Micron 38 (2007) 214–217
www.elsevier.com/locate/micron
A standard protocol for obtaining fish chromosomes
under post-mortem conditions
Maria Rita de Cáscia Barreto Netto a, Erica Pauls b,
Paulo Roberto A. de Mello Affonso c,*
a
Laboratório de Biologia e Genética de Peixes, Instituto de Biociências,
Universidade Estadual Paulista, 18 618-000 Botucatu, SP, Brazil
b
Departamento de Zootecnia, Universidade Federal Fluminense, 24 210-470 Niterói, RJ, Brazil
c
Departamento de Ciências Biológicas, Universidade Estadual do Sudoeste da Bahia, UESB,
Rua José Moreira Sobrinho s/n, Jequiezinho, Jequié-BA, CEP 45 200-000, Brazil
Received 15 June 2006; received in revised form 12 July 2006; accepted 13 July 2006
Abstract
In order to improve cytogenetical analyses on fish, especially focusing on delicate and rare species, we have adopted a new in vitro methodology
using dead animals. The results obtained from 24 neotropical freshwater and marine fish species demonstrate that chromosomes can be obtained
under post-mortem conditions. Significantly, the samples analyzed provided reliable cytogenetical data in nearly all cases. Other advantages of this
new methodology are also discussed.
# 2006 Elsevier Ltd. All rights reserved.
Keywords: Chromosomes; Freshwater fish; Marine fish; In vitro cytogenetical technique
1. Introduction
Cytogenetical studies on fish have been useful to provide
information concerning evolutionary and taxonomic studies, as
well as for the genetic improvement of commercial fish stocks
(Gold, 1979).
Several techniques have been devised to obtain mitotic
chromosomes in fish, ranging from direct preparations to longterm cell culture (Denton, 1973; Ojima, 1982; Alvarez et al.,
1991, among others). Among these methodologies, in vivo
procedures, usually time- and cost-saving, have been the most
widespread (Egozcue, 1971; Gold, 1974; Rivlin et al., 1985).
However, to perform direct techniques, it is necessary to
carry out a previous colchicine treatment of live animals for
about 1 h. Very often, this is a not a feasible method, as in the
case of delicate/fragile species after lengthy transportation or
species requiring specially suited tanks (e.g., marine species).
Consequently, most of the available cellular material can be
* Corresponding author. Fax: +55 3525 6683.
E-mail address: [email protected]
(P.R.A. de Mello Affonso).
0968-4328/$ – see front matter # 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.micron.2006.07.019
lost (Ozouf-Costaz and Foresti, 1992; Maddock and Schwartz,
1996).
The development of a new methodology, based on the
procedures previously described by Gold et al. (1990),
Fenocchio et al. (1991), and Foresti et al. (1993), which
allows post-mortem cytogenetical analyses contributes to
chromosomal research on fish, particularly by increasing
sample size. This technique, formerly focused on the
improvement of chromosomal studies in very large or tiny
species, has now been extended and applied successfully to
neotropical freshwater and marine fish species. In this report,
we describe in detail the new methodology and present some
representative data.
2. Material and methods
The species studied comprise 15 Perciformes, 2 Mugiliformes, 1 Clupeiformes, 1 Lophiiformes, 1 Pleuronectiformes, 2
Characiformes, 1 Siluriformes and 1 Gymnotiformes (Table 1).
The marine species were collected on the Atlantic shore, along
the coast of the states of Bahia and Rio de Janeiro (northeastern
and southeastern Brazil, respectively). Freshwater fishes were
collected in the Itabapoana River, in southeast Brazil.
M.R. de Cáscia Barreto Netto et al. / Micron 38 (2007) 214–217
215
Table 1
Analyzed fish species and respective fishing methods
Order
Family
Species
Fishing method
Anostomidae
Prochilodontidae
Leporinus sp.
Prochilodus vimboides
Set gill net
Set gill net
Sternopygidae
Cichlidae
Eigenmannia sp.
