Orkney. When we have all these complete sequences we will perform detailed phylogenetic reconstructions, design new probes for the most represented groups, and apply these probes to environmental samples (both from the coastal sites and cruises).

May 2002

• Download Year 2 report

Pigment analysis by Mikel

Thirteen cultures of the class Prasinophyceae isolated from Roscoff and Blanes have been analyzed for pigment composition. The species were classified according to their pigment (normalized to chlorophyll a) characteristics (open figure as PowerPoint file). Two clear groups could be distinguished: one with chlorophycea-like pigment composition; and another one with more typical Prasinophyceae-like composition.

Within this general classification, there were some striking features in some particular cultures. The most important exceptions in terms of pigment composition were RCC136, which had prasinoxanthin and lutein (and not zeaxanthin) as main accesory carotenoids; and BL7817 with two quantitatively important peaks with the absorption spectra of prasinoxanthin. The chemical structure of these two “prasinoxanthin-like” compounds, however, must be very different from that of the pure prasinoxanthin because their retention time was very different. In addition, several cultures presented “vaucheriaxanthin-like” peaks (vaucheriaxantin has been only described before in Esutigmatophyceae and Xantophyceae), but also with different retention times from that of the typically found vaucheriaxanthin ester.

We will proceed to select and reanalyze some of the most special cultures and check for contaminants and physiological status to confirm these preliminary results.

February 2002

1. Cultures. We have now 154 cultures of picoeukaryotes, isolated from six different samples, four from the Blanes coastal site, one from the Barcelona harbour and one in the open Mediterranean Sea offshore Blanes (collected during the Canyon cruise). The phylogenetic affiliation of most cultures has been identified by RFLP and sequencing analysis (see Figure 1). We are currently focusing more effort in isolating heterotrophic picoeukaryotes, aiming to retrieve cultured representatives of the novel phylogenetic lineages that appear abundant and widespread in genetic libraries.

2. Genetic libraries. We have completed the fourth genetic library from the Blanes site (sampling on BL_010320). The sequences and the BLAST results will be soon send to Daniel. This fulfils our commitment to obtain four genetic libraries from different times of the year in the Blanes site.

Before Christmas I send a message sharing a problem that we found in our sequencing analyses. In a significant number of 18S rRNA clones, there was a region (usually positions 650-750) that was not recognized by the BLAST during the first search. When I submitted only these 100 bp, the BLAST search did not find any 18S rRNA sequence similar. I knew that corresponded to a very variable region, but I was surprised that there was any sequence remotely similar and I wondered if this could have been caused by a sort of PCR artefact. Now I am convinced that this is indeed a very variable region.

3. Coastal sampling. It is proceeding as scheduled. The collection of inorganic nutrients, samples for cell counts, phytoplankton pigments and environmental DNA was continued in the framework of the time series of monthly samples off Blanes.

4. Microscopic counts. Analyses of nano- and microphytoplankton samples (cells bigger than 3 and 20 microns, respectively) was started. The first results reveal a high diversity of nano- and microphytoplankton as well as a high variability of the assemblages from month to month. The comparison to the picophytoplankton (<3 um) will reveal possible relations regarding the temporal changes of the respective assemblages.

5. Novel Stramenopile probes. We developed probes against two clades of novel stramenopiles that have been very successful to identify these phylotypes through FISH in natural and enrichment samples (see Figure 2). We have a manuscript submitted with this data. The plans for the future are to develop probes against the other novel stramenopile clades (at least 8 different clades detected) and the application of these probes to different sets of marine samples.

December 2001

1. Cultures. See list of culture from Laure.

2. HPLC (from Mikel). We keep up with the annual cycle in Blanes and with Laure's cultures of Prasinophyceae, but just sampling, not a single analysis since we moved to the new building (as I was afraid of). Here everything is still quite slow, today we finally had our Helium on and we can start finishing the setting up of the HPLC. In the meantime I've been playing with CHEMTAX with some Med samples and I hope this experience will be useful to run CHEMTAX on the PICODIV samples.

3. Libraries. We have completed the third library. The fourth one has been a problem. Now we have done it again and things are going better. We hope to get the 100 clones (or close to it) probably before Christmas.

4. Coastal sampling. It is proceeding according to schedule (you can have a look at some of the data in Fig 3 of the paper).

5. New Stramenopile probes. We have probes for three clusters. They work beautifully on filters. A paper on their application to coastal samples has just been submitted. The third one targets a cluster that apparently is not present in the Blanes waters and that is why we have not included it in the paper. We will try to get probes for the remaining clusters in the next few months.

September 2001

Cultures (Laure)

Ramon
The analysis of the first two genetic libraries from the Blanes site (samplings BL000921 and BL001221) has been completed. A text file with the sequences has been sended to Daniel, together with an Excel file with the results from a BLAST analysis of each new sequence. We are currently doing the last two genetic libraries, from samplings BL010320 and BL010625. After some problems in the PCR step, we got very good products after purifying the DNA extract. Next week we will clone these products.

During the month of July we moved to the new building and we expended the first weeks of August in seting-up the new lab. We are still working of fixing some techniques that have to get used to the new environment. That has implied some delay on the lab work schedule. Nevertheless, the monthly sampling in Blanes has been carried out with minor problems.

