memo - ilvo.vlaanderen.be€¦ · keygene patent on workflow: re -cut >size selection >...
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Mnemiopsis leidyi: Ecology, Modelling and Observation
MEMO
ACTIVITY 1
IDENTIFICATION, MIGRATION AND FYLOGENY
OF MNEMIOPSIS LEIDYI INDIVIDUALS AND
POPULATIONS
STEFAN HOFFMAN
November 2013
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Mnemiopsis leidyi: Ecology, Modelling and Observation
The Use of Genomic Tools in
Finding Answers for Research Questions
Within the MEMO Project
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Correct and unambiguous Identification of ctenophores
Mnemiopsis leidyi
Bolinopsis infundibulum
Identification of ctenophore DNA in fish stomachs
Migration patterns
Fylogeny and Population grade of organisation
Chromosomal organisation & Genome size
?
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Mnemiopsis leidyi: Ecology, Modelling and Observation
1. Identification of Mnemiopsis leidyi and other ctenophores/jellyfish using molecular analysis.
Protocol
1: Extract DNA
2: PCR
3: Sequence
4: Blast & Results
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Mnemiopsis leidyi: Ecology, Modelling and Observation
1. Identification of Mnemiopsis leidyi and other ctenophores/jellyfish using molecular analysis.
Results (on individual stored samples, not in bulk/water samples):
o Mnemiopisi leidyi: • Primers Fuentes (2010) ITS1 marker -> OK • 2011-2012-2013 439 indiv were tested, from 27 locations, +/-163 tested positive
for ML. o Other Ctenophores:
• Primers Fuentes (2010) worked for Beroe sp. and Pleurobrachia pileus. (not for Bolinopsis sp.) -> problem: little reference sequences in Genbank.
o Lovenella sp. • Different primers for ITS1-18S and COI were tested
* amplification ok for some, sequencing failed -> contaminated DNA? o Jellyfish (e.g. Cyanea lamarckii, Tima bairdi, …)
• Low importance, ITS1 primers Fuentes (2010) did not work -> no further effort
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Mnemiopsis leidyi: Ecology, Modelling and Observation
1. Identification of Mnemiopsis leidyi and other ctenophores/jellyfish using molecular analysis.
- ML was confirmed on the following InterregIVa2Seas region locations:
• Continental shelves of FR, BE, NL • Baie de Seine, Gravelines, Boulogne-sur-Mer, Ile de Tahitou, Le Havre • Port of Zeebrugge and Spuikom Oostende • Scheldt estuary
- ML individuals from other locations were also confirmed: (reference) • German Bight and North Eastern Atlantic, • Waddensea (NL) • Villefranche-sur-mer (Fr) • Baia BLU Santa Theresa (It) • Chesapeak Bay
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Overview of samples used for migration experiment (SNP development). (Chesapeake Bay, Villefranche and Baia Santa Theresa not shown)
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Mnemiopsis leidyi: Ecology, Modelling and Observation
1. Identification of Mnemiopsis leidyi and other ctenophores/jellyfish using molecular analysis – partim fixative test.
Test:
• Find a suitable fixative that preserves morphological features • Find a preservative that allows long term storage and further analysis (isotope
analysis, molecular analysis, ….) Results:
• Different methods of fixation were tested and scored for DNA extraction and amplification of ITS1:
Ethanol (70%, 100%), freezing (-20°C, -80°C), freezedrying, Acid lugol
solutions, Battaglia sauce, RNA later, TCA, RCL2 (Alphelys) and HOPE® (DCS Diagnostics)
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Mnemiopsis leidyi: Ecology, Modelling and Observation
1. Identification of Mnemiopsis leidyi and other ctenophores/jellyfish using molecular analysis.
- Only Acid lugol’s solution (2-5%) in seawater preserved morphological features (some discoloration and shrinkage was present) and allow simultaneous DNA extraction and molecular identification.
- TCA preserved morphology but did not allow DNA extraction, all other fixatives allowed DNA extraction and ITS amplification (Battaglia sauce: low DNA conc.) but gave poor morphological feature preservation.