Geophagus brasiliensis
Set gill net
Set gill net
Engraulidae
Lophiidae
Cetengraulis edentatus
Lophius gastrophysus
Drift net
Drag-of-fund net
Mugiliformes
Mugilidae
Mugil curema
Mugil incilis
Drift net
Drift net
Perciformes
Carangidae
Alectis ciliaris
Chloroscombrus chrysurus
Selene setapinnis
Selene vomer
Trachinotus goodei
Drag-of-beach
Drag-of-beach
Drag-of-beach
Drag-of-beach
Drag-of-beach
Centropomidae
Centropomus undecimalis
Hook and line
Gerreidae
Diapterus rhombeus
Eugerrus brasilianus
Set net
Set net
Haemulidae
Anisotremus virginicus
Haemulon aurolineatum
Diving fishing
Hook and line
Mullidae
Mullus argentinae
Upeneus parvus
Drag-of-fund net
Drag-of-beach net
Pomacanthidae
Sparidae
Pomacanthus paru
Pagrus pagrus
Snorkeling
Drag-of-fund net
Soleidae
Ariidae
Gymnachirus melas
Genidens genidens
Drag-of-fund net
Hook and line
Freshwater species
Characiformes
Gymnotiformes
Perciformes
Marine species
Clupeiformes
Lophiiformes
Pleuronectiformes
Siluriformes
Chromosomal preparations were performed as follows:
(a) Extract small pieces of kidney, gills and/or spleen, and
rinse in RPMI 1640 culture medium.
(b) Cut the pieces into small tissue fragments in a small plate,
containing 5 ml of cooled RPMI medium (4 8C), and
transfer the solution to a centrifuge tube, adding RPMI to a
final volume of 9.5 ml. The material immersed in RPMI
might be kept cool for several hours (i.e., up to 12 h) prior
to performing the following steps.
(c) Add five drops of colchicine 0.05% (w/v) or 0.016% (w/v).
(d) Mix the solution well and keep the centrifuge tube for 30–
35 min at room temperature (20 8C).
(e) Centrifuge the material at 1000 rpm for 10 min and discard
the supernatant.
(f) Add 10 ml of hypotonic solution (KCl 0.075 M).
(g) Mix the solution and keep it for 20 min at room
temperature.
(h) Add five drops of Carnoy’s fixative (methanol:acetic acid
3:1 at 4 8C).
(i) Centrifuge the material at 1000 rpm for 10 min and discard
the supernatant.
(j) Add 6 ml of fixative (freshly prepared) to each centrifuge
tube at room temperature.
net
net
net
net
net
(k) Centrifuge the material at 1000 rpm for 10 min and discard
the supernatant.
(l) Repeat steps (j) and (k) twice.
(m) After the last centrifugation, discard the supernatant and add
Carnoy’s fixative at a ratio of 1:1 (v/v) to the pellet. Mix the
solution until a homogeneous cell suspension is obtained.
(n) Put three drops of the cell suspension onto a glass slide,
covered with a thin water layer at 60 8C.
(o) After air-drying, stain the material with 5% Giemsa
solution for 8 min and then wash the slides under tap water.
3. Results and discussion
The number and quality of metaphase chromosomal spreads
obtained were variable among individuals and species. Such
variation indicates that species-specific technical requirements
must be developed or adapted for each fish group. On average,
however, independent from the fishing method, satisfactory
results were obtained using the new protocol for different
species, as suitable chromosomal preparations were obtained
for 83.1% of the 190 specimens analyzed (N1) (see data in
Table 2 and Fig. 1).
The post-mortem time of individuals analyzed ranged from
some few minutes to few hours (usually about 1 h). However, in
216
M.R. de Cáscia Barreto Netto et al. / Micron 38 (2007) 214–217
Table 2
Evaluation of mitotic chromosomal preparations from different fish species, obtained under post-mortem conditions
Species
N1
Tpm
N2
Material quality
2n
Leporinus sp.
Prochilodus vimboides
Eigenmannia sp.