We have just started to play with FISH for picoeukaryotes. We have prepared filters from seawater and from a set of heterotrophic and phototrophic cultures. We will try first the Bremen protocol, that includes a formaldehyde fixation and the use of 5’-CY3 labeled oligonucleotide probes. We will start with an eukaryotic probe to assess the chracteristic of the positive signal.

Pigment analysis (Mikel)
1.- Samples for HPLC have been taken monthly from the Blanes site (analyses have not been performed yet).
2.- Analyses of samples sent from the Rosoff site have been analyzed. The results have been checked and sent back to Rocoff for public availability

June 2001

Blanes cultures (Laure)
Serial dilutions have been made on 21 cultures (6, 8/1, 34, 36, 37, 38, 43, 44, 48, 57, 59, 70, 74, 78, 82, 105, 107, 120, 123, 131, and 132). Extraction, clone library, RFLP patterns and sequencing is now processing on 14 probably pure cultures (Flagellates: 2, 20/5, 44/4, 49/5, 73, 83, and 146. Coccoides: 39, 33/3, 76, 77, 122, 130, and 147).
This month we process the last isolation from natural samples.

Clone libraries (Ramon)
We have almost completely analyzed two genetic libraries from the Blanes coastal site. The first library on 21 September 2000 (BL000921) comprises 78 clones and 44 RFLP patterns. Two of these resulted to be chimeras. Of the remaining 42 RFLP patterns we have sequenced 34. As soon we have all the data it will be sent to the web page. The most abundant clones were group-I novel alveolates, novel stramenopiles and dinoflagellates. Many other phylogenetic groups were also present.
The second library on 21 December 2000 (BL001221) is completely sequenced and the data has been sent to Daniel to be included in the tree with the other sequences. This library includes 112 clones and 43 RFLP patterns. The most abundant clones were group-I novel alveolates (40 clones), dinoflagellates (29 clones) and prasinophytes (8 clones). Many other phylogenetic groups were also present. (Download Excel file with BLAST hit)
During the next months, we will perform two more genetic libraries from Blanes (samples of 20 March 2001 and 22 June 2001)

Pigment analysis (Mikel)
Regarding pigments, the samples from Roscoff (second year) have been run successfully. The results need to be processed and digested but I will need the volumes filtered. Thanks. We keep going on with the monthly sampling in Blanes. On the other hand, we'll start our moving to the new building in a couple of weeks. Let's see.

Two points regarding the web page: 1) when you enter through "experimental protocols" and go to "HPLC for pigments" it shows Ramon's protocol. 2) if you keep scrolling down on the Table with the analyzed parameters the sample volume shows 1000 mL when in the protocol it says 2000 for GFF filters and 5000 for the 3 um filter. Also, when you click the remark column where it says "see Mikel protocol" a different document shows up (Note: this has been fixed June 26, 2001).

Sampling in Blanes (Ramon)
The monthly sampling started last March. We collected samples as decided in the PICODIV protocol without having any major problem. Samples were collected for pigments (chlorophyll and HPLC), fixed samples to count microorganisms (phytoplankton by inverted microscope, bacteria and picoalgae by flow cytometry, and heterotrophic flagellates by epifluorescence), and samples for molecular analyses (DNA extraction, and FISH for eukaryotes and prokaryotes). Most of these samples are kept refrigerated and will be processed at the end of the yearly sampling. DAPI-stained flagellates (heterotrophic and phototrophic) are observed by epifluorescence the same day of sampling. In this way, the autofluorescence of even dim picoeukaryotes are easily observed. During the last sampling date (23 May 2001) we collected additional samples for FISH.Euk (a filter) and FISH.Prok (50 ml-fixed seawater) and these samples were sent to Roscoff and Warwick, respectively. During the next months we will continue sampling in the coastal site.

December 2000

1. We are finishing two papers which will acknowledge both MIDAS and PICODIV:

one on DGGE of eukaryotes one on the five clone libraries of eukaryotes (the one that was rejected in Nature). We hope to send one tomorrow and the second on Monday. We will send preprints.

2. We have completed the second sampling in our coastal station.

3. Organization of the HPLC workshop proceeds ahead. Mikel will send you the state of the question today or tomorrow. We have HPLC, microscopic, and molecular data from Mediterranean samples that will fit nicely in PICODIV and that we will present at the workshop.

4. Laure has quite a few cultures growing. She will tell you about this.

November 2000

Cultures

Two different stations were sampled the 21 september 2000, one coastal (BLA1, 25 m) and the other more oligotrophic (BLA2, 200 m).

For each station, the sea water was prefiltrated through a 0.6 and 3 um millipore filters.

The first 16 precultures, made in 50 ml, were prepared the same date, using the following media:
- sample, without any adding
- F/2, diluted in 9/10 of sample
- old sea water (older than two months), diluted in 9/10 of sample
- Rice medium, diluted in 9/10 of sample, placed in dark.

From sample without any adding, a second series of 12 precultures were prepared the 11 october 2000 by innoculate 1 mL of the stock solution using the following media (50 mL final volume):
- F/2
- old sea water
- Rice medium, placed in ligth by error.

