CompCytogen 9(1): 79-87 (2015) COMPARATIVE — Aveerrerewet open-access over

doi: 10.3897/CompCytogen.v9i |.8587 Kas Cyto genetics

http://compcytogen.pensoft.net International journal of Plant & Animal Cytogenetics,

Karyosystematics, and Molecular Systematics

B chromosomes in the species Prochilodus argenteus
(Characiformes, Prochilodontidae):
morphologicalidentity and dispersion

Manolo Penitente!, Fausto Foresti*, Fabio Porto-Foresti!

| Departamento de Ciéncias Biolégicas, Faculdade de Ciéncias, Universidade Estadual Paulista (Unesp), Campus
de Bauru, Bauru, SR Brazil 2 Departamento de Morfologia, Instituto de Biociéncias, Universidade Estadual
Paulista (Unesp), Distrito de Rubiao Junior, s/n, 18618-970, Botucatu, SP Brazil

Corresponding author: Fabio Porto-Foresti (fpforesti@fc.unesp.br)

Academic editor: G. Furgala-Selezniow | Received 15 September 2014 | Accepted 28 January 2015 | Published 20 March 2015
http://zoobank.org/83103179-4367-42A9-8621-ACA7043AA67F

Citation: Penitente M, Foresti FE Porto-Foresti F (2015) B chromosomes in the species Prochilodus argenteus
(Characiformes, Prochilodontidae): morphological identity and dispersion. Comparative Cytogenetics 9(1): 79-87. doi:
10.3897/CompCytogen.v9il.8587

Abstract

B chromosomes have attracted the attention of Neotropical fish cytogeneticists in recent years, both for
their remarkable occurrence in this group and also because of the interest in studies of the genetic structure
and role played in the genome of these organisms. The aim of this study was to report the first occurrence
of supernumerary chromosomes in Prochilodus argenteus (Agassiz, 1829), this being the fifth carrier species
among thirteen within the genus Prochilodus (Agassiz, 1829). The extra elements identified in this species
are small sized heterochromatic chromosomes characterized by a low mitotic instability index, being very
similar to other supernumerary chromosomes described in the species of the genus Prochilodus. Morphol-
ogy, structure and dispersion of the supernumerary genomic elements which occur in species of this genus
are discussed aiming to better understand aspects involved the origin of supernumerary chromosomes and

the differentiation process and relationships among species of this family.

Keywords

Curimbata, cytogenetic, neotropical fish, supernumerary chromosomes

Copyright Manolo Penitente et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC
BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

80 Manolo Penitente et al. / Comparative Cytogenetics 9(1): 79-87 (2015)

Introduction

Among the Characiformes fish, representatives of the family Prochilodontidae are dis-
tinguished by their abundance in the environments in which they occur, by their
wide distribution in South America and the high migratory capacity displayed by
the species of this group of organisms (Castro 1990, Sivasundar et al. 2001, Turner
et al. 2004). According to Castro and Vari (2004) the family Prochilodontidae con-
sists of three genera: Prochilodus (Agassiz, 1829), Semaprochilodus (Fowler, 1941), and
Ichthyoelephas (Posada Arango, 1909), which include thirteen, six and two species,
respectively. This family can be easily distinguished from other Characiformes families
through a distinct set of morphological characters (Castro 1990), and fishes of the
genus Prochilodus can be highlighted as a pioneer group in studies of B chromosomes
among Neotropical fishes.

Besides the conservative karyotype formulae found among representatives of the
genus Prochilodus, variation in the chromosome number can be observed among spe-
cies due to the occurrence of supernumerary chromosomes. Among seven species of
this genus, the occurrence of B chromosomes has been identified and described in four
species, present in Prochilodus lineatus Valenciennes, 1836 (Pauls and Bertollo 1983),
Prochilodus nigricans (Agassiz, 1829) (Pauls and Bertollo 1990, Venere et al. 1999),
Prochilodus brevis (Steindachner, 1874) (Pauls and Bertollo 1990) and Prochilodus
mariae Eigenmann, 1922 (Oliveira et al. 2003), but not occurring in cells of Prochilo-
dus argenteus (Agassiz, 1829), Prochilodus costatus (Valenciennes, 1850) and Prochilo-
dus vimboides (Kner, 1859), as described by Pauls and Bertollo (1990).

