MEMO - ilvo.vlaanderen.be€¦ · Keygene patent on workflow: RE -cut >size selection > genomic analysis. (license expensive, VIB will not purchase this) -Solution: -Perform size

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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The Use of Genomic Tools in

Finding Answers for Research Questions

Within the MEMO Project



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

?


1. Identification of Mnemiopsis leidyi and other ctenophores/jellyfish using molecular analysis.

Protocol

1: Extract DNA

2: PCR

3: Sequence

4: Blast & Results





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





- 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



Overview of samples used for migration experiment (SNP development). (Chesapeake Bay, Villefranche and Baia Santa Theresa not shown)


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)





- 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.




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




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




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.





- 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)





- 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





- 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)




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.





- 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.




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.




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.




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)




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



Documents

MEMO - ilvo.vlaanderen.be€¦ · Keygene patent on workflow: RE -cut >size selection > genomic analysis. (license expensive, VIB will not purchase this) -Solution: -Perform size