These precultures were screened by flow cytometry the 23 October 2000 (except the 8 preculture with rice medium). We have detected chlorophyll-containing species in 14 of the 20 precultures, by the presence of mixte or isolated populations of:
- 2 different Synechococcus-like populations, with low Fl2??? in 8 precultures
- Population of small Cryptophyceae in 5 precultures
- Population of picoeukaryotes in 11 precultures

Maximum abundance is found with the F/2 medium. The populations with orange fluorescence (Synechococcus like and Cryptophyceae) are abundant with the F/2 medium, since picoeukaryotes are more abundant in old sea water.

Serial dilutions have been made on these 14 precultures in order to isolate pure culture, using both F/2 and old sea water media. These precultures are also maintained in tube of 10 mL and will be transferred in new medium every two weeks.
The rice medium is not screened to date.

October 2000

• The first sampling in Blanes Bay was performed in 20 September 2000. Two stations were sampled at surface, one at 1 mile of the coast (depth 25 m) and the other at about 4 miles of the coast (depth around 200 m). Samples for DNA extraction, flow cytometry counts, HPLC measurements and electron microscopy were collected. Precultures of picoeukaryal organisms were initiated.
• Laure Guillou started to work in the Institut de Ciències del Mar on September 2000, as a recipient of a two-years EU postdoctoral fellowship. Her experience in culturing picoeukaryal organisms will be very valuable.
• The protocol to study picoeukaryal diversity using Denaturing Gradient Gel Electrophoresis has been fully optimized. We obtain sharp bands and complex band patterns from marine picoplankton. The patterns obtained from each sample are reproducible, indicating that they are characteristic of the picoeukaryal composition of the assemblage. We investigated different statistical approaches to use the DGGE fingerprints in the comparison of different eukaryal assemblages. We are preparing a manuscript with this information that will be submitted very soon.
  

April 2000

• The first sampling in Blanes Bay for obtaining picoeukaryotic isolates (Workpackage 1) and environmental SSU rDNA picoeukaryotic sequences (Workpackage 2) is scheduled in mid July, after the first PICODIV meeting, when protocols for the different tasks will be discussed and unified.
• DGGE (Denaturing Gradient Gel Electrophoresis) with eukaryal primers is being currently optimized in our laboratory. This technique plays a key role in the monitoring of diversity of picophytoplankton in different coastal systems (Workpackage 5). The results of different primer combinations, which amplify different regions of the SSU rRNA gene, are compared. The performance of these primer pairs are tested with phytoplankton cultures of various phylogenetic affiliations and with several natural samples. A method to analyze and compare DGGE fingerprints has been developped, and current efforts are devoted to obtain DNA sequences from DGGE bands.
• Microbial samples for DNA extraction have been collected in several cruises performed in different regions of the NW Mediterranean, in the Alborán Sea (cruise MATER99, 13-25 September 1999) and in the Catalan Sea (cruise HIVERN00, 29 January to 8 February 2000). During these cruises, different stations were visited (10 stations in MATER99 and 17 stations in HIVERN00) and sampled at different depths in the water column. Normally, the microbial fraction between 0.2 and 5 µm was collected on filters, although other prefilters were also used in particular samples. DNA extracts for some of these samples have already been obtained. These samples will be in the future analyzed by DGGE with eukaryal primers, and some samples will be chosen to construct genetic libraries of eukaryal SSU rRNA genes

Oslo

January 2003

Manuscripts

The Florenciella ultra (I have checked that there is no genus in the botanical system called Florenciella yet) ms is approaching its final stage. I have received both the molecular and pigment data. We still lack the final mounting of the figures, a final ”beautification” of the tree and a latin diagnosis. We have a professor at the Univ. of Oslo that will do the translation. Also, of course the ms needs improvements.
Lately, I have been working to find a fixation that will make the hairs of Florenciella stay on the flagella, I have not succeeded, but I have made some improvements. I think now is the time to put the figures together. At the moment I am learning the program Illustrator and I am using it on Florenciella and Telonema (Schalcian-Tabrizi et al. paper). The next figures will be the Prasinophyte paper that Laure is leading.

Worpackage 1 Cultures

Oslofjord: I have many cultures growing. Many of them are species of Micromonas, Mantoniella, Imantonia, Pseudopedinella, Chlorella ie species that we are not very interested in. I have a Pyramimonas (ca 3-4 microns) that I have not found in the literature yet. Also a Chrysochromulina of the same size that I am not sure of.

Arctic cruise: The cultures are raw cultures and I have started with some purifications. I have some diatoms like Pseudo-nitzchia and cf.Navicula that are too big for PICODIV. Otherwise there are many Chrysochromulina (of the saddle-shaped type), Phaeocystis, Imantonia, Micromonas, Pseudopedinella, several heterotrophs most of them I do not know what are, but I have something that I think is Colpodella (Apicomplexa). I have made some whole-mounts of many of the crude cultures. Also, there seem to be some differences in species composition between the stations.

RCC 505:A flagellate very similar to Florenciella, but bigger and a Monosiga sp. (ca 3-5 microns).