In this paper we describe for the first time the occurrence of supernumerary chro-
mosomes in P. argenteus, identifying its similarity with the extra genomic elements
characteristic of this group. The description of the occurrence of supernumerary chro-
mosomes in this species, besides addressing structural and morphological aspects, has
also broadened the knowledge of aspects involved in the differentiation, evolution and
relationships among species of the genus Prochilodus.

Material and methods

In this study 29 individuals of the species P argenteus (Table 1) resulting from crosses
performed in the fish farm Projeto Peixe, Cravinhos — SP, Brazil, involving matrices
derived from natural population of the Sao Francisco River, captured near Trés Marias
— MG, Brazil, were analyzed. The procedures for collection, maintenance and analysis
of the fish were performed in accordance with the international protocols on animal
experimentation followed by the Universidade Estadual Paulista. Morphometric and
meristic data were taken following Castro and Vari (2004) and specimens were de-
posited at Laboratério de Biologia e Genética de Peixes fish collection, Universidade
Estadual Paulista, Botucatu, SP. Vouchers of the used material are described in Table 1.

B chromosomes in the species Prochilodus argenteus (Characiformes, Prochilodontidae)... 81

Table |. B chromosome frequency and Mitotic Instability index (MI) of somatic cells in
P. argenteus.

Specimen Number of B per cell MI
denim [op Tap [aw Tap

4170 a OB 30 0.000
4172 1 12 1B 13 0.157
ee ee eee ee eee: 30 0.000
wu 4175 30 ee ee ed | ee 30 0.000
4176 3 27 : 1B 30 0.200
4177 1 29 , 1B 30 0.034
4178 2B 30 0.036
4180 1B 30 0.337
4181 1B 30 0.000
4182 2B 30 0.065
4183 1B 14 0.294
4184 1B 15 0.000
4188 OB 30 0.000
4189 OB 30 0.000
4190 1B 16 0.133
4250 1B 30 0.274
4251 OB 30 0.000
4252 1B 14 0.000
4253 OB 30 0.000
4254 1B 12 0.173
4256 2B 17 0.111
4257 25 ae |S bel | eee | Ms 25 0.000
4258 2B 14 0.072
4259 OB 30 0.000
4260 1B 30 0.136
4261 3B 30 0.082
4262 1B 18 0.337
4263 OB 30 0.000
4264 1B 14 0.152
2 0,136

(MB) modal number of B chromosomes, (N) number of metaphases analyzed, (MI) Mitotic Instability

index, (X,,,) average MI among individuals with supernumerary chromosomes.

Chromosome preparations involved previous use of mitosis stimulation (Lozano
et al. 1988, Oliveira et al. 1988) followed by cell suspension preparations using kidney
tissue fragments of the individuals, according to the protocol proposed by Foresti et al.
(1981). The karyotype organization was performed according to the method of Levan
et al. (1964), using images processed by Adobe Photoshop CS5 program. To quantify
the Mitotic Instability index (MI) the method of Pardo et al. (1995) was used. Active

82 Manolo Penitente et al. / Comparative Cytogenetics 9(1): 79-87 (2015)

nucleolar regions (NOR) in metaphase chromosomes were identified by using silver
nitrate staining (Howell and Black 1980) and the detection of constitutive heterochro-
matin (C-banding) was performed according to Sumner (1972).

