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ICES 1966
MARICULTURE COMMITTEE
CM 1996/ F: 8
RAPID FLOW CYTOMETRY METHOD FOR
TRIPLOIDY DETERMINATION IN TURBOT
(Scopthalmus maximus L.)
Vazquez, E.; Fernandez*, C.; Martinez*, I.; Blanco, G.; and Sanchez, J.A.
Universidad de Oviedo.
Departamento de Biologia Funcional. Genetica.
Julian Claveria s/n. 33071 Oviedo. SPAIN
*Instituto Espaöol de Oceanografia.
Centro Oceanografico de Santander.
39080 Santander. SPAIN
•,
ABSTRACT
Aceurate methods for assessing triploidy are important because treatments designed to
induce poliploidy are not 100% and offspring from treated eggs are usually a mixture
oftriploids and diploids.
The most common method is kariotype preparation and ehromosome eounting. Flow
cytometry was more recently applied to triploidy determination and usually carried out
using erythrocytes.
This paper deseribes a rapid and simple method for triploidy determination from the
larval stage ofturbot (Scopthalmus maximus L.) by fIow cytometry.
High-quality histograrns were obtained from turbot larvae and the very good
reproduetibility provided unambigous diserimination of diploids and triploids even in
early stages of development.
Keywords: fIow cytometry, larval stage, triploidy, turbot.
I. INTRODUCTION
Produetion of sterile fish has become a major coneern in aquaculture,
because as sterility in commercial fish stocks has the potential to inerease production
yields, as metabolie energy whieh would otherwise be used for gonadal development
is redireeted to somatic growth (Nagy, 1987). Another advantage is the avoidanee of
•
reproduction in farmed species whieh beeome sexually mature before reaehing an
econorruc Slze.
Triploids are generally expeeted to be sterile due to the likely failure of
chromosomes to pair correcdy during meiosis. (Benfey and Sutterlin, 1984). Triploidy
can be indueed by disruption of developmental events post-fertilization, to prevent
extrusion of the second polar body, thus retaining a diploid matemaI set within the
ovum. A variety of teehniques has been developed to induee triploidy in fish (for
review see Thorgaard and Allen, 1987; Thorgaard, 1992; Tave, 1993).
Aceurate methods for assessing triploidy are important because treatments
designed to induce poliploidy are seldom 100% effective (Allen and Stanley, 1979;
Chourrout, 1980; Thorgaard et aI., 1981) and offspring from treated eggs are
usu~ly
a mixture oftriploids and diploids.
The most common method is kariotype preparation and chromosome
counting (Chourrout and Hape, 1986). Other current methods involve microscope
cytophotometry (Gervai et a1., 1980) or indirect evaluation from erythrocyte nucleus
diameters or volume (Chourrout et a1., 1986; Child and Watkins, 1994). All these
methods are tedious and time-consuming, and determination is made from a limited
number of cells.
Flow cytometry was more recently applied to triploidy determination and
usually carried out using erythrocytes (Allen, 1983; Crozier and Moffett, 1989). This
is only possible when well-grown fish are used, but early knowledge of triploidy
induction results can be advantageous.
The present research describes a rapid and simple method for triploidy
determination from the larval stage of turbot (Scopthalmus maximus L.) by flow
cytometry.
11. MATERIAL AND METHODS.
Reproductive1y mature fish were obtained from the Centro Oceanogräfico de
Santander, where a domesticated turbot stock is maintained.
•
Eggs and milt were stripped from females and males and fertilization was
carried out in plastic basins. The result of fecundation were divided into experimental
batches. Triploidy was induced by exposing eggs to heat shock.
Treatments were administered by transferring batches as required to a
temperature regulated water bath equipped with mechanical water circulation to assist
temperature equilibration. In each experiment one batch was not subjected to heat
shock and was used as the control group.
After treatment, eggs were immediately transferred to incubation tanks in the
laboratory and reared at room temperature until hatching. Tanks were checked
frequently and dead eggs removed.
•-
On day 5 or 6 post-fertilization (at the moment of hatching), sampIes of 20
larvae were collected in order to determine the ploidy level. The method used was
based on the measurement ofnuclear DNA content by flow cytometry.
The procedure used in the sampIe preparations was previously developed in
human tissues by Vindel0V (1983).
Larvae of each sampIe were gently disrupted into 1 1nI. of citrate buffer (pH
7.6) and 500 pI of the rnixture were then centrifuged for 10 minutes at 1,200
revolutionsImin. . The supernatant was discarded and the pellet was resuspended in
300 pI of a solution with NP-40, spermine and trypsin during 10 minutes at room
temperature. A second solution with trypsin inhibitor and Rnasa was added forIO
minutes. At last, nucleus were stained for 10 minutes using a solution of the
propidium iodide and spermine light protection.
These preparations were then analysed with a Cytorom absolute (Ortho
diagnostic systems).The apparent ploidy of each sampIe was calcuIated from the
relative proportion of triploid ceIIs to the total number analyzed by the curve-fitting
program ModFit (Verity Software House).
III. RESULTS AND DISCUSSION
Analysis of larval sampIes yielded data from a population of approximately
•
2,000 larvae. The calculation of apparent ploidy assurnes that each larva, diploid and
triploid, makes an approximately equal contribution of ceIIs to the overall pool of
ceIIs, a reasonable assumption as the larval size is very similar.
To check this, a known sampIe of 5 individuals (3 diploids and 2 triploids)
was analyzed by flow cytometry (FC!v1). The results (Figure lA), indicated that there
was a good agreement between the frequency of each cell population (58.77 and
41.23% respectively) and the number of individuals belonging to the different cIasses
present in the sampIe.