RCC 404: Telonema. To complete the ms on Telonema I need to make some serial sections, to be able to elucidate the flagellar apparatus which is important knowledge for this new class of organisms.

RCC 386: Prymnesiophycean according to its pigments. Covered with elongated scales that are not known from the literature and resembles scales found in RCC 392 and natural samples from the Oslofjord.

RCC 391: New prasinophyte of the order Mamiellales. We have a collaboration with A. Zingone (who also has it in culture) on the description of this new species.

October 2002

Arctic cruise (Aug-Sept 2002)

• Download report by Carlos

Description of Telonema ultrastucture

• Download early draft version of paper

Description of a novel Dictyochophyceae species

• Download early draft version of paper (Florenciella ultra, RCC 446)
• New TEM pictures

Cultures belonging to the order Mamiellales in the Class Prasinophyceae (with Laure).

The order is characterized by flagellates or non-motiles covered by spider-web like organic scales. The order also contains flagellates and non-motiles lacking the scaly covering. The cells usually contain a nucleus, a dictyosome, a single mitochondrion and a chloroplast. The chloroplast of some some species have a pyrenoid in addition to a starch grain and some of the flagellate species have a stigma. According to molecular data we have several groups of organisms belonging to the Mamiellales. Below a description of their fine-structure is given.

• 1: Ostreococcus sp. clade II (O. oceanicus). Cultures RCC141, 143, 393, EUM 18 BL. Cells less than 2 microns containing one chloroplast, a dicthyosome, a single mitochondrion and a nucleus. No surface structures cover the cells. RCC 143 has peculiar budding structures (new TEM pictures).
  
• 2: Ostreococcus sp. clade IV. Cultures RCC 356, 393, 371, 343.  Cells less than 2 microns. One chloroplast, one mitochondrion, a nucleus and a dictyosome. No surface structures.
• 3: Mamiella sp./Mantoniella sp. RCC 391. Cells 2-4 microns possessing two flagella ca 10 microns in length, containing a nucleus, a dichtyosome, a single mitochondrion and a chloroplast with a pyrenoid surrounded by a starch shield. Two scale types are present. One small and one larger. The cell surface is covered by two types of scales. The flagella are covered by one type. The genus Mamiella is distinguished from the genus Mantoniella mainly on the basis of differences in flagellation and swimming behavior. Mamiella has two long flagella and Mantoniella one long. Also, the scales covering the flagella differ somewhat. The scales covering the flagella of Mamiella gilva, which is the only formally described species of the genus (observations have been made of other related organisms) has a small spine that the more square scales of Mantoniella squamata lacks. The scales covering the flagella are most similar to those covering the flagella of M. squamata. RCC 391 has two long flagella and swims like a Mamiella.
  
• 4: Micromonas-like. RCC 434, BLA 122. The latter is indistinguishable from Micromonas pusilla and has been studied both in section and whole-mounts.The former is not examined.
  
• 5: Micromonas-like. RCC 114, BLA 105/7. The former has not been examined, the latter is indistinguishable from Micromonas pusilla in whole-mounts.
  
• 6: Micromonas-like. RCC 372. Swimming cells.
  
• 7: BLA 82/7. Non-motile cells ca 2 microns without scales. One mitochondrion, a chloroplast with a conspicuous chloroplast lumen. No dictyosome has been observed, but the cell possess vacuoles.
  
• 8: RCC 287 and Noum 15. Non-motile cells with a varying fine-structure. Usually with a delicate cell wall sometimes covered by “hairs”. Inside the cell a mitochondrion, a chloroplast sometimes with a starch grain and a nucleus may be distinguished, but no dictyosome has been observed hitherto. The organism also produce cells that may have several chloroplasts and a wavy surface. This may be resting stages.

May 2002

• Download Year 2 report
• Cyano. This far I have sectioned one Cyano from Roscoff RCC 351 and two from Dave. They are called (Dave's lab) Dim and Bright. I have embeded another 5 species and all the strains Dave has sent me are genetically far apart. The Dim did not fix as well as the bright and this far I can not really se any other differenc them in size and shape (one is more round and smaller). The pictures are still in the microscope. The paper on the ultrastructure of some selected cyanoes and something on their genetic relationship to each other should still be on if the results turn out to be interesting.
  
• Prymnesiophytes. I have section RCC 400 and I am getting an idea of the scales. From whole-mounts I have only been able to verify that it probably has scales. It seem like it has two kinds of delicate scales, the outer layer with an upright rim. I will have to fix and section it again with only osmium to see if that resolves the scales better. I am also, considering negative staining. RCC 390 also has very delicate scales that I have not been able to resolve in whole-mounts. Finaly, it has survived the trip to Norway and it is now growing well. I made a inclusion of it 3 weeks ago, but the pellet is so small that I do not think it is worth sectioning it. I will make a new inclusion. I know that Marie-Joseph is describing two new Chrysochromulina species, do you know if they may be any of "ours"? Also, I know that Adriane and Jahn have some new ones from Naples and I will compare with their material .
  
• I have been trough some whole-mounts of natural samples lately and it is still disapointing. The Helgoland whole-mounts from may 2002 were dominated by the heterotrophic Paraphysomonas cf. imperforata.
  