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Mnemiopsis leidyi: Ecology, Modelling and Observation
2. Identification of Mnemiopsis leidyi in fish stomachs.
- Goal:
- Identification of ctenophore (i.c. ML) DNA in fish stomachs for the study of predation of fish on jellyfish.
- Strategy:
- Development of a protocol for the identification of ML DNA in fish stomachs. - Extract DNA from all caught individuals and confirm identity as Mnemiopsis leidyi for use
in later studies.
- Results:
- A protocol for the identfication of ML DNA in fish stomach was drafted.
- Cut stomach from complete fish stored in Ethanol, pool stomachs per location - Grind stomach in liquid Nitrogen - Extract DNA with invisorb, amplify ITS 1 marker, sequence and BLAStn
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Mnemiopsis leidyi: Ecology, Modelling and Observation
2. Identification of Mnemiopsis leidyi in fish stomachs (cnt’d).
- Ctenophore DNA could be detected by sequencing amplified ITS1 markers in DNA from fish stomachs in (samples from LVS: NorthSea/Scheldt estuary):
- Herring (Clupea harengus)-> ML - Sprat (Sprattus sprattus)->ML - Horse mackerel (Trachurus trachurus)->PP - Greater sand eel (Hyperpolus lanceolatus)->ML, PP
(ML: Mnemiopsis leidyi, PP: Pleurobrachia pileus)
- A control experiment should be conducted to confirm the analysis. A positive and
negative control is needed. -> rearing or keeping sprat or herring in a tank and feeding them with ML is difficult (according to aquaculture specialist-> these fish live in groups)
- Samples from IFREMER are in process
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Mnemiopsis leidyi: Ecology, Modelling and Observation
3. Migration patterns of Mnemiopsis leidyi individuals.
- Goal:
- To study migration pathways of ML individuals (re-introduction or new introduction), traceback of place origin.
- Strategy:
- Use of existing microsatellite protocol.
- Development of SNP’s (single nucleotide polymorphisms) based on DNA from caught M individuals.
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Mnemiopsis leidyi: Ecology, Modelling and Observation
3. Migration patterns of Mnemiopsis leidyi individuals.
- Protocol:
- Microsatellite analysis: - Repeated DNA sequences (2-5nt), different per individual - Workflow: DNA extraction -> amplification of µSAT loci -> scoring length of fragment
- Protocol from Reusch et al (2010) - 7 microsatellite loci, divided over 2 pools (pool1: 3 loci and pool 2: 4 loci)
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Mnemiopsis leidyi: Ecology, Modelling and Observation
3. Migration patterns of Mnemiopsis leidyi individuals.
- Protocol:
- SNP Development: - Single Nucleotide polymorfisms, need more SNP’s then µSAT Loci
- Development tool: Reduced Representation Sequencing (RRS) or Restriction site Associated DNA sequencing (RADtag)
- Work flow for RRS:
DNA extraction -> pool DNA #indiv per loc/timepoint (RRS) – no pooling for RADtag 1 indiv per location/timepoint -> RE- digest -> size selection -> library prep &
Sequencing -> compare to reference genome and check for SNP’s per location
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Mnemiopsis leidyi: Ecology, Modelling and Observation
3. Migration patterns of Mnemiopsis leidyi individuals.
- Results :
- Microsatellite analysis:
- 7 primer couples for 7 loci were tested in 2 pools (Pool 1: 3 loci, pool 2: 4 loci) - 2 separate PCR’s and Fragment analysis’ were performed - Allel number analysis per loci was analysed with Genemapper software (ABI) - Results:
- data in chromatograms not fit for analysis. - No reference data from Reusch was obtained. (Reusch sampled ML individuals
from Oostende Spuikom, no data was obtained from his analysis).
