GENETIC IDENTIFICATION OF FISH EGGS IN FORMALDEHYDE-

FIXED PLANKTON SAMPLES

MARINEGGS

5FP- Reference QLK5-CT1999-01157

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, Spain2 AZTI, Basque Country, Spain

3 IPIMAR, Portugal4 Aristotle University of Thessaloniki, Greece

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

Mackerel eggs

Hake eggs

Atlantic hakeMerluccius merluccius

Four spotted megrimLepidorhombus boscii

MegrimLepidorhombus whiffiagonis

MackerelScomber scomber

Spanish mackerelScomber japonicus

Atlantic horse-mackerelTrachurus trachurus

Snipe fishMacrorhamphosus scolopax

WhitingMerlangius merlangus

SardineSardina pilchardusNine species of

commercial interest

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

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

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

Comparing DNA extraction protocols (formaldehyde-fixed eggs)

Protocol Time required

Cost % success

Others

Chelex 120 min 25 € 97.1% Manual

QIAmp 360 min 200 € 90% Possible robotic

extraction

A sequence within the 16S rDNA gene, amplified with the primers:

• 16S-A:

5’-TGTCTTCGGTTGGGGCGA-3’

• 16S-B:

5’-GCTGTTATCCCTGGGGTAAC-3’

The marker

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)

Merluccius merluccius: 155 bp (154-156)Lepidorhombus whiffiagonis: 168 bp (167-170)Lepidorhombus boscii: 171 bp (171-174)

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

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

SSCP patterns obtained at the complete 16S

rRNA genes for different fish species 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)

Another technique for egg identification

SSCP technique works in formaldehyde-fixed eggs

1-2 3-5 6-12 13-19

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

Identification of 69 test eggs (Bay of Biscay)Fragments N Species identification Identification with

another marker

168 9 Lepidorhombus

whiffiagonis

-

155 2 Merluccius merluccius -

156 43 Macrorhamphosus

scolopax

-

160-164 3 Scomber spp 3 S. scomber

154 + 177 + 200 1 Unknown

160 + 164 + 169 5 Unknown

135 1 Unknown

178 1 Trachurus trachurus -

No amplification 4 - -

1 First marker: partial 16S rDNA gene, primers 16S-A & 16S-B

We have the markers

Do we really need genetic markers for egg identification?

Hake eggs

Overlapped spawning areas for M. merluccius, L. boscii and L. whiffiagonis

Visual vs genetic identification of hake/megrim eggs

Stage Sampling year

Reference Visual identification N Success Coincidence visual-genetics

Eggs 2002 Walter-Herring

M. merluccius 38 78.9% 63.3%

Eggs 1995 Lough Foyle M. merluccius 17 88.2% 100%

Eggs 1995 Tridens M. merluccius 15 93.3% 100%

Eggs 1995 Walter-Herring

M. merluccius 43 86% 94.6%

Eggs 1995 Johem Hjurt M. merluccius 12 75% 88.9%

Eggs 1995 Heinke M. merluccius 19 94.7% 50%

Eggs 1995 Cirolana M. merluccius 9 77.7% 85.7%

Eggs 1995 Scotia M. merluccius 24 91.7% 90.9%

Larvae 2002 LW batch B L. whiffiagonis 31 83.9% 0%

Larvae 2002 LB batch B L. boscii 12 75% 0%

Total 220 85% 67.3%

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” )

Acknowledgments

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

Thanks for your attention