• My plans before the Roscoff meeting;
  
  • Write drafts on Telonema and Dictyochophyte papers.
  • Examine additional Telonema cultures from Roscoff.
  • Section natural samples from Blanes and cyanos from Dave.
  • Examine heterotrophic cultures sent by Laure "in totto".
  • Make inclusions of Chrysochromulina (RCC 400 and 390) and some additional Prasinos from Laure.
  • Make SEM preparations of RCC 287, RCC 303 (to compare it to the newly described Prorocentrum nux) and some cyanos (including RCC 351).
    
• I have quite a few pre cultures going from the Oslofjord that I have not examined properly. I would like to catch up on that.

March 2001

Novel TEM pictures from Wenche have been added to the web site (Link to EM pictures).

TEM examination of prasinophytes

RCC 141 and 143. These two cultures look very similar to each other. One chloroplast with thylokoids in bands off 3-6. Pyrenoid seem to be lacking. No starch observed. Small dichtyosome and a nucleus. Central mitochondrion with flat cristae. No scales. No microtubular structures observed. Look very much like a Ostreococcus.

RCC 356. Contains a nucleus, a dichtyosome and a mitochondrion with flat cristae. One chloroplast with thylakoids in bands of three and with starch. No scales. No microtubular structures observed. Looks much like Ostreococcus.

RCC 371. Contains a nucleus, a dichtyosome and a central mitochondrion with flat cristae. The single chloroplast has thylakoids in bands of 3-6. A vacuole empties its content to the exterior. No scales. No microtubular structures observed. Resembles Ostreococcus a lot.

RCC 287. This algae looks different and either fixes poorly or is about to go into a resting stage? Contains a nucleus and chloroplast with irregulary arranged thylakoids and may have a pyrenoid. No observations yet of mitochondrion or dichtyosome. The cell surface is irregular. No microtubular structures observed. No scales. Will be embedded and section again. Will be prepared for SEM.

RCC 391. Mamiella/Mantoniella sp. nov. Morphologically this is a Mamiella because of its two long flagella. (Mantoniella has one long and one very short). Two types (perhaps three) of scales cover the cell and flagella. Will be embedded and sectioned.

RCC 434 (=BL 122) is a Micromonas

I still have a big problem with the natural samples. I have prepared some of the natural samples for SEM and examined some of them in the microscope that we have in Oslo (see pictures), which is not a field emission microscope (new type that very few institutions have yet). This means that the resolution is not good enough for the reckognition of scales and other delicate structures. As we all know filtration is a risky business and I guess that it is no surprise that some larger cells and parts of large organisms comes into our “less than 3 microns fraction”. Also in the SEM I find far less organisms then the rest of you find in your samples with your methods. At the moment the UiO workshop is helping me to design a filtering device that will facilitate the preparation of SEM preps. I am hoping to examine many of our samples in the field emission microscope that colleges in Denmark have just installed.

It still seems very difficult to get whole-mounts of natural samples with cells on them.

January 2001

Novel TEM pictures from Wenche have been added to the web site (Link to EM pictures).

December 2001

This month I have been teaching 3 days a week as a “stand in” for Jahn, who is working in Naples at the moment.

Bl 122 has been studied in LM, as whole-mounts and sections. The fix and staining is wonderful, but morphologically it is very similar to Micromonas pusilla. I cannot distinguish them.

I have studied the transition zone of the flagellar apparatus of RCC 355. As Apedinella radians it has two rings underneath the transvers septum. The chloroplast has an embedded pyrenoid penetrated by a tylakoid and the nuclear envelope is connected to the outer chlorolast membrane. The flagellar hairs are tubular and are produced in the perinuklear compartment. Weather they is tripartite or not, I do not know yet. I have found no microtubular roots and I think I can see some fibres stretching from the basal bodies to the surface of the nucleus. Judged from ultra-structure evidence. I think this is a heterokontophyte that may be related to the pedinellids. I would like to embed this culture again to see if I can get more details out of it, eg flagellar hair details. RCC 332 has a different type of chloroplast and pyrenoid. This species also needs a better fixation and a closer inspection. Telonema is sectioned, but I have not looked at it yet. I will send Jahn em-graphs and he has started the outline for the paper. He knows Telonema well from before.

Also, I have some bad news. Due to the poor development of the interest rate in various market places the Nansen foundation is not granting any money this year! To my knowledge this has never happened before. I will send the same application to the Norwegian research council, but my hopes are low. The council has had a dramatic reduction of its funds this year due to a new conservative government. I think I can handle my own expenses for the arctic cruise, but will have problems with additional costs. I have been informed that the cruise schedules will probably be ready before Christmas, which means that those who need an invitation for the cruise may have one (funding applications etc).

Natural samples; I still find very little material in the whole-mounts. I use both silicon and pluronic. In the sections of natural samples from Blanes in June I have found well preserved prasinophytes and cyanophytes so far. The addition of pluronic add some dark staining spots to some of the cells, but this has not been too disturbing yet. For the samples that I receive from Helgoland and Roscoff I think we should try to double the concentration of the fixatives. I have completed preparations for scanning microscopy and will start on them next week. I really hope that this will help.