- Development of SNP’s:
- Literature: Reduced Representation Sequencing (RRS) was chosen above RADtag (RRS individual variation between ML specimens is reduced, population markers are made visible)
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Mnemiopsis leidyi: Ecology, Modelling and Observation
3. Migration patterns of Mnemiopsis leidyi individua
- Development of SNP’s:
- # Tests: - 5 dna ext from 5 indiv was pooled and cut with different RE - HaeIII (4-cutter, blunt) was selected - A BioAnalyser analysis of the size distribution was done at the VIB-Genomics
Core - Conditions were OK
- Final Analysis: - DNA from different indivuals from 27 locations was pooled into 27 separate
samples covering all sampling locations / different time points. - HaeIII restriction digest was performed.
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Mnemiopsis leidyi: Ecology, Modelling and Observation
3. Migration patterns of Mnemiopsis leidyi individuals.
- DNA from different indivuals from 27 locations was pooled into 27 samples. (samples were confirmed as ML)
- HaeIII restriction digest was performed and shipped to VIB-Genomics core on dry ice.
- No results upon till now. - Problems:
- Size distribution could not be verified on BioAnalyser. - Ampure Bead selection of desired fragments (300-700bp) failed. - Keygene patent on workflow: RE-cut ->size selection -> genomic
analysis. (license expensive, VIB will not purchase this)
- Solution: - Perform size selection with Gel purification (1000-1500b is still ok
for Illumina Sequencing) at ILVO -> MiSeq Run at VIB.
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Mnemiopsis leidyi: Ecology, Modelling and Observation
4. Population structure and fylogeny of Mnemiopsis leidyi populations.
- Goal:
- To develop an insight in the structure of ML populations within the InterregIISeas area..
- Strategy:
- Sequence mitochondrial DNA marker genes (cytochrome b, cytochrome oxidase I). - Use NGS data from migration pattern study.
- Results:
- Mitochondrial markers: - Primers for cytochrome b and cytochrome oxidase I were designed and tested on 80
samples - A high degree of conservation was observed from the data.
- NSG data not available due to technical problems. - Literature: high reproduction rate in ML is not in favor of high within population variation.
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Mnemiopsis leidyi: Ecology, Modelling and Observation
5. Study of the genomic and chromosomal organisation within Mnemiopsis leidyi indivuals.
- Goal:
- Link whole mt and nuclear genome sequence data (present from literature) to chromosomal organisation
- Strategy:
- Karytoyping: Take 10.000 eggs from ML, transfer them as soon as possible (they hatch within 24hr), arrest cellular division, lyse cells gently by mixing in a hypotonic solution -> spread on glass plate and colour DNA -> view chromosomes on microscope.
- Genome size estimation: on lobe tissue, obtain individual cells, colour DNA with fluorescent dye, measure and separate on the FACS (fluorescence activiated cell sorter), estimate amount of DNA on a standard (known size) series.
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Mnemiopsis leidyi: Ecology, Modelling and Observation
5. Study of the genomic and chromosomal organisation within Mnemiopsis leidyi indivuals.
- Karyotyping: - Difficult to get eggs in the right condition and of good quality - Last results showed small chromosomes but spread on the glass plate is still not
optimal to get a good result.
- Genome size estimation: - First tests show a genome size of 250MB (genetic sequence: 150MB-> difference due to
repetitive and centromeric DNA?).
- Problems: genome was smaller then the smallest standard, extrapolation was done on a logaritmic scale.
- Linear scale testing with new animal standard needs to be repeated (resulst were not consistent)
- Flow cytometry on gDNA from B. gracilis, M. leidyi and Pleurobrachia pileus against C.elegans and tomato standard, showed that ML has a DNA content half the size of B. gracilis but double the size of PP. (preliminary results)
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Mnemiopsis leidyi: Ecology, Modelling and Observation
6. Still to do in December:
- Analyse the remaining fish stomachs from IFREMER
- SNP-development: - Try size selection with gel-fractionation (ILVO) - If successful: MiSeq run on limited samples (proof of working principle)
- Input of reference sequences for Mnemiopsis leidyi and Pleurobrachia pileus.
- Finish article on findings of genome size with flow cytometry
- Finish DNA analysis of new Lovenella sp. sample