COST Action FA1004, 4th Conference on Conservation Physiology of Marine Fishes, Thessaloniki, Greece
Population analyses (cox1 gene) and bile PAH
metabolites content of commercial fishes
along Adriatic coast
Ana BARIEVI1, Victor S. PERUSCO2, Nikola MEDI2, Bojan HAMER1
1 Ruer Bokovi Institute, Center for Marine Research, LMMB, Giordana Paliage 5, 52210 Rovinj, Croatia
2 Juraj Dobrila University of Pula, Preradovieva 1, 52100 Pula, Croatia
E-mail: baricevic@cim.irb.hr, hamer@cim.irb.hr
Introduction
Bile PAH metabolites content in selected
commercial species Mugil spp., Sarpa salpa
and Mullus barbatus purchased from local fish
markets (Fig. 1): Ploe (F2-Pl), Split (F4-St),
ibenik (F5-i), Rijeka (F8-Ri) and Rovinj (F9-
Ro) were analyzed as a biomarker of organism
exposure to oil and possible indicator of marine
environment quality (1, 2). Local fish markets
were used because was not possible to catch
chosen species at selected sites during sea
water and mussel sampling campaign in the
frame of our national biomonitoring pilot project
Coastal cities water pollution control project 2
(3).
Fishes were selected according their food
web positions: Mugil (water column, carnivore),
Sarpa (water column, herbivore) and Mullus
(benthic, detritovore) (Fig. 2).
PAH metabolites were analyzed in fish
bile by means of fixed wavelength fluorescence
(FF) measurement using fluorimeter excitation/
emission wavelength pairs 290/335, 341/383,
and 380/430 nm. By FF290/335, mainly
naphthalene type of metabolites, typically
associated with petroleum products are
detected, benzo[a]pyrene type of metabolites
are best detected by FF380/430 and by
FF341/383 mainly pyrene-derived metabolites.
The population analysis of mitochondrial
reference marker gene cytochrome oxidase
subunit 1 (CO1) was applied as tool for fish
specimens identification (barcoding) and
determination of abundance of possible
haplotypes (4).
Figure 1. Map of investigated areas covered by fisheries
monitoring (dot - fish market, circle fishing area)
C
Figure 3. Spatial distribution bile PAH Naphthalene-type
metabolites determined in fish Mugil spp., Sarpa salpa
and Mullus barbatus.
Methods
-Fluorimetric fish bile PAH metabolites
determination (FF290/335, 341/383 and
380/430 nm)
-genomic DNA preparation of liver tissue Figure 5. Mugilidae species seatled in the Adriatic Sea.
-P C R a m p l i f i c a t i o n , p u r i f i c a t i o n a n d
sequencing of CO1 gene using universal fish
barcoding primer pair: F 5'-TCGACTAA
Results & Discussion
TCATAAAGATATCGGGAC-3 and R 5-ACTT C D
CAGGGTGACCGAAGAATCAGAA-3' The results sort out NAPH-type metabolites as
-NCBI BLAST service major measures exposure of fish to two-ring PAHs
-Sequence alignments and analysis (Geneious where the majority of chemicals are NAPH and its
7.1.7, Biomatters Ltd.). derivatives (Fig. 3). Further total sum values of all
three fish species ranked fish contaminant
exposure at investigated areas as follows:
F5>F9>F4>F2>F8 (without values of fish Sarpa
salpa: F5> F4>F8>F2>F9). So, we can say Rovinj
area (F9) is less contaminated and ibenik (F5)
has a higher pollution load on the basis of NAPH-
type metabolites analyses.
Total fish species average NAPH-type
metabolites values indicated that fish Sarpa salpa
has in general highest contaminant loads
(36790), Mullus barbatus (32352), and then
A) Mugil spp. (22062). Further, results indicate
B)
C)
mostly naphthalene-type fish exposure (pollution)
cca 10-30 times higher values for FF290/335
(30491), than two other types FF341/383 (246)
and FF380/430 (144).
The barcoding analysis of CO1 gene was
applied to identify purached fish species, because
identification is difficult in case of close related
fishes, and especially when morphological
characters have been removed during sampling.
The PCR amplification of CO1 gene of the Red
Mullet (Mullus barbatus) was not succesful. The
Salema (Sarpa salpa) was proved as single
species with few haplotypes at whole investigated
area, but fishes purchased as Mugil spp. were
identified by sequencing and DNA analyses
(blastn, NCBI) as 3 species (Liza aurata, L.
ramada and Chelon labrosus) (Fig.4). The
morphometric characteristics of family Mugilidae
Figure 2. Comercial fishes purached from local fish markets species settled in Mediterranean (8) are minimal
along Adriatic coast: A) Mullus barbatus, B) Sarpa salpa and and only 6 of them inhabited the Adriatic Sea (Fig.
C) Mugil spp.. 5).
.
Figure 4. Philogenetic tree and CO1 sequence alignment of fishes purached as Mugil spp. with NCBI blastn match: Liza
aurata KC500813, EU392235, L. ramado KC349864, Cheon labrosus JF911706 and outcamer Mugil cephalus KC500933.
References
1) Coastal cities water pollution control project 2, Part 3.2 Sea water quality monitoring, MENP/M-C-1 Consulting services for the
definition of the Monitoring and Observation system for ongoing assessment of the Adriatic Sea, 2 IBRD 7640/HR, 2013 2014.
2) Aas E., Baussant T., Balk L., Liewenborg B., Andersen O.K. (2000) PAH metabolites in bile, cytochrome P4501A and DNAadducts as
environmental risk parameters for chronic oilexposure: a laboratory experiment with Atlantic cod. Aquatic Toxicology 51: 241258.
3) Lin E.L.C., Cormier S.M., Torsella, J.A. (1996) Fish biliary polycyclic aromatic hydrocarbon metabolites estimated by fixed-wavelength
fluorescence: comparison with HPLC-fluorescent detection. Ecotoxicology and Environmental Safety 35: 16-23.
4) Ward R.D., Hanner R., Hebert P.D.N. (2009) The campaign to DNA barcode all fishes. FISH-BOL, Journal of Fish Biology 74: 329-356.