The rest of the month I will proceed with Telonema and Blanes cultures and I will process the natural samples that I receive. Also, cultures arriving from Roscoff will be prepared for sectioning.

November 2001

Natural samples (my headache);

• Received samples from Helgoland and Roscoff (June-October), Blanes (June) prepared for TEM.
• Pictures from natural samples (March-May sent to Daniel on CD)
  • March sample from Roscoff
  • May sample from Roscoff dominated by organisme in picture RCmai01M394x8 (station,month,year,number of picture, magnification).
  • May sample from Helgoland contained the undescribed scaly picobug that we know from the Oslofjord see picture Hcmai01M380x17
  • June sample from Oslofjord

Cultures;

• Embeddings; RCC 356, 141, 343, 371, 344, 287, 143 and 404 (Telonema). Plus Blanes 122. I have sectioned Blanes 122 and Telonema. I have checked the fixation in 122 and it looks promising. At a first inspection this species seems to be devoid of special characters and more work needs to be done.

TEM inspection;

• RCC 390 delicate scales, needs to be embedded. Not in culture with me. Very interesting
• RCC 400 delicate scales, needs to be embedded. Growing well in culture with me. Very interesting.

I have been in contact with all the Norwegian Institutions that go on cruises to the Arctic. None of them have fixed their cruise schedule yet, but they are all interested in giving us ship time for 2-4 persons. I have sent an application to the Nansenfoundation for some money approx 1700 Euros (they do not grant large sums), which should cover the air fares for ca 3 people. In the application I have used the CVs of Mikel, Carlos an myself since I reckon we all want this cruise to involve pigment analysis, molecular methods and traditional taxonomy. Mikel has a lot of cruises next summer and we have to find a replacement for him. Carlos will try to apply for some Spanish money in addition. Also, I know that Fabrice and Dominique Marie from Roscoff are interested too.

• Sectioning of prasinophyte cultures and Telonema
• Examination of natural samples in TEM.

September 2001

Material processed, and taxa revealed if possible to identify in whole mounts

Roscoff cultures

RCC 391 Mamiella sp. (RCC391M443x10edopt), scales are similar to Mantoniella and Mamiella scales
RCC 392 non-motile with scale (see picture RCC392M445x10edopt) with flagellate (see picture RCC392M444x08edopt)
RCC 389 as 392 possibly with one additional flagellate
RCC 386 as 392, and Paraphysomonas sp.
RCC 382 Mesopedinella arctica
RCC 395 Mantoniella (no EM yet)
RCC 400 Chrysochromulina (no EM yet)
RCC 404 Imantionia and Telonema (see picture)

The "Laure" cultures (RCC 343, 344, 371, 141, 143, 356, and 287) grow well (they survived without problems our culture room accident were the temperature reached 30 degrees Celsius). Unfortunately, our standard fixing protocol does not yield nice fixations of their chloroplast so we will have to reprocess with some alterations and additions.

Dead cultures
RCC 382, 391, 392, 386, 389,
RCC 390 was dead upon arrival

Cultures from the Oslofjord
Green scaleless coccoid ca 1.5x2 micrometer . It seems to be without contaminants.
Our culturing efforts have not given the results we were hoping for. We tried semi-continous culturing (adding 1 ml of medium every day) in medium of half strength (Erd-Schreiber), and some serial dilution cultures. The June samples have grown very well and we have had all the little bugs that we know so well from before, coming up. The July samples were killed in our culture room accident. In August we made no new cultures

Natural samples

Blanes: June sample, whole-mount; Chrysochromulina throndsenii, C. cf. leadbeateri, cells of Chrysochromulina with spiny scales resembling C. brevifilum. Scales from C. mactra and C. ericina, Unidentified scales probably from Chrysochromulina species, setae from several Chaetoceros species. Micromonas pusilla, Pyramimonas sp. cf. Bolidomonas, cf Ochromonas and cf. Phaeocystis. Many types of bacteria and some of the small balls may be eukaryotes. The latter if present may be revealed in the sections.
Helgoland: no new results
Roscoff: no new results.

Plans for the future

• Section May and June natural samples from Blanes. Fix and section Laure's cultures again.
• Work with natural samples from Roscoff and Helgoland.
• Continue on paper about RCC 355
• Sample (September) and start cultures from the Oslofjord.

June 2001 from Wenche

CULTURES

Oslofjord
May 2001: Collected 1m, 5m, 10m, 15m and inoculated serial dilution cultures (SDC in Erd-Schreiber), plated on agar and made semi-continous cultures (As medium we used 50% sterile filtered seawater and 50% Erd-Screiber). At the moment I have registered growth in SDC from 1m (15 degrees C, the others are kept at 3 and 10, because of the lower temperatures at those depths at the time of collection), the others have little growth yet.
June 2001: Collected surface water and inoculated SDC and semi-continious culture.
From the March sample we have a flagellate that resembles RCC 341 in addition to the not very interesting Imantonia and Micromonas. In addition we have small green balls growing on agar together with lots of bacteria.

Plans: I have made more sections of RCC 341 and RCC 355, but I need to do some more microscopy and have started on two separate papers that I will pass on to Daniel/Kadidhja and Mikel to fill in molecular and pigment data.