Geophagus brasiliensis
Cetengraulis edentatus
Lophius gastrophysus
Mugil curema
Mugil incilis
Alectis ciliares
Chloroscombrus chrysurus
Selene setapinnis
Selene vomer
Trachinotus goodei
Centropomus undecimalis
Diapterus rhombeus
Eugerrus brasilianus
Anisotremus virginicus
Haemulon aurolineatum
Mullus argentinae
Upeneus parvus
Pomacanthus paru
Pagrus pagrus
Gymnachirus melas
Genidens genidens
7
7
3
1
20
2
1
1
4
8
18
5
4
12
13
1
14
5
4
31
17
9
1
2
20 min
1h
20 min
1h
1h
1h
1h
>2 h
20 min
40 min
40–50 min
30 min
2h
2h
1 h and 30 min
>1 h
1–2 h
1–2 h
1h
40 min
1 h–2 h
1–2 h
40 min
20 min
7
5
3
1
5
2
1
1
2
4
9
2
3
12
6
1
9
4
4
13
14
5
1
2
++++
++++
++++
+++
++
++++
+
++++
++
+++
++++
++
++
++++
++
+++
+++
+
+++
+++
++++
+++
++
++++
54
54
38
48
48
48
28
28
48
48
46
48
48
48
48
48
48
48
44
44
48
48
36
54
(N1) Number of collected specimens; (Tpm) mean post-mortem time; (N2) number of individuals with satisfactory results. (+) Reduced number of well defined
metaphases per slide; (++) about 10 metaphases per slide; (+++) 20–30 metaphases per slide; (++++) more than 30 metaphases per slide.
some cases, as in Mugil incilis, this period could not be clearly
defined since, when caught from the net, the specimen was
already dead and the post-mortem period was not precisely
estimated. Even so, such sample provided some of our best
results (Table 2 and Fig. 1a).
We were unable to establish any correlation between postmortem time and cytogenetical results, and statistical analysis
would probably not be helpful, due to the variation in sample
size between the different fish groups and species. Moreover,
the majority of the data came from animals analyzed 1 h after
death. Nevertheless, the intention of this work is to
demonstrate that satisfactory chromosomal preparations can
be obtained from dead animals. This might be expected, since
the cells of some tissues are still viable, in short- or long-term
culture for a few hours post-mortem (Maddock and Schwartz,
1996).
It must be emphasized that, using this protocol, cytogenetical studies can be performed in field, as long as the material is
kept in cool RPMI medium for up to 12 h, with no significant
cell death. Thus, solid tissues from dead fish can easily be
collected from their natural habitat and transported, on ice, to
the laboratory. We have successfully performed this with
several species, such as Anisotremus virginicus, Prochilodus
vimboides and Pagrus pagrus (Fig. 1b–d). Thus, it is possible
to increase significantly the number of analyzed animals,
permitting one to perform cytogenetical studies on nearly
every specimen collected.
Furthermore, some metaphases figures obtained by this
method were even superior in quality when compared to those
obtained by conventional air-drying technique (e.g., carangid
and mullid species). This is probably caused by the more
precise control of some steps in this modified methodology,
mainly the colchicine and hypotonic treatments, crucial to
obtain good chromosomal preparations (see Maddock and
Schwartz, 1996).
In conclusion, we highlight some of the major advantages of
this methodology as follows:
(1) The utilization of most fish specimens for chromosomal
preparations, with a consequent increase in sample size.
This leads to an extended characterization of fish species
and populations, with the production of more reliable
cytogenetical information, particularly on those species that
have previously presented difficulties for obtaining good
cytogenetical results, such as marine species (Galetti et al.,
2000).
(2) The possibility of working with large fish or very fragile
specimens, without keeping such animals alive in the
laboratory.
(3) The protocol is inexpensive, able to provide very successful
results and is flexible for any researcher’s schedule, as it is
not necessary to perform it immediately following tissue
extraction.
We hope to encourage fish cytogeneticists to adopt this
methodology, with adaptation to their specific laboratory
conditions, which should lead to the improvement of data from
each fish group.
M.R. de Cáscia Barreto Netto et al. / Micron 38 (2007) 214–217
217
Fig. 1. Somatic metaphase spreads of Mugil incilis with 28 chromosomes (a), Pomacanthus paru with 48 chromosomes (b), Prochilodus vimboides with 54
chromosomes (c), and Anisotremus virginicus with 48 chromosomes (d).
Acknowledgments
The authors are grateful to fishermen from Niterói, Maricá
and Bom Jesus do Itabapoana and staff from IEAPM and
Department of Marine Biology-UFF for their support in
collecting animal samples. We would also like to thank Dr.
L.A.C. Bertollo and Mr. L.D.S. Abel for reviewing the
manuscript. Funds supporting this study were provided by
CNPq and IED-BIG.
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