GENETIC IDENTIFICATION OF FISH EGGS IN FORMALDEHYDEFIXED PLANKTON SAMPLES

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GENETIC IDENTIFICATION OF FISH EGGS IN
FORMALDEHYDE-FIXED PLANKTON SAMPLES MARINEGGS
5FP- Reference QLK5-CT1999-01157
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Garcia-Vazquez E.1, Perez J.1, Martinez J. L.1,
Alvarez P.2, Lopes P.3, Gomes L.3, Teia A.3,
Karaiskou N.4, Triantafyllidis A.4 and
Triantaphyllidis C.4
1 University of Oviedo, Spain 2 AZTI, Basque
Country, Spain 3 IPIMAR, Portugal 4 Aristotle
University of Thessaloniki, Greece
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The problem Spawning of commercially important
fish species overlap in time and space Stock
biomass estimation is likely biased by ambiguous
identification of eggs and larvae
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Mackerel eggs
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Hake eggs
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Nine species of commercial interest
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Handling formaldehyde-fixed eggs

• Protocol A
• Rinse eggs in PBS twice
• Put individual eggs in a filter paper. Squash
  with another paper
• Cut the paper around the egg and put it in an
  Eppendorf
• Follow to Chelex or to Qiagen (QIAamp DNA Mini
  Kit)
• Protocol B
• wash individual eggs in PBS for 2-3 min
• squash the egg with a pipette tip

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DNA extraction protocols for egg samples

• Proteinase K and phenol/chlorophorm (Taggart et
  al. 1992)
• 2 x CTAB/proteinase K
• Resine extraction (12 Chelex)
• Bilatest DNA kit
• QIAmp mini Kit

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Our Chelex protocol

• Embed the egg in 150 µl of 12 Chelex 20 µl
  proteinase K at 55ºC for at least 1 h
• Inactivate proteinase K at 100ºC for 20 min

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Comparing DNA extraction protocols
(formaldehyde-fixed eggs)
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A sequence within the 16S rDNA gene, amplified
with the primers
The marker

• 16S-A
• 5-TGTCTTCGGTTGGGGCGA-3
• 16S-B
• 5-GCTGTTATCCCTGGGGTAAC-3

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Methods

• PCR amplification (conditions detailed in Perez
  et al. 2005)
• Fluorescent fragment detection by capillary
  electrophoresis in a 3100 Genetic Analyzer
  (Applied Biosystems) with a 36 cm capillary and
  POP 4 polymer
• Fragment sizes established using the GeneScan 3.7
  Analysis Software (Applied Biosystems)

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Merluccius merluccius 155 bp (154-156)Lepidorhom
bus whiffiagonis 168 bp (167-170)Lepidorhombus
boscii 171 bp (171-174)
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Fragment sizes obtained for partial 16S rRNA
sequence in different species employing the
primers 16S-A and 16S-B

• Species Fragments (bp)
• Trachurus trachurus 178 ( 163-164)
• Macrorhamphosus scolopax 156
• Scomber scomber 160-164 ( 148-150)
• Scomber japonicus 160-164 ( 148-150)
• Sardina pilchardus 156 142
• Merlangius merlangus 157-158
• Merluccius merluccius 155
• Lepidorhombus whiffiagonis 168
• Lepidorhombus boscii 171

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Additional markers for Scomber spp.

• SST F 5 TGTCATCACTAACCTACTCTCA 3
• SST R 5 GGTGGAGAACCGCTGCCGCTAA 3
• SJT F 5 ATTCGTTATCCTGGCAGCAACAA 3
• SJT R 5 TGCGAGAGAGGAGAGGGCCACG 3

SST F R, S. scomber SJT F R, S. japonicus
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SSCP patterns obtained at the complete 16S rRNA
genes for different fish species
Another technique for egg identification
1 Macrorhamphosus scolopax 2 Scomber scomber
3 Trachurus trachurus 4 Lepidorhombus boscii
5 L. whiffiagonis 6 Merluccius merluccius 7
Merlangius merlangus 8 Molva molva 9
Pollachius virens 10 Pollachius pollachius 11
Gadus morhua 12 formaldehyde-fixed egg
(Trachurus trachurus)
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SSCP technique works in formaldehyde-fixed eggs
PCR-SSCP patterns found for Lepidorhombus
whiffiagonis eggs fixed in ethanol (6-12) or in
4 formaldehyde (13-19). Control adults 1-2,
Lepidorhombus whiffiagonis 3-5, Merluccius
merluccius
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Identification of 69 test eggs (Bay of Biscay)
First marker partial 16S rDNA gene, primers
16S-A 16S-B
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We have the markers

• Do we really need genetic markers for egg
  identification?

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Hake eggs
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Overlapped spawning areas for M. merluccius, L.
boscii and L. whiffiagonis
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Visual vs genetic identification of hake/megrim
eggs
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Future perspectives

• Project FISH CHIPS (6FP) Towards DNA chip
  technology as a standard analytical tool for the
  identification of marine organisms
• Cordis Technology Marketplace (http//www.cordis.
  lu/marketplace/home.html Offer in
  Biology/Medicine Species-specific fish
  assessment )

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Acknowledgments

• Ivan G. Pola (University of Oviedo) helped in
  laboratory tasks

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Thanks for your attention