WHOLE-MOUNTS (all the samples that you have sent are on grids);

Roscoff May 2001: Dominated by non-flagellated cells covered by a skin. In addition; Mamiella/Mantoniella (scales only), Pyramimonas cf orientalis, Chrysochromulina simplex and scales of C. hirta, Choanoflagellate. It seems like both the filtering process and the concentration have been quite successful.

Helgoland May 2001: cf. Hemiselmis, Mamiella/Mantoniella scales and Pyramimonas scales. The preservation of the cells seem to be better since the Hemislmis I found was with flagella. Probably I have not been able to concentrate the sample enough. I have been trough one grid and examining 5 more will give more results.

Oslofjord June 2001: Dominated by Imantonia rotunda. In addition; Chrysochromulina brevifilum and C. polylepis. Pyramimonas grossi, Mamiella gilva, Pterosperma swarmers, Choanoflagellate, cf. Hemiselmis, diatom.

On the June samples from the Oslofjord I tried the silicone and pluronic and I did get more cells in the samples were I added the surfactants. This also means that I can centrifuge a smaller volume and I may also turn down the speed. I have been a little worried about whether the pluronic would coat the scales so they can not be recognized, so I tried it on a culture of Bathycoccus . I think it clogs the structures of the very tiny scale of Bathycoccus a little bit, but for bigger less delicate scales in the June samples from the Oslofjord it did not seem to matter so much. One disadvantage is that it adds more debris to the grids. I have ordered pluronic and silicone and want to use it for the whole-mounts that will arrive from Helgoland, Roscoff and Blanes in the future.

I have been in contact with Tromsø (Svein Kristiansen) asking about the cruise to Svalbard next May. They were very positive, but they have not starting the planning yet. 2-3 people would be OK. The ship will cruise Kongsfjorden and we will probably not get into the open ocean. I will come back with more info about the ship, dates etc. when I hear more.

February 2001 from Wenche

At the moment I am working with RCC 355. In the sections I have found some cyanobacteria, but I do not see them anymore in the transferred culture (LM). On the other hand, there are some heterotrophic flagellates that get abundant when the culture is old. I am a little puzzled by that photosyntetic organism. For some reason (wrong fixation?) I do not detect flagellar hairs in the whole-mounts, but in the sections I find something that resembles tubular shafts in the perinuclular compartment. RCC 341 resembles Pelagomonas in thin-sections, but also here I have problems with hairs. RCC 362 has scales that resembles Isochrysis and the motile stage of Emiliania. It has been sectioned and so far I have not found any sign of a haptonema. This in combination with a non-motile stage may point to Emiliania, but there are other features that I do not find.

December 2000 from Jahn and Wenche

Work is proceeding, not exactly as we anticipated, but rewarding even so.
Wenche has been through a large number of cultures with LM, direct preps in
TEM, and many have been embedded, some also sectioned. TEM graphs were
obtained for species which did not appear just as electron dense spheres of
different size. Her summary report is enclosed. The filling into the data
base is proceeding, but some information more than the pure taxonomical
will have to be gathered over some time. A file of todays status is
included, but there are more inclusions to follow. For the higher taxa we
have applied the botanical system in accordance with van den Hoek et al.
(1995), using Heterokontophyta instead of the "new" term Stramenopiles for
the division comprising classes Chrysophyceae, Bacillariophyceae,
Eustigmatophyceae.

Link to new Culture page to read Wenche report on Roscoff and AWI cultures.

UIO

Results from natural samples (Oct 00):

Cultures; serial dilution cultures. We have cultures of Chrysochromulina and Micromonas pusilla. They all have heterotophic flagellates in them.

Whole mounts (okt 00): Emiliania huxleyi, Phaeocystis sp. cf Dicrateria, Chrysochromulina cf. apheles, C. cf. scutellum, Chrysochromulina sp. Micromonas pusilla, Mamiella cf. gilva, cf.. Pelagococcus, Paraphysomonas cf. imbricata. Unidentified non-motile pico cell with scales. Pico flagellate with one very long and smooth flagellum and ejectosomes. Picoflagellate with one flagellum with mastigonemes and blunt end.

The Osfj is an undescribed species that I keep finding in Oslofjord samples. I hope to isolate it.

EM neg: L446-451.

Thin-sections (embeddings okt. 00). Cf. Pyramimonas. Synechococcus.

EM neg: L 432, 433, 438, 440, 441, 443.

Natural samples collected dec. 00

Whole mounts were made and embeddings are in progress. Serial dilution cultures were inoculated.

October 2000 from Wenche

Cultures examined in the LM upon arrival, 01.10 and 14.10.00 and in TEM.

The whole-mounts were made on the cultures upon arrival from AWI:

"Drebes 3 high light (h.l.)" - looks like a Phaeocystis. Confirmed in TEM
"Drebes 1 h.l." - non motile pico (cyanobacterium?)+ colourless flagellate. TEM gave no additional info. I will embed and section this culture when it is growing well.
"Drebes 2 h.l." - non motile pico. Looks like Micromonas, but do not swim (but jumps), no flagellum observed in the TEM. - Will be embedded and thin-sectioned.
"Drebes 2 low light (l.l.)." - cf. Emiliania + flagellate + cf diatom. In TEM: also Bathycoccus prasinos was observed.
"Drebes 6 l.l." - non motile pico. Identified in TEM as B. prasinos
"IMR 2 l.l." - non motil pico + 2 types of colourless flagellates. In TEM: also Phaeocystis was observed, the non-motile did not seem to carry scales.
"IMR 4 l.l." - cf Micromonas. This culture is not growing well and in the TEM preps no Micromonas cells were observed.