Flow cytometry was carried out for 100 sampIes, of which 65 were shown to
be mixtures oftriploid and diploid individuals.
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The least squares curve-fitting methods of ModFit produced estimates of
mean relative fluorescence (relative DNA content), coefficients of variation (CV) of
fitted curves, and relative number of events in each population.
Examples of histograms from diploid and triploid of turbot larvae and their
statistical values are shown in Figures 2A and 2B respectively. In these sampIes only
one cell population was found at channels 51.56 and 75.51.
Two cell populations, well differentiated, were evident from FCM analysis of
some treated sampIes (Fig. IB). Channel values observed for peak means (76.72 for
triploid, 51.18 for diploid) correspond to a fluorescence ratio of 1.5, identical to the
ploidy ratio (3
~
2). This calculation was possible as the ordinate (channel number) is
a sufficiently linear representation ofDNA content (Allen, 1983).
The mean model channel number of the diploid cell populations examined
was 52.06
± 0.68, while that ofthe triploids was 78.05 ± 0.27, giving a DNA ratio
1.488 ±. 0.016, elose to the expected 2:3 ratio of diploids-triploids, corfirming the
latter group as triploid.
One of the most important criteria for determining if a histogram is possible
to evaluate for ploidy status is CV ofthe mean DNA content cell population. Hs value
corresponds to the ability to resolve two different DNA content populations in the
same sampIe. Histograms with values of 8% are generally acceptable to most
research ( Allen, 1983; Johnson et al., 1987; Crozier and Moffett, 1989; Allen and
•
Bushek, 1992;). In our work the Cv range ofvariation was from 3.17 to 5.15% in
diploid cell populations and from 1.48 to 4.33% in triploid cell populations.
In conelusion, cytofluorometry proved to be a fast procedure for evaluating
polyploidy. High-qtiality histograms were obtained from turbot larvae and the very
good reproductibility provided unambigous discrimination of diploids and triploids
even in early stages of development.
REFERENCES
Allen, S.K. 1983. Flow cytometry: assaying experimentat polyploid fish and shellfish.
Aquaculture,33:317-328.
•
Allen, S.K. and Stanley 1G. 1979. Polyploid mosaics induced by cytochalasin Bin
landlocked Atlantic salmon. Transac!ions o/the American Fisheries Soeiety, 108:
462-466.
Allen, S.K. Jr. and Bushek, D. 1992. Large-scale production oftriploid oysters,
Crassostrea virgillica (Gme1in), using "stripped gametes". Aquaculture, 103:241251.
Benfey, T.J. and Sutterlin, AM.1984. Growth and gonadal development in triploid
landlocked Atlantic salmon (Salmo salar). Can. J. Fish Aquat Sei. 41: 1387-1392.
Crozier, W.W. and Moffett, 1.1.1. 1989. Experimental production oftriploid hrown
trout, Salmo tmtta L., using heat shock. Aquaculture and Fisheries Management,
20: 343-353.
Child, AR. and Watkins, RP. 1994. A simple method to identify triploid molluscan
bivalves by the meas~rement of cell nucleus diameter. Aquaculture, 125: 199-204.
Chourrout, D. 1980. Thermal induction ofdiploid gynogenesis and triploidy in the
eggs ofthe rainbow trout (Salmo gairdneri Richardson). Reprod Nutr. Develop.,
20: 727-733.
Chourrout, D. and Happe, A 1986. Improved methods of direct chromosome
preparation in rainbow trout, Salmo gairdneri. Aquaculture, 52: 255-261.
Chourrout, D.; Chevassus, B.; Kreig, F.; Happe, A; Burger, G. and Renard, P. 1986.
Production of second generation triploid and tetraploid rainbow trout by mating
•
tetraploid males and triploid females- Potential oftetraploid fish. Theor. Appl.
Genet.,72: 193-206.
Gervai, 1; Peter, S.; Nagy, I.A; Horvath, L. and Csanyi, V. 1980. Induced triploidy
in carp, C>prinus carpio L. Joumal 0/ Fish Biology, 17: 667-671.
Johnson, O.W.; Utter, F.M. and Rabinovitch, P.S. 1987. Interspecies differences in
salmonid cellular DNA identified by flow cytometry. Copeia, n° 4: 1001-1009.
Nagy, A1987. Genetic manipulation performed on warm water fish. In: K.
Tiews(Editor). Proc. World Symp. Selectioll, Hybridization and Genetic
Engineering in Aquacllltlire. Bordeaux, 27-30 May 1986. Heenemann, Berlin,
pp. 127-145.
Tave, D. 1993. Genetics for Fish Hatchery Managers, 2nd. Edn, Van Nostrand
Reinhold. New York.
•
Thorgaard, G.H 1992. Aplication of genetic technologies to rainbow trout.
Aquaculture, 100: 85-97.
Thorgaard, G.H. and Allen, Jr.,S.K. 1987. Chromosome manipulation and markers in
fishery management. In: N. Ryman and F.M.Utter (Editors). Population Genetics
and Fishery Management. University ofWashington Press, Seattle, pp. 319-331.
Thorgaard, G.H.;Jazwin, M.E. and Stier, AR. 1981. Polyploidy induced by heat
shock in rainbow trout. Transactions 0/ the American Fisheries Society, 110: 546550.
Vindel0V, L. 1983. A detergent-trypsin method for the preparation ofnuc1ei for flow
cytometry analysis. Cytometry, 3: 323-327.
•
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