Chromosomal mapping of ribosomal genes was performed with the technique
of fluorescent in situ hybridization (FISH) according to Pinkel et al. (1986) using 5S
and 18S rDNA probes obtained by PCR (Polymerase Chain Reaction) from genomic
DNA of P. argenteus. Primers A (5_-TACGCCCGATCTCG TCCGATC-3_) and B
(5_-CAGGCTGGTATGGCCGTAAGC-3_) (Pendas et al. 1994) were used to obtain
the 5S probe, and NS1 (5_-GTAGTCATATGCTTGTCTC-3_) and NS8 (5_-TC-
CGCAGGTTCACCTACGGA-3_) according White et al. (1990), to obtain the 18S
rDNA probe. The 5S probe was labeled with biotin-dUTP and the 18S probe was
labeled with Digoxigenin-dUTP (Roche) by PCR, according to the manufacturer’s in-
structions. [he preparations were stained with DAPI (4-6-diamidino-2-phenylindole)
and examined in a fluorescence light microscope (BX 50, Olympus) equipped with an
Olympus Q-color 5 digital camera. The photomicrographs were obtained using Q-
Capture Pro 5.1.1.14 software.

Results and discussion

Cytogenetic analysis performed on specimens of Prochilodus argenteus revealed the
diploid number of 2n = 54 and fundamental number of 108 for this species, with a
karyotype composed of meta and submetacentric chromosome types (Figure 1). No
morphological differentiation between males and females was detected, confirming
data published by Pauls and Bertollo (1983, 1990), Hatanaka and Galleti Jr. (2004)
and, more recently by Voltolin et al. (2013). This karyotype identity, also present in
other species already described is a conserved feature in the genus Prochilodus, and it
may be identified also among the components of related groups (Galetti Jr. et al. 1994,
Arai 2011). Among the 29 individuals analyzed the presence of up to three B chro-
mosomes was observed, with a modal number in metaphases analyzed between zero
and one B chromosome (Table 1, Figure 2), being that 14 individuals presented one B
chromosome, four presented two B chromosomes and a single specimen carried three
B chromosomes. Ten individuals were not carriers of supernumerary chromosomes in
their cells in this sample.

One of the first descriptions of B chromosomes in Neotropical fish refers to ad-
ditional chromosomes found in P. /ineatus by Pauls and Bertollo (1983). Since then
this species has been one of the most widely used in studies on the origin, inheritance
and maintenance of these chromosomes in fish. Moreover, the occurrence of supernu-
merary chromosomes was also described in other species within the genus Prochilodus
(Pauls and Bertollo 1990, Venere et al. 1999, Oliveira et al. 2003), adding to this list
the species P. argenteus taken in this work. The number of studied species thus high-
lights this genus as one of the most representative and significant among Neotropical
fishes concerning with the occurrence and study of these extra elements.

B chromosomes in the species Prochilodus argenteus (Characiformes, Prochilodontidae).... 83

WAR BA HAG KR BA RA KK KH AR KR

7 8 9 10

msm | 2K BM KA RR AM KK KE WB BN KA

11 14 15 16 1

7 9 20
21 22 23 24 25 26 27

Figure |. Karyotype of P. argenteus (2n=54 chromosomes). In the inset, three B chromosomes and the
NOR bearing pair.

Figure 2. Metaphase plates of P. argenteus showing a cell of individual without B chromosomes (a); in
individual presenting one B chromosome (b); cell presenting two B chromosomes (€); and cell presenting

tree B chromosomes indicated by arrows (d). Bar = 10 pm.

84 Manolo Penitente et al. / Comparative Cytogenetics 9(1): 79-87 (2015)

The supernumerary genomic elements found in this species can be easily identified
in metaphasic cells due to their small size when compared with the chromosomes of
the standard complement, usually heterochromatic, but presenting diverse and com-
plex patterns of heterochromatin distribution (Figure 3). Such microchromosomes
generally representing the metacentric type revealed no NOR mark after silver nitrate
staining. Similarly, analysis of in situ hybridization using 5S and 18S rDNA probes
did not reveal the presence of these genes in the supernumerary chromosomes (Figure
4). Visible signs of hybridization were observed in sinteny in one pair of chromosomes
in the normal complement, as previously reported by Hatanaka and Galleti Jr. (2004)
and Voltolin et al. (2013).