Samples from Blanes.

1.Thin-sections made from natural samples collected in Blanes. The fixation seems OK, but something must have happened during the filtration, because the sample is dominated by nano and microplankton.
2. Whole-mounts made from natural samples collected in Blanes. The sample was dominated by nano and microlankton and also the cells were very much glued together.

Picocells identified this far; Bathycoccus prasinos, Synechococcus sp., and a lot of heterotrophic bacteria. I will spend more time on the sample, but information from direct preps is likely to be meagre; I will need to make more sections.

We think that for the taxonomic study of picoplankton we meet two major obstacles. Fixing and concentration of the organisms. Jahn and I have collected material in the Oslofjord, and concentrated the picofraction in different ways. On fixed prefiltered (3 microns) material we have filtered (on to 0.4 micron filters) and centrifuged in tubes and with a continuous centrifuge. In September and October 2l of water has been sufficient to give material for whole-mounts and thin-sections. We have also concentrated 2l of live prefiltered (3 microns) seawater with tangential flow. Whole-mounts have been compared and it seems like tagential flow concentration is a workable method. We will do a tangential flow session on a Micromonas culture to check retention efficiency, the viability of cells and so forth.

October 2000

1. We have received crude cultures from AWI and prepared whole mounts that have been examined in the EM. The cultures are still growing, and we try to make some unialgal cultures from the ones that are not already so.

2. Laure has sent some concentrated natural material from Blanes, from which Wenche has made whole mounts as well as embeddings.
A) the whole mounts are not well preserved/fixed and few species are recognizable in the TEM.
B) Wenche has sectioned the embedded material, it will be stained onThursday and she will go for a TEM session on Friday.

3. Beside this we are looking into the Oslofjordplankton to refine the techniques, and possibly also get new strains into culture. For concentration of material we have tested a new continuous centrifuge (made in the institute workshop) and the results are promissing. We will pursue the concentration efforts by trying also the tangential flow filtration.

4. Wenche is now exploiting the possibilities of applying a freeze substitution technique on picoplankton cultures, for thin sectioning and TEM . Our Electronmicroscopy laboratory for Biological Sciences has developed advanced cryotechniques for TEM, so I hope we can draw from their expertise to enhance the picoplankton fine structure studies.

Wenche will prepare a short progress report when she has more results from the latest EM sessions on the material from Blanes and AWI cultures. The access to the EM-lab has been periodically hampered by ongoing courses.

June 2000

PROSOPE grids have been sent to Wenche.  Here is a report from her findings: "I have shadow-casted and looked at grids from station Med 1 and Med 2. I do not really find much to look at. According to your graphs I would have expected more cells. Most of the ones I do find are little round cells with no scales or other features that may help me identify them. I have found a few flagellates of which I can only identify Micromonas. I think it would have been a good idea to fix and thin section this kind of samples (that would give more info, but as you know there would still be many things that we could not identify). The rest of the week I will spend on the other two stations. I had planned to spend more time on the Prosobe grids, but I just learned that the EM lab is giving a cryo course next week and the week after which means that they will need all three TEM microscopes. If you want to I can spend more time on them this comming fall. Some of the grids do not look to good, some have holes in them and others have a precipitate. Since we are going to work with very small cells I suggest we use 100 mesh grids. That will save me a lot of time on the microscope. To be able to orientate my self and make sure I examine all the meshes I need to switch between low magnification and high mag for every mesh and this procedure is a little time consuming. Thus using a bigger mesh will save time."

Other work going on: "I have fixed and sectioned a Micromonas that Jahn has isolated from the Mediterranean and a Synechococcus from the Skagerrak in order to check out our "new facillities" and our fixing protocol on pico plankton. Things have worked out well and I think we are well equipped and ready for all the new cultures that the rest of you are going to isolate. I have also spent some time getting familiar with a (to me) new electron microscope and ultramicrotome."

April 2000

In Oslo we have spent time on library studies, rearranging laboratory facilities and trying out digital handling of TEM graphs.

Some of our time in this first month of PICODIV has been used on literature studies in order to expand our library on taxonomic picoplankton papers. We have equipped ourselves with a scanner that is capable of scanning TEM negatives in addition to 35mm negatives and slides. The resolution appears to match the negatives well, and it will certainly save a lot of darkroom hours. Some problems we experience with the 35 mm feeder will hopefully be solved by the supplier.
We are setting up a new lab for preparation work, and will start testing and developing techniques using a picoplankton species (it is similar to Micromonas) that is isolated from the Skagerrak. Our setup is ready to receive PICODIV cultures at the earliest convenience. Experience has shown that fixation procedures may have to be modified for the individual strains. Hence, we look forward to start this period of trial and error as soon as possible.

Last updated 12 February 2003