An interesting feature of B chromosomes present in the carrier species of this genus
is the identification as completely or partially heterochromatic microchromosomes,
which exhibit expressive inter and intra-individual number variation (Table 1). A
higher frequency of variation is found in P. lineatus, which includes individuals
without B chromosomes as well as others with up to nine supernumerary elements
(Voltolin et al. 2011). In other carrier species the frequency of occurrence is less
wide-ranging, with zero to three in P. mariae (Oliveira et al. 2003) and from zero to
two in P. brevis and P. nigricans (Pauls and Bertollo 1990, Venere et al. 1999). It can
be considered that the particularity of these genomic elements that do not follow the
Mendelian segregation laws in meiosis could determine their independent nature,
providing different ways of influence to their accumulation or disappearance during
the evolutionary process.

The occurrence of homoplasy could be considered to explain the origin of mor-
phologically similar genomic elements in species of the same biological group. Thus,
the existence of specific chromosomes bearing structural elements capable of originat-
ing extra chromosomes in an ancestral form could act in an independent way and give
rise to supernumerary chromosomes found today in some of these species. However,
it can be also considered the idea of a common origin of these elements in all species
of the genus from an ancestral carrier, followed by the loss in some species during the
diversification process.

The rate of mitotic instability (MI) calculated from individuals with supernumer-
ary revealed a mean value of 0.136 (Table 1). In studying the variation of MI in P. lin-
eatus, Cavallaro et al. (2000) observed a decrease of 0.486 to 0.004 among individuals
between the years 1979 to 1989, suggesting the occurrence of a stabilizing trend in the
population studied. Comparison of the above results with the mean value of MI found
in P. argenteus in the present study (MI = 0.136) permits the inference that the low
values of instability in the sample analyzed in this work presents could be an indicative
that the species is developing the stabilization of B chromosomes in somatic cells in the
course of next generations.

The great similarity found in morphology, size, heterochromatic nature and fre-
quency of B chromosomes of P. argenteus with those of other species in the genus
Prochilodus, described as microchromosomes and, in general, totally heterochromatic

B chromosomes in the species Prochilodus argenteus (Characiformes, Prochilodontidae)... 85

c
7 * ° , e ~e
4
’ . . -
- 7 ‘
. + . * ss _
i __ ow °
+ .* _ . : -.* 7 4
| . - "ss
= - o — - *
7 aA °
_ * _-
7 » . . . P “ — r . @
>
e 7 > 7 ’ s +. bh
+ 7 . . .
+:
e. . ° ¥ : —
‘+ e e a »®?
— > »

Figure 3. C-banding in metaphases plates of P. argenteus showing the distribution of heterochromatin
in B chromosomes. Different cells showing one B chromosome (a); two B chromosomes (b); and tree B

chromosomes indicated by arrows (c). Bar = 10 um.

Figure 4. Fluorescent in situ hybridization using probes of 5S (green) and 18S (red) rDNA in metaphasic
cells of P. argenteus, carrying one B chromosome (a) and two B chromosomes (b), indicated by arrows.

FISH marked segments are visualized only on the NOR bearing chromosomes. Bar = 10 um.

(Pauls and Bertollo 1983, 1990, Venere et al. 1999, Oliveira et al. 2003, Voltolin et al.
2013) can identify patterns of origin and dispersion of these genomic elements in this
group of organisms. [he morphological and structural analyses of these extra elements
in the genus Prochilodus could suggest a possible common mechanism of origin which
would manifest independently in the species of this genus, and then would follow their
own paths of differentiation and evolution.

Acknowledgements

The authors are grateful to Suely Marlene Rodrigues from Projeto Peixe fish farm for
providing fish samples used in the present work. Funds supporting this study were

provided by Fundacao de Amparo 4 Pesquisa do Estado de Sao Paulo (FAPESP).

86 Manolo Penitente et al. / Comparative Cytogenetics 9(1): 79-87 